Epigenetics

*Changes in gene expression or phenotype that don’t involve

changes to the DNA sequence()

*Its defined as heritable changes in gene activity and

expression that occur without alteration in DNA sequence

*Modern definition is non-sequence dependent inheritance.

*How do different adult stem cells know their fate?

*Myoblasts can only form muscle cells

*Keratinocytes only form skin cells

*Hematopoetic cells only become blood cells

*But all have identical DNA sequences.

*Identical Twins with

Different Hair Color

High

ecdysone

Low

ecdysone

Endocrine system mediating temperature-dependent

polyphenism: Bicyclus

*Mechanism of EpigeneticsChromatin Remodelling & Histone Modification

DNA Methylation

Non-coding RNA mediated pathway

Prion

Epigenetic code:

Consist of Both DNA methylation and histone modifications

Genetic code in each cell is the same

Epigenetic code is tissue and cell specific

*

Gene Expression

RNA Interference

Histone ModificationsDNA Methylation

*

*De/Acetylation

*Methylation

*Phosphorylation

*Ubiquitination

*ADP-Rybosilation

*Swi/Snf complex, which, in vitro, uses the energy of ATP hydrolysis to disrupt histone-DNA interactions

* Chromatin modifications with

impact on gene expression

*K9 histone H3 and H4 acetylation

*K9 H3 methylation

*K4 H3 methylation

*Ser 10 phosphorylation

*Ubiquitination

*Association of chromatin remodeling complexes

Acetylation/deacetylation of DNA

Condensation of DNA involves coiling around proteins called

histones. Acetylation is when acetyl groups (-COCH3) are

attached to lysines in the histone tails. This reduces

condensation and promotes transcription because the

transcription machinery has better access to the DNA.

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*Histone deacetylases (HDAC)s

Removal of acetyl groups by histone deacetylases (HDAC)s is generally associatedwith re-packing of chromatin and a lack of gene expression or gene silencing

Lysine acetylation is reversible and is controlled by the opposing actions of HATs and HDACs in vivo

Adcock IM, Ford P, Ito K and Barnes PJ. Respiratory Research 2007; 7 :21.

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Lysine residues in the tails of histone H3 and H4 may be acetylated forming bromodomains enabling the association of other co-activators such as

*TATA box binding protein (TBP),

*TBP-associated factors,

*chromatin modifying engines and

*RNA polymerase II.

Gene transcription occurs when the chromatin structure is opened up, with loosening of the tight nucleosomal structure allowing RNA polymerase II and basal transcription complexes to interact with DNA and initiate transcription.

MBD protein

Me Me Me

DNA

HMTMe Me Me

*Chromatine silencing

*Transcriptional

repressors such as

REST, Rb or by methyl-

CpG bindingproteins

induces deacetylation

of histone tails by

HDACs

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*

Chromatin Immunoprecipitation

Cross Link

Sonicate

Primary Ab & Protein G Beads

Ab Enriched DNA

Supe

after IgG

IP

INPUT

IP

Quantitate by

Real Time PCR

Identify proteins that interact with DNA

* Non-coding RNAs and their

epigenetic regulatory mechanisms

*It is widely accepted that ncRNAs (non-coding RNAs),

as opposed to protein-coding RNAs, represent the

majority of human transcripts; and the regulatory roles

of many of these ncRNAs have been elucidated over

the past decade. One important role so far recognized

for ncRNAs is their participation in the epigenetic

regulation of genes. Indeed, it is becoming increasingly

apparent that most epigenetic mechanisms of gene

expression are controlled by ncRNAs.

*RNA silencing and its

transmission

*

*siRNA-mediated suppression of transcription

associated with histone and DNA methylation of

mammalian cells that targets the promoter

region has been reported by several

independent laboratories

*DNA Methylation

*DNA methylation occurs at the Cytosine bases of DNA after

which the Cytosine dinucleotide is converted to 5-

methylcytosine by the activity of DNA methyltransferase

(DNMTs) enzymes

*Silencing:

Methylation of CpG sites within the promoters of

genes can lead to their silencing, a feature

found in a number of human cancers (eg.

silencing of tumor suppressor genes)

* Activation:

In contrast, the hypomethylation of CpG sites

has been associated with the over-expression of

oncogenes within cancer cells.

*

*technique used for the analysis of methylation at CpG island

in a significant sequence. In bisulphite sequencing, bisulphite

converts methylated Cytosines into methylation-dependent

SNPs. The advantage of bisulphite sequencing is that DNA

methylation patterns are detected in a single cell.

Study of DNA

methylation

**Bisulfite Conversion is the most widely used technique for

studying DNA methylation

*Converts non-methylated cytosines to uracil

* Methylated Cytosine remains a C.

* After PCR amplification all Uracils are converted Thymine.

* Comparing the sequences of the native DNA to the

bisulfite treated DNA will show you the methylated bases.

*

Bisulfite conversion

PCR

*

Wide range of techniques used to study DNA post-bisulfite conversion

*Methylation Specific Restriction Enzymes

*Sanger Sequencing or Pyrosequencing

*Microarrays

*PCR Techniques

Bisulfite Specific PCR (BSP)

COBRA – Combined Bisulfite Restriction Analysis

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