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1
Gene Regulation amp Epigenetics
Concepts and Applications to Drive Discovery
Dandan Zhong PhD
Senior Field Application Scientist
Merck Millipore Bioscience
2012 December
2
Millex filter
Amicon Ultra
Immobilon PVDF
Millipore
2
About Merck Group
Established in 1668 in Germany
The oldest pharmaceutical and chemical company in the world
40000 employees in 67 country worldwide
Total revenues of EUR 93 billion in 2010
Acquired Millipore in 2010
wwwhistorymerckde
Headquarter
Darmstadt Germany
Merck amp Millipore A Perfect Fithellip
3
Millipore becomes the Life Science Division of Merck in the Chemicals Sector
Chemicals
(Business Sector)
Pharmaceuticals
(Business Sector)
Merck KGaA
Merck Millipore
(Division)
Performance Materials
(Division)
Merck Serono
(Division)
Consumer Health Care
(Division)
6
Star Product lines
bull Epigenetics
bull Drug Screening
bull Stem Cell
bull Neuroscience
bull Metabolic disease
bull (ELISA amp Milliplex)
bull Apoptosis
bull wwwapoptosiscom
now a part of Merck Millipore
4
7
Merck Millipore is a division of
bull Small Molecule Inhibitors
bull Oncology
bull Prokaryotic Expression(PET)
bull Insect Expression
bull Protein Extraction amp Purification
now a part of Merck Millipore
8
Drug Discovery
Cell Signaling
Epigenetics
Stem Cells
Neuroscience
Antibodies
Assay Kits
Instruments
Biotools
Cell culture media amp plates
Cells
Merck Millipore Today Total Solutions for Life Science Research
Cancer Research
Protein Research Inhibitors amp Kinases
5
Epigenetics vs Genetics
Central Dogma
DNA Protein RNA
Classical genetics
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
2
About Merck Group
Established in 1668 in Germany
The oldest pharmaceutical and chemical company in the world
40000 employees in 67 country worldwide
Total revenues of EUR 93 billion in 2010
Acquired Millipore in 2010
wwwhistorymerckde
Headquarter
Darmstadt Germany
Merck amp Millipore A Perfect Fithellip
3
Millipore becomes the Life Science Division of Merck in the Chemicals Sector
Chemicals
(Business Sector)
Pharmaceuticals
(Business Sector)
Merck KGaA
Merck Millipore
(Division)
Performance Materials
(Division)
Merck Serono
(Division)
Consumer Health Care
(Division)
6
Star Product lines
bull Epigenetics
bull Drug Screening
bull Stem Cell
bull Neuroscience
bull Metabolic disease
bull (ELISA amp Milliplex)
bull Apoptosis
bull wwwapoptosiscom
now a part of Merck Millipore
4
7
Merck Millipore is a division of
bull Small Molecule Inhibitors
bull Oncology
bull Prokaryotic Expression(PET)
bull Insect Expression
bull Protein Extraction amp Purification
now a part of Merck Millipore
8
Drug Discovery
Cell Signaling
Epigenetics
Stem Cells
Neuroscience
Antibodies
Assay Kits
Instruments
Biotools
Cell culture media amp plates
Cells
Merck Millipore Today Total Solutions for Life Science Research
Cancer Research
Protein Research Inhibitors amp Kinases
5
Epigenetics vs Genetics
Central Dogma
DNA Protein RNA
Classical genetics
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
3
Millipore becomes the Life Science Division of Merck in the Chemicals Sector
Chemicals
(Business Sector)
Pharmaceuticals
(Business Sector)
Merck KGaA
Merck Millipore
(Division)
Performance Materials
(Division)
Merck Serono
(Division)
Consumer Health Care
(Division)
6
Star Product lines
bull Epigenetics
bull Drug Screening
bull Stem Cell
bull Neuroscience
bull Metabolic disease
bull (ELISA amp Milliplex)
bull Apoptosis
bull wwwapoptosiscom
now a part of Merck Millipore
4
7
Merck Millipore is a division of
bull Small Molecule Inhibitors
bull Oncology
bull Prokaryotic Expression(PET)
bull Insect Expression
bull Protein Extraction amp Purification
now a part of Merck Millipore
8
Drug Discovery
Cell Signaling
Epigenetics
Stem Cells
Neuroscience
Antibodies
Assay Kits
Instruments
Biotools
Cell culture media amp plates
Cells
Merck Millipore Today Total Solutions for Life Science Research
Cancer Research
Protein Research Inhibitors amp Kinases
5
Epigenetics vs Genetics
Central Dogma
DNA Protein RNA
Classical genetics
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
4
7
Merck Millipore is a division of
bull Small Molecule Inhibitors
bull Oncology
bull Prokaryotic Expression(PET)
bull Insect Expression
bull Protein Extraction amp Purification
now a part of Merck Millipore
8
Drug Discovery
Cell Signaling
Epigenetics
Stem Cells
Neuroscience
Antibodies
Assay Kits
Instruments
Biotools
Cell culture media amp plates
Cells
Merck Millipore Today Total Solutions for Life Science Research
Cancer Research
Protein Research Inhibitors amp Kinases
5
Epigenetics vs Genetics
Central Dogma
DNA Protein RNA
Classical genetics
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
5
Epigenetics vs Genetics
Central Dogma
DNA Protein RNA
Classical genetics
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
6
Typical Example of Epigenetics Regulation
12
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
7
What is Epigenetics
13
Definition of Epigenetics
Changes gene expression
No Change in DNA sequence
Heritable but potentially reversible
DNA Protein RNA
Epigenetic Potential for Therapeutic Intervention
Most dynamic Most permanent
Deletion
(amplification)
Chromosomal
Translocation
Point mutations
Genetics
Chromatin
Changes
Promoter
Methylation
Silencing
Cell-cycle
Regulated
Changes
Transcription
Factors
Response To
Environmental
Stimuli
Epigenetics
Adapted from James Herman
DNMT1i
Decitabine-Dacogen 2006
HDACi Vorinostat-Zolinza 2006
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
8
16
How to Study Epigenetics
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
9
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
Chromatin Packaging
Eukaryotic DNA is packaged as chromatin
Histone octamer
ndash 2 molecules each of H2A H2B H3 amp H4
Histone Octamer + DNA
ndash Nucleosome
H1 + Nucleosome
ndash 30 nm fiber
ndash Scaffolding Proteins (Nuclear Matrix Attachment)
ndash Topoisomerases (Supercoiling)
Multiple levels of compaction
Heterochromatin vs Euchromatin
Felsenfeld et al Nature 2003
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
10
20
Histones
H1 H2A H2B H3 H4
bullRich in Arg amp Lys (Stablize the electrostatic
interaction with DNA)
bullMost conserved proteins
The Universe of Histone Modifications
Regulate biological function either by
bull Recruiting other protein ldquoreaders writers erasersrdquo
bull Changing chemical structure to allow DNA access or nucleosome
repositioning or remodeling
bull ldquoHistone coderdquo
Bhaumik et al NSMB 2007
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
11
22
Histone Covalent Modifications
bull Both N- and C- terminals
bull Most Modifications on N-terminal
Acetylation
Phosphorylation
Methylation
Ubiquitination
Sumoylation
Citrullination
Ribosylation
Crotonylation
Biotinylation
The Universe of Histone Modifications
23
Acetylation
bull Histone aceytltransferase (HAT)
bull Histone deacetylase (HDAC)
bull Reversible amp Dynamic
bull Acetylation activates gene transcription
bull Deacetylation acts contrarily
P300
PCAF H2B
PEPAKSAPAPKKGSKKAVTKAQKK 5 12 15 20
Ac Ac Ac Ac
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 5 8 12 16
H4
Ac H2A
SGRGKQGGKARAKAKSRSSRAGLQ 5 9
Ac
H3
ARKSTGGKAPRKQLATKAARKSAP 9 14 18 23 27
Ac Ac Ac Ac Ac
Ac Ac Ac Ac
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
12
24
HATsHDACs amp CancerCancer therapy
o Many tumor suppressor genes are silenced by a mechanism involving DNA methylation and histone
deacetylation contributing to many types of cancer HDAC inhibitors currently show tremendous early promise as
treatment agents
o The 1st drug of this type suberoylanilide hydroxamic acid (Verinostat MSD) was approved by the FDA for the
treatment of cutaneous T-cell lymphoma in 2006
33
Methylation
JHDM3 LSD1 PAD4
JHDM3
bull On Arg Lys
bull Reversible
bull Single- di- tri- methylation
bull Activate or repress gene expression
Regulate
bull Cell cycle
bull Cell proliferation
bull Chromatin structure
bull Early development
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
13
Disease Associated Genetic Alterations in Protein Methyltransferases
Copeland Drug Discovery
Today In Press 2011
36
Phosphorylation
Main function
bullCell cycle
bullMeiosis
bullApoptosis
bullDNA damage repair
bullDevelopment
bullCell division
H2A
SGRGKQGGKARAKAKSRSSRAGLQ
P
1
H2B
PEPAKSAPAPKKGSKKAVTKAQKK 14
P
H3
ARTKQTARKSTGGKAPRKQLATKAARKSAP 3 1011 28
P P P P
SGRGKGGKGLGKGGAKRHRVLRDNIQGIT 1 18
H4 P P
bull On Ser Thr
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
14
38
Histone H2AX Phosphorylation Ser139
H2AX variant of H2A has unique C-term
sensor for DNA double-strand breakage
involve in DNA damage repair
H2AX deficiency related to tumorigenesis
bullGood marker for early apoptosis
bullSensitive readout for DNA damage
H2AX Phosphorylation assay (Cat 17-327)
Cell-based ELISA (CELISA)
Log phase NIH3T3A31 cells were treated for 2
hours with increasing Etoposide
40
H2AX Phosphorylation Assay Kit (17-327)
bull Chemiluminescent
bull Detect signals in cells
bull Avoiding lysing cells to protect Phosphorylation signal
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
15
42
H2B Serine 14 Phosphorylation Apoptosis marker
Detection of H2B phosphorylated at serine 14 in
HL-60 cells stimulated to undergo apoptosis by
treatment with VP-16 Left anti-phospho
histone H2B (Ser14) (07-191) staining Right
DAPI staining Courtesy Dr David Allis The
Rockefeller University
Asynchronous HeLa cells stained with anti-
phospho H3 (Ser10 ) (cat 06-570) in red
and anti-beta tubulin (cat 05-661) in green
Serine 10 phosphorylation of H3 is almost
exclusively observed on condensing mitotic chromosomes (arrows)
Phosphorylation of H3 ( Ser10)
cell mitosis marker
Left anti-phospho
histone H2B (Ser14)
(07-191)
Right DAPI
44
Ubiquitinization
Single Ubiquitin ligation(different from Hydrolysis)
H2A(5-15)和H2B(1-15)
Change Chromatin structure expose DNA start transcription
Recruit transcription factor
Regulation other Histone modifications
eg H2BK123 Ubiquitination by Rab6 amp H3K4 Methylation
SGRGKQGGKARAKAKSRSSRAGLQ
H2A
119
u
120
H2B
PEPAKSAPAPKKGSKKAVTKAQKK
u
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
16
45
Sumoylation
Small ubiquitin-related modifier (SUMO)
Not hydrolysis
H2AH2BH4
Inhibit gene transcription
Merck Millipore Provide
bull anti-SUMO-1 anti-SUMO-23 抗体
bull SUMO1 SUMO2 SUMO3 SUMO Activating Enzyme
46
Citrullination
bull Rheumatoid athritis
bull MS
bull Alzheimerrsquos disease
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
17
Histone Code Where To Start
H3 N- hellipK4hellipK9S10helliphellipK27hellipK36
me2 me3
Ac
me3
Ac
Ph
Ac
me3
- active promoters -repressed genes (PRC2)
bivalent promoters
- transcribed exons - heterochromatin
me3
me1 - active enhancers
me3 - bivalent promoters
- active genes
- active genes - active enhancers
- mitosis
Distinct Chromatin Architectures Demarcate Regulatory Elements in Genome
Example of the actively transcribed gene
Bell et al Nature Reviews Genetics 2011
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
18
High Low Probability
Enhancers
BivalentSilence
Initiation
Elongation Desert
Heterochromati
n Insulator
Ernst et al Nature Biotechnology 2010
Genomic Elements and Chromatin State
TSS
UTR
Exons
Intron Promoter Enhancer
LINEs SINEs
Heterochromatin
H3K4me1
AcH3K27
H3K9me3
H3K36me3 AcH3K9
AcH4
H3K4me3
Active Promoter
H3K27me3
Inactive Promoter
H3K36me3
H4K20me1
AcH3K9
Centromere
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Chromatin Associated Non-Coding RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
19
53
Pro
DNA
Why we need ChIP analysis
Transcriptional Regulation
Transcription factors chromatin modifiershellip
ChIP is the most common in vivo method used to study
endogenous proteins associated with chromatin
ChIP can show the actual gene regulation under physiological
status
Chromatin IP (ChIP)
A method to identify specific proteins associated with a region of DNA or to identify regions of DNA associated with specific proteins
56
ChIP Workflow
N - + I
Experimental Design +- chemical cytokine hormone RNAi transfection etc Fixation
+- crosslinking harvest and count cells
Fragmentation Sonication enzymatic treatment
Ab selection amp Immunoprecipitation Polyclonalmonoclonal protein AG
agarose or magnetic beads
DNA Recovery Digest protein remove beads
reverse crosslinks extractpurify DNA
Detection PCR qPCR Microarray
1
2
3
4
5
7
6
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
20
Critical Considerations for Successful ChIP
bull Experimental design
ndash Epitope abundance biology of ChIP targets planning for controls (control Ig and primers for positive or negative loci)
bull Chromatin preparation
ndash Evaluate chromatin with validated assays optimize protocol for each cell line amp keep consistent
bull Antibody quality and IP strategy
ndash Use well characterized antibodies
ndash Consider all available applications for unknowns
bull Data Analysis
ndash Utilize controls consider assay performance (qPCR standard curve)
ndash Test enrichment by qPCR before running genome wide experiment
WB
IP
IFIHC
ChIP
Pre
dic
tive
Ch
IPa
bil
ity
59
Experimental Designcell number
Examples In one cell type (HeLa epithelial carcinoma) assaying an active promoter
Target
Abundance Target
Cells
ChIP
ChIPs
15 cm Plate
High Total Histone H3 1 X 105 100
Medium-High Acetyl Histone H3 5 X 105 20
Medium RNA Polymerase II 1 X 106 10
Low Transcription Factor X 2 X 107 05
Extremely Low Liver specific Txn Factor infin NA
1
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
21
Standard ChIP Experiment with Biological Validation
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3
(Lys27) with human α-Satellite (Fold
Enrichment relative to mock IgG control)
ChIP Ab+ Antibodyprimer set (Cat 17-622)
specifically detects Trimethyl-Histone H3 (Lys27)
with human Hela cells (Fold increase relative to
mock IgG control)
Mock IP vs Specific IP is useful
but location controls are valuable toohellip
Experimental Design 1
Mock IP vs Specific IP is useful but location controls are valuable toohellip
002 004 003 003001 002
043
051
035038
028033
000
025
050
075
C-M
yc
B-G
lobin
Myo
D
GAPD
H In
tron
SYN R
E-1
GAPD
H C
oding
Pe
rce
nt
of
Inp
ut
001006
002
060
000
020
040
060
080
IgG TCF4 IgG TCF4
Pe
rce
nt
Inp
ut
GAPDH
Promoter
Sp5
Promoter
17-10109 ChIPAb+ Tcf-4
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
22
Fixation
Does not require fixation
ndash Gentler but less robust method
ndash May require more cells
ndash Often used with enzymatic digestion instead
of sonication
ndash Formaldehyde crosslinking may mask epitope
Requires high affinity interactions
ndash Histones and Histone modifications
ndash Requires fixation (ie
Formaldehyde 1 10min at
37degC)
ndash More common for detecting
weaker interactions
ndash Interrogation of complexes
ndash Over-crosslinking can
increase artifactual
interactions
ndash Crosslinking can mask the
epitope
Native ChIP (N-ChIP) ChIP (X-ChIP)
2
Chromatin Fragmentation
Required to solubilize chromatin
Produce fragments from 300 bp to 1 Kb
Largely random fragmentation
Many instrument models with varying probe sizes
power settings hellip
Performance varies with volume of sample and
cell density
Foaming denaturation shearing of target protein
Can be challenging requires optimization
Sonication
bull Micrococcal nuclease
bull Can achieve single nucleosome resolution
bull Restriction endonucleases
Enzymatic Treatment
Nucleosomal
Ladder
Increasing Cycles of Sonication
Cat 17-375 EZ-Zyme
Cat 12-704 ChIPable Chromatin HEK293
Cat 12-705 ChIPable Chromatin HT29
Cat 12-687 ChIPable Chromatin HeLa
ChIPable Chromatin Sonicated formalhehyde-fixed
chromatin
TroubleShooting
Has the DNA shearing been
checked on an agarose gel
3
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
23
69
Antibody
Not all antibodies are suitable for ChIP
Specificity of epitope is critical
bull Epitope(s) must be exposed in native conformation
Epitope(s) must not be masked by crosslinking
Antibody Selection Polyclonal vs Monoclonal Purified vs Crude
Use ChIP validated Abs where possible
If unavailable consider IHC EMSA IP validated antibodies
ChIP validated Abs
4
70
IP Magnetic vs Agarose Beads
Agarose Beads
More cost effective
High background need blocking
(BSA carrier DNA)
Pre-clear samples
Perform -+ antibody controls
Magnetic Beads
No centrifugation Fast
Smooth surface No need blocking
Less unspecific binding
Less Sample amp less Ab
Ability to automate Ideal for high
throughput
ChIP on chip where salmojn sperm DNA
can be problematic
5
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
24
DNA Recovery And Detection 6
Safety and disposal issues
Perceived as cost effective
Useful for low amount of DNA
Easily have protein contaminant
Improved Purity
Easier to handle
Faster processing time
PhenolChloroform Column Purification
In the classical ChIP Kit In the EZ-ChIP Kit
72
Detection method after ChIP
PCRqPCRGene specific known binding sequence
ChIP-CloningFind unknown binding sequence
ChIP-SequencingFind unknown binding sequence
ChIP reChIPDetect the co-binding sequence by two
proteins
Genomic tiling micro-array (ChIP chip)Genome-wide
analysis
7
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
25
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Repeatable results
Presentation title in footer | 00 Month 0000 73
An Assessment of Histone-Modification Antibody Quality by modENCODE Group
Egelhofer et al Nature StructuralampMol Biol 2010 httpcompbiomedharvardeduantibodies
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
26
Antibody Quality--Validation Data
H3K4me3 affinity purified
Polyclonal Ab Cat 07-473
No
ne
Un
mo
dif
ied
H3K
4m
e1
H3K
4m
e2
H3K
4m
e3
H3K
9m
e3
H3K
27m
e3
Single band on WB
Peptide Blocking
H3K4me3 Recombinant Rabbit
Monoclonal Ab Cat 05-745R
H3K4me3 H3K4me3gtH3K4me2
40 ng
4 ng
Dot Blot
All peptides contain H3K4me23
Peptide Microarray
Biological Specificity in ChIP
AbSurancetrade Histone Antibody Specificity Arrays
Cat 16-665 Histone H2A H2B H4 Array
Cat 16-667 Histone H3 Array
Cat 16-668 Complete set (H2A H2B H3 H4 Array)
AbSurancetrade
Arrays
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
27
Concerns of Researchers doing ChIP
Successful ChIP
Lower background
Specificity
Reproducible results
Presentation title in footer | 00 Month 0000 78
79
How to get reproducible ChIP results
Accurate Cell counts
超声周期的增加 酶消化
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
28
bull Speedy 14sec
bull Accurate
Cell Count Accuracy mdashmdash the basis for good and reproducible results
82
超声周期的增加 酶消化
How to get reproducible ChIP results
Accurate Cell counts
Same assay condition
Chromatin Fragmentation
bull Sonication optomization same
sonication condition for same
sample amount and sample
volume
bull Enzymn digestion
bull DNA input
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
29
83
Merck Millipore provides 3 Generation of ChIP kits
17-295 ChIP Kit
17-245 ChIP Acetyl H3
17-229 ChIP Acetyl H4
17-371 EZ-ChIP
17-375 EZ-Zyme
ChIP (bare bonesmdashexperienced users)
EZ-ChIP (controls buffers spin filtersmdashNew users)
Magna ChIP (faster cleaner higher throughput)
17-61011 Magna ChIP Protein AG (buffers spin filters)
17-4089 EZ-Magna ChIP AG (complete controls)
1
2
3
The first ChIP kit in the world
Most citation
Magna ChIPtrade G Tissue Kit
Presentation title in footer | 00 Month 0000 84
New
300 microm of coronal mouse brain section with region specific
microdissection of the hippocampus (A) and cerebral cortex
(B) The isolated tissue was placed above the dissected region
(a hippocampus b cortex)
bullOptimized protocol for Tissue ChIP
Reproducible amp reliable ChIP from
small amounts of tissue
Site specific ChIP analysis using
Microdissection of 1mm3 tissue
sections using included tool
Includes tissue stabilization and
lysis reagents
Proven performance in a variety of
tissues
Expert support from the leader in
chromatin immunoprecipitation
bullTissue samples are more complex and the cells are
heterogeneous thus making ChIP analysis less amenable
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
30
Magna ChIPtrade HT96 17-10077 17-10078
85
17-10071 Magna GrIP HY96 Rack
New
bull High throughput
bull Less sample
- 104~105 cellswell
bull Less Antibody
bull Better reproducibility
bull Easier to perform
- Single buffer system
- Protein AG blend
86
ChIP Ab+
bull Include negative control Ab
bull qPCR control primers
bull Strict QC Every lot gets ChIP and qPCR tested
Merck Millipore provide ChIP validated Antibody bull gt166 Histone and Modified Histone Abs
bullgt49 Histone-Modifying Enzyme Abs
Donrsquot forget high quality ChIPable Abs
gt20000 Epigenetic papers using MM antibodies
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
31
ChIP Related Products and Accessories
EZ-Zyme Chromatin Prep Kit (cat 17-375)
Enzymatic approach
Alternative to sonication
Compatible with cross-linked or native chromatin
Magna ChIP Protein A Beads (cat16-661 and 16-661X)
Magnetic beads optimized for ChIP
Magna ChIP Protein G Beads (cat 16-662 and 16-662X)
Magnetic beads optimized for ChIP
Magna ChIP Protein A+G Beads -New (cat 16-663 and 16-663X)
Magnetic beads optimized for ChIP
Magna GrIP 8-well Rack (Cat 20-400 )
For Magna ChIP beadskits only Low cost option
PureProteometrade Magnetic Stand (Cat LSKMAGS08)
Universal Rack for protein IP or ChIP
Feature rich rack
Protein A Agarose Beads (Cat 16-157)
Blocked for ChIP
Protein G Agarose Beads
Cat 16-201 blocked for ChIP
EZ-Zyme Digested Chromatin
Nucleosomal
Ladder
90
Histone amp Histone modifying Enzyme Products
Antibodies166 Histone and Modified Histone Abs
49 Histone-Modifying Enzyme Abs
Kitsp300CBP Immunoprecipitation HAT Assay Kit (17-284) HAT
Assay Kit (17-289) HDAC Assay Kit (17-320 17-372 17-374)
Histone Methyltransferase Assay Reagent Kit (17-330) Phospho-
Histone H3 (Ser10) Assay (HCS204) etc
Proteins amp Enzymes62 Histone (Histone H3 H4 Acetyl-H4 (Lys5)
Acetyl-H4 Site Specificity Pack etc) and Histone-Modifying
Enzymes (p300 HDAC8 SET9 PRMT1 etc)34 Control
Histone Protein and Peptides
InhibitorNicotinamide (19-171) Trichostatin A (19-138) Sodium
Butyrate (19-137) 等
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
32
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene Regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
92
What Happens After Transcription Post Transcriptionhellip
ChIPTranscriptional Regulation
Transcription factors chromatin modifiershellip
Post-transcription regulation
has pivotal roles for development metabolism neuronal function and aging
Splicing transport localization translation degradation of mRNA and non-
coding RNAs
temporally and coordinately influence the rate of protein synthesis
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
33
96
Various function of RNA
RNA Function Messenger RNAs
(pre-mRNA mRNA)
Protein Coding
Small Nuclear RNAs
(snRNA snoRNA)
Splicing
Ribosomal RNAs
(rRNA)
Translation (sequence recognition)
Transfer RNAs
(tRNA)
Translation (amino-acyl peptide formation)
7S xist piwi others Specialized Functions
(Telomeres X inactivation)
Non-coding RNAs
(ncRNA miRNAmicroRNA siRNA shRNA)
RNA Stability and Translation
RNA-binding proteins (RBPs)
bull Play a key role in post-transcriptional regulation of expression
bull Involved in every step of mRNA and ncRNA metabolism
bull mRNA Processing (capping polyadenylation)
bull RNA splicing
bull Stability
bull Transport
bull Translational regulation (rate amount of protein synthesis
bull ncRNA processing and function
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
34
98
~10 or more of disease associated
point mutations affect splicing
Examples of Diseases involving RBPs and RNA
Disease Genemutation Function
Fragile X Mental Retardation FMRP mRNA Transport
Spinal Muscular Atrophy (SMA) SMNs Splicing
Myotonic Dystrophy 1 (DM1) DMPK Sequestors RNA regulators
Cancer miR-372 miR-373 RNA interference
Cancer miR-17-92 cluster RNA interference
Cancer SFRS1 Splicing translation export
Cancer RBM5 Splicing
RIP Analysis of RNA-Binding Proteins in a ChIP-Like Way
RNA-binding protein immunoprecipitation (RIP) RNA analog of ChIP
Immunoprecipitate endogenously formed complexes of RBPs and RNAs and isolate the RNAs associated with the RBPs
The isolated mRNAs (and non-coding RNAs) can be directly measured using down stream applications including qRT-PCR microarray analysis (RIP-chip) and 2nd-generation sequencing (RIP-Seq)
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
35
101
Transcription
Processing
Transport
Stability
Localization
Translation
Post-transcription
nucleus
cytoplasm
RNA-Binding Protein
Regulated (RIP)
DNA-Binding Protein
Regulated ( ChIP)
102
Merck Millipore provides RIP kits amp RIPAb+
03-101 RIPAb+ PABPC1
03-102 RIPAb+ HuR
03-103 RIPAb+ SNRNP70
03-104 RIPAb+ CUGBP1
03-105 RIPAb+ Lin28
03- 248 RIPAb+ pan Ago
03- 246 RIPAb+ FXR2
03- 245 RIPAb+ Hexim 2
03- 205 RIPAb+ hnRNP C1C2
03- 206 RIPAb+ hnRNP U
03- 116 RIPAb+ STAU1(Staufen1)
03- 107 RIPAb+ EF1alpha
amp more 100
30469
0
50
100
150
200
250
300
350
400
IgG PABPC1
Fo
ld E
nri
ch
me
nt
B-Actin RT-PCR HeLa RIP IP
Part Product Reaction
17-701 EZ-Magna RIP Kit 12
(includes positive controls)
17-700 Magna RIP Kit 12
17-704 Magna RIP Quad Kit 48
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
36
103
RBP (RNA Binding Protein) Antibodies gt80种 covering many neuro targets ncRNA biogenesis splicing regulatorshellip
04-321 Anti-4E-BP1 Rabbit Monoclonal
05-490 Anti-ACTRAIB1 clone AX15
07-599 Anti-Ago 1
07-590 Anti-Ago 2
04-085 Anti-Ago family
04-083 Anti-Ago1 clone 6D82
04-642 Anti-Ago2 clone 9E82
04-084 anti-Ago2 clone 9E82
05-967 Anti-Ago4 clone 5F92
09-456 Anti-AIRE 100 microg
07-210 Anti-AKAP 150 200 microL
07-235 Anti-AKAP 79 200 microL
06-417 Anti-AKAP 95 200 microg
07-260 Anti-AUF1
AB10004 anti-BARD1 100 microL
AB16507 Anti-Bnip3L internal 100 microg
05-641 Anti-BRAF35 clone 421
07-306 Anti-CtBP-1 200 microg
05-621 Anti-CUG-BP1 clone 3B1 200 microg
04-047 Anti-CuGBP2 Clone 1H2 100 microg
04-721 Anti-Dicer1 clone 5D122
07-717 Anti-Drosha
07-397 Anti-Ebp1
05-235 Anti-EF1α clone CBP-KK1
07-589 Anti-EF2 Kinase
AB3251 Anti-eIF4E Binding Protein
04-347 Anti-eIF-4E CT Rabbit Monoclonal
07-1568 Anti-ESETSetDB1
07-378 Anti-ESETSetDB1
MAB2160 Anti-Fragile X Mental Retardation Protein clone 1C3 100 microL
07-504 Anti-hnRNP A0
04-088 Anti-hnRNP K clone F45P9C7
05-620 Anti-hnRNP M1-M4 clone 1D8
05-689 Anti-HP1α clone1519s2 200 microg
AB5969 Anti-HuB
AB5829 Anti-HuC
AB15882 Anti-HuC
AB5971 Anti-HuD
AB5831 Anti-HuR
07-1735 Anti-HuR
07-468 Anti-HuR
AB15506 Anti-Iron regulatory protein 1 50 microg
AB15508 Anti-Iron Regulatory Protein 2
MAB5582 Anti-Iron Regulatory Protein 2
07-1385 Anti-Lin28
04-048 Anti-MBNL clone 3A4
07-575 Anti-MDM2
MAB3776 Anti-MDM2 clone SMP14
MAB10085 Anti-MSI2 clone 1F2
AB5977 Anti-Musashi
AB15648 Anti-Musashi
07-637 Anti-Nova-1
MAB1287 Anti-Nuclear Ribonucleoprotein clone 58-15 100 microL
05-565 Anti-Nucleolin
AB15244 Anti-NXF2
05-257 Anti-p300 CT clone RW128 200 microg
05-950 Anti-p54nrbNonO clone 78-1-C6 100 microg
05-850 Anti-p68 clone PAb204 200 microg
05-847 Anti-PABP clone 10E10
MAB11015 Anti-PABPC4 clone 6E12
AB3242 Anti-PGC-1
07-823 Anti-phospho eIF4E (Ser209)
07-822 Anti-phospho-eIF2Bε (Ser539)
07-760 Anti-phospho-eIF-2α (Ser51)
04-342 Anti-phospho-eIF-2α (Ser51) rabbit monoclonal
07-824 Anti-phospho-eIF4G (Ser1108)
07-566 Anti-phospho-hnRNP A0 (Ser84)
MAB10104 Anti-PUM2 clone 1E10
AB9904 Anti-QKI-5
AB9906 Anti-QKI-6
AB9908 Anti-QKI-7
MAB10105 Anti-RBMS3 clone 1H6 100 microL
05-839 Anti-RNase L clone 2G5
AB4146 Anti-RO52 100 microg
MAB385 Anti-S100L Protein clone S100-14 100 microg
06-891 Anti-SMRTe 200 microg
AB5781 Anti-Staufen
MAB3672 Anti-TATA-Binding-Protein-Associated Factor II68 aa 175-414 100 microL
104
The first RIP kit user in China published RIP result on Nature Immunology
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
37
RNA Detection in Live Cells
SmartFlareTM RNA detection Probe
Presentation title in footer | 00 Month 0000 105
Novel
bull Simple overnight mix with cells
bull Stable 1-3 days
bull Non-toxic Cells reusable
bull Sensitive
Now you can know Whatrsquos Really Happening
Presentation title in footer | 00 Month 0000 106
Applications
bull RNA detection in live cells
bull miRNA or mRNA quantification
bull Real time analysis
bull Detection of different biomolecules in
the same sample (ie protein + RNA)
bull Cell Sorting based on an RNA target
bull Isolation of pure cell populations
bull Monitoring of transfection efficiencies
bull Multiplex up to 3 RNA targets
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
38
Agenda
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
bull Cancer hypomethylation of repetitive regions (reactivation) and aberrant hypermethylation of CpG islands (silencing)
bull CpG islands ndash unmethylated GC-rich regions found upstream of ~60 of human genes
bull Most methylation occurs in repetitive genomic regions (SINE LINE retroviruses)
bull 70-80 of all 5rsquohellipCpGhellip3rsquo dinucleotides are methylated
bull m5C accounts for ~1-8 of total DNA bases
Mammalian DNA methylation patterns vary in time and space
DNA Methylation Modifications
bull Cellular Differentiation stem cell neuronal hydroxymethylation
Methylation Oxidation
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
39
Functions of DNA Methylation and Connection to Chromatin Remodeling
bullImprinting
bullX-Chromosome Inactivation
bullAging
bullNutrient Sensing
bullCellular Differentiation
bullGenomic Diversity (Transposons)
Feinberg amp Tycko Nature Reviews Cancer 2004
DNA Methylation Technologies
How is it studied
Bisulfite modification followed by MSP or sequencing
Methylated DNA enrichment
ndash Methylated DNA immunoprecipitation (MeDIP)
ndash Enrichment using methylated DNA binding proteins (MBD)
Methylation-specific restriction digestion
ndash Methylation-sensitive (cuts only unmethylated DNA)
ndash Methylation-specific (cuts only methylated DNA)
Many Epigenetics researchers use bisulfite modification as the
technique of choice to study DNA methylation because of high
resolution
Harris et Al Nat Biotechnol 2010 Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic
epigenetic modifications
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
40
A Toolbox for Cytosine Methylation Analysis
Method Advantages Disadvantages
Sodium
Bisulfite
Conversion
Resolution at the nucleotide level bull Harsh chemical treatment of DNA can
cause its damage
bull Potential incomplete conversion or
over-conversion artifacts
bull Not 5-mC and 5-hmC specific
MeDIP bull Fast
bull Applicable for NGS and arrays
bull Requires specific antibody
bull May require more than on mC for Ab
binding
bull Requires DNA denaturation
bull Medium level resolution
Methylated
DNA-binding
Proteins
bull No DNA denaturation required
bull Applicable for NGS and arrays
bull Medium level resolution
Methylation
Specific
Restriction
Enzymes
bull Easy to use
bull Availability of recombinant
enzymes
bull Limited by the enzyme recognition site
(low resolution)
bull Lower throughput
Platform Technologies for DNA Methylation
Laird Nat Rev Gen 2010
Applications by
Detection
Platform
Resolution amp Sample
Quantity by
Appplication
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
41
113
MSP
BLAH U U U U U
Challenge 1 Bisulfite Conversion is a Balancing Act
Schumaker methologixcom
Genereux et al NAR 2008
Original ATCGATCATCGATCCAT
Converted ATCGATTATCGATTTAT
Incomplete conversion ATCGATCATCGATTTAT
Inappropriate conversion ATCGATTATTGATTTAT
mC hmC
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
42
CpGenome Turbo Bisulfite Kit Fast Efficient and Easy Speedy amp Simple
ndash The fastest most efficient and easiest bisulfite
modification kit from the epigenetics market leader
bull Features and Advantages
ndash Complete protocol in lt90 minutes
ndash 999 conversion efficiency without over-conversion
ndash Convert from 1 ng to 1 mg of DNA
ndash Recover modified DNA in as little as 25 ml
ndash Proven performance in multiple downstream
applications
0
2
4
6
8
Ho
urs
Protocol length 15 3 6
Millipore Supplier Z Supplier Q
18
22
26
30
34
38
Cycle
Th
resh
old
M DNA 3365 3385
Millipore Supplier Z
DNA Quality Influences The Extent of Bisulfite Conversion
Warnecke et al Methods 2002
Complete protein removal is essential for avoiding artifacts
Non-CpG methylation
observed in crude genomic
preparations in GSTPI promoter
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
43
119
httpwwwurogeneorgmethprimerindex1html
MSP primer design
Challenge 2 MSP Prime Design
Common methods of DNA methylation
Methylation-Specific PCR(MSP)
DNA Microarray
Restriction Landmark Genomic Scanning
Whole Genome Bisulfite Sequencing
Methylated
DNA of interest
Downstream
Applications
DNA Microarray
DNA Sequencing
Bisulfite Modification
CpGenome
MethylQuest
Methyl-DNA
IP
Mag Bead
+
Mag Bead
Mag Bead
Mag Bead
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
44
Summary
Concepts in Epigenetics
Chromatin
Histone Modifications
Gene regulation by RNAs
DNA Methylation
Epigenetics Tools and Applications
Critical considerations and experimental design
(experimental successes and potential pitfalls)
ndash ChIP
ndash Bisulfite
ndash RIP
ndash SmartFlare
128
Lastest Epigenetics Brochure
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom
45
Online Tools For Epigenetics Research
To Learn More Please Visit Us Online or contact your local
Representative
wwwmilliporecomepigenetics
130
Chromatin Immunoprecipitation (ChIP)
Magna ChIPtrade Kits (Protein A G AG ChIP-seq ChIP-chip HT96)
Magna ChIPtrade Beads (Protein A G AG)
RNA Immunoprecipitation
Magna RIPtrade (Protein AGwith and without controls)
Bisulfite Modification and Analysis
CpGenometrade Turbo Bisulfite Modification Kit (90 min protocol)
CpG Wizreg Methylation Specific PCR Kit
CpGenometrade Methylated and Unmethylated DNA controls
Isolation and Enrichment of Methylated DNA
CpG MethylQuesttrade Methylated DNA isolation kit
MeDIP antibodies against 5meC 5hmeC
Beta-Glucosyltransferase
Chromatin Modification SIRTaintytrade Class III HDAC Activity Assay
HAT assay HDAC Assays H2AX and H3S10 phosphorylation assays
Recombinant Histones dimers tetramers and octamers
Epigenetics Antibodies
ChIPAb+trade and RIPAb+trade Validated Antibody Primer sets
gt200 Histone Antibodies Chromatin Modifiers amp Transcription Factors
Absurancetrade Histone Peptide Arrays (H3 and H2AH2BH4)
EMD Millipore Kits Assays Antibodies amp Proteins for Shaping Epigenetics Discovery
46
131
Questions
Merck Millipore
Unleash the Potential of Life Science
132
asiatechservmerckgroupcom