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ROLE OF EPIGENETIC EVENTS IN RESPONSES TO TOXIC AGENTS
Igor P. Pogribny, M.D., Ph.D.Division of Biochemical Toxicology
U.S. FDA-NATIONAL CENTER FOR TOXICOLOGICAL RESEARCH
October 4, 2011
GLOSSARY
CpG sites: Regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. "CpG" stands for cytosine and guanine separated by a phosphate, which links the two nucleosides together in DNA. The "CpG" notation is used to distinguish a cytosine followed by guanine from a cytosine base paired to a guanine.
DNA methylation: Methylation of cytosine residue at CpG sites.
Hemimethylated CpG site:
Unmethylated CpG site:
Histone methylation: The modification of certain lysine or arginine residues in a histone protein by the addition of one, two, or three methyl groups.
Histone acetylation: The modification of certain lysine residues in a histone protein by the addition of the acetyl group.
-CG--GC-
M
-CG--GC-
EPIGENETICS (from the Greek “epi” - “on” and genetics) – the developmental processes by which genotype gives rise to phenotype.
Waddington C., PNAS, 1939
EPIGENETICS – heritable changes in gene expression mediated by methylation of DNA, modifications of histone proteins, nucleosome positioning along DNA, or by non-coding RNAs that are not due to any alteration in the DNA sequence.
EPIGENETICS
GENETICS VERSUS EPIGENETICS
EPIGENETICS
Epigeneticchanges
Heritable transmission of information in the absence of changes in DNA
sequence
GENETICS
SNP
C/T
Heritable transmission of information based on differences in DNA sequence
MTHFR, C677TGCC→GTC
Allis et al., 2007
Cytosine DNA methylation is a covalent modification of DNA, in which a methyl group is transferred from S-adenosylmethionine (SAM) to the 5 position of cytosine by a family of cytosine (DNA-5)-methyltransferases
CYTOSINE DNA METHYLATION
Methylation of cytosine
Establishment and propagation of methylation patterns
SAM SAH
DNMTs
DNA DNA
CpGm
CpG
GpCm
GpC
CpGm
CpG
GpGpC
CpGCpG
GpCGpC
DNMT3A/B
UHRF1
DNMT1
de novo Maintenance
C
(i) (ii)
METHYLATION LANDSCAPE OF THE MAMMALIAN GENOME
Simple tandem repeat
DNA transposon
LTR – endogenous retrovirus
Methylated domains (repeated DNA sequences)
SR SR SR SR
IR IRTransposon
5’LTR 3’LTRgag envpol
TSDR TSDRMSC P ORF2ORF1 A/T RichSite
TSDR TTTTDR DRPoly(A)SVA Element
Non-LTR autonomous retrotransposon: LINE
Non-LTR non-autonomous retrotransposon: SINE
Sequence compartment CpG G + C (%) CpG
Obs/Exp (%)
Genome 29,848,753 41 24
Promoter 1,876,802 62 89
First Exon 508,553 56 65
Other Exons 1,337,271 48 40
DNA Transposons 565,601 29 23
Line Transposons 3,242,225 32 18
LTR Transposons 1,958,798 37 19
SINE Transposons 7,479,682 38 41
Alpha Satellite ~766,000 38 33
Classical Satellite ~1,140,000 34 67
Other 8,358,888 42 15
Distribution of CpG sites in the human genome
Wilson et al., 2007
Rollins et al., 2006
- unmethylated CpG sites
- methylated CpG sites
CpG island:DNA regions >500 bp with GC content >55% and observed CpG/expected CpG of 0.65
POST-TRANSLATIONAL HISTONE MODIFICATIONS
H2AH3
H4H2B
CHROMATIN NUCLEOSOME
POST-TRANSLATIONAL HISTONE MODIFICATIONS
Histone modifications
Role in transcription
Histone-modified sites
Acetylation activation H3 (K9, K14, K18, K56)
H4 (K5, K8, K12, K16)
H2A
H2B (K6, K7, K16, K17)
Phosphorylation activation H3 (S10)
Methylation activation H3 (K4, K36, K79)
repression H3 (K9, K27)
H4 (K20)
Ubiquitination activation H2B (K123)
repression H2A (K119)
Sumoylation repression H3 (?)
H4 (K5, K8, K12, K16)
H2A (K126)
H2B (K6, K7, K16, K17)
TYPES AND ROLES OF HISTONE MODIFICATIONS
ROLE OF EPIGENEIC MODIFICATIONS IN NORMAL CELLS
•X-CHROMOSOME INACTIVATION – balances gene expression between males and females
•GENOMIC IMPRINTING – ensures parental-specific gene expression in diploid cells essential for development
•SILENCING OF REPETITIVE ELEMENTS – ensures maintenance of proper chromatin order
•TISSUE-SPECIFIC DNA METHYLATION – controls gene- and tissue-specific epigenetic patterns
•CHROMATIN ORGANIZATION – controls proper expression of genetic information
APPROACHES TO DETECTING DNA METHYLATION
Global DNA methylation• High performance liquid chromatography (HPLC)• Immunocytochemistry for 5-methylcytosine• High performance liquid chromatography-
electrospray ionization tandem mass spectrometry (HPLC-ES-MS/MS)
• Methyl-acceptance assay• Cytosine extension assay• Analysis of LINE-1 and AluI methylation
Gene-specific methylation• Methylation-sensitive PCR• Bisulfite genomic sequencing• Methylation-specific PCR (MCP)• Methylation-sensitive single-nucleotide primer
extension (Ms-SNuPe)• Combined bisulfite restriction analysis (COBRA)• CpG island microarray• Methylated DNA immunoprecipitation• Pyrosequencing
Search for new hypermethylated genes• Demethylating drugs and expression microarrays• Amplification of intermethylated sites (AIMS)• Methyl-PCR arbitrary techniques• Restriction landmark genomic scanning (RLGS)
CYTOSINE METHYLATION
APPROACHES TO DETECTING DNA METHYLATION: METHYLATED DNA IMMUNOPRECIPITATION AND PYROSEQUENCING
Marlowe J. et al., Mol. Clin. Environ. Toxicol., 2009
Genome-wide profiling (MeDIP) Locus-specific profiling (Pyrosequencing)
Methylation status of the gene
Correlation with gene expression
Comprehensiveview of genome
function & activity
Bis-DNA modificationDNA fragmentation
Genomic DNA
APPROACHES TO DETECTING HISTONE MODIFICATIONS
Liquid chromatography-electrospraymass spectrometry
Immunohistochemistry
HISTONE MODIFICATIONS
Chromatin immunoprecipitation analysis
Control Treatment
H4K20me3 H4K20me3
Western immunoblotting
Loading
Control Treatment
Modification 2
Modification 1
MECHANISMS LINKING ENVIRONMENTAL EXPOSURES TO EPIGENETIC ALTERATIONS
Exposure
DNA methylation
Histone codemiRNA
Altered gene expression andchromatin condensation
Disease risk
Inflammation
Endocrine disruption
Clonal selection
Methyl‐groupavailability
Methyltransferasesactivity
Oxidativestress
Baccarelli and Bollati, 2009
CYTOSINE DNA METHYLATION
DNMT3aDNMT3b
DNMT1
SAM SAH
DNA alterations
Cytosine (C) 5-methylcytosine (m5C)
TOBACCO SMOKE AND DNA METHYLATION CHANGES
• Methylation-associated carcinogen-induced mutations
• Gene-specific DNA hypermethylation (e.g., CDKN2A, MGMT, DAPK, RASSF1A, CDH1)
• Altered global and gene-specific histone modifications
• Altered expression of microRNAs
TOBACCO SMOKE AND EPIGENETICS: CARCINOGEN-INDUCED MUTATIONS
ACROLEIN
Feng et al., 2006
Denissenko MF et al., Science, 1996
Denissenko et al., 1997
B[a]P
CHILDHOOD ASTHMA AND EPIGENETICS: EPIGENETIC ALTERATIONS INDUCEDBY PRENATAL EXPOSURE TO POLYCYCLIC AROMATIC HYDROCARBONS
Perera et al., 2009
• Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide.25-45% of patients with COPD have never smoked.
• 3 billion people, half of the world population, are exposed to smoke from different sorts of fuel (i.e., biomass fuel, transportation-related fuel, etc.).
• This suggests that exposure to fuel smoke is the biggest risk factor for COPD globally.
EPIGENETIC ALTERATIONS INDUCED BY LOW-DOSE BENZENE EXPOSURE
Bollati et al., 2007
LINE-1
p15
AluI
MAGE-1
Zhang et al., 2010
MECHANISM OF ARSENIC-INDUCED EPIGENETIC ALTERATIONS
DNA hypomethylation
Oxidativestress
OxidativeDNA lesions
Conjugationwith glutathione
ARSENIC-INDUCED EPIGENETIC ALTERATIONS
Zhao et al., 1997; Chen et al., 2004; Reichard et al., 2007
ER-α
ER-α
ER-α methylation
In vitro studies
In vivo studiesTreatment
Perc
enta
ge o
f con
trol
(%)
LIST OF SELECTED ENVIRONMENTAL CHEMICAL AGENTS THAT ALTER CELLULAR EPIGENETIC PATTERNS
Agent EffectArsenic Global DNA hypomethylation
Gene-specific hypomethylation (Er-α, cyclin D1)Gene-specific hypermethylation (p53, p16INK4A, RASSF1A)Inhibition of DNMT1 and DNMT3a expression↓Histone H3K27 trimethylation↑Histone H3K4 trimethylation
Cadmium Global DNA hypomethylation Inhibition of DNMT activity Gene-specific hypermethylation (p16INK4A, RASSF1A)
Hydrazine Global DNA hypomethylationGene-specific hypomethylation (p53, c-myc, HMG CoA reductase)
Benzo[a]pyrene Global DNA hypomethylationGene-specific hypermethylation (CYP1A1) Promoter-specific histone H3 lysine 9 hypo- and hyperacetylationCpG-methylation-associated mutations (p53)
Aflatoxin B1 Gene-specific hypermethylation (GSTP, MGMT, RASSF1A, p16INK4A) 2-Acetylaminofluorene Gene-specific hypermethylation (p16INK4A)
Increased DNMT1 expression↓Histone H4K20 trimethylation
Bisphenol A Agouti gene, IAP elementGene-specific hypomethylation (PDE4D4)
Benzene Global DNA hypomethylationGene-specific hypermethylation (p15)Gene-specific hypomethylation (MAGE-1)
Dibromoacetic acid Global DNA hypomethylation
EPIGENETIC ALTERATIONS IN CANCER CELLS
•GLOBAL DNA HYPOMETHYLATION
•GENE-SPECIFIC DNA HYPERMETHYLATION
•GENE-SPECIFIC HYPOMETHYLATION
•ALTERED HISTONE MODIFICATIONS
•ALTERED NUCLEOSOME POSITING ALONG DNA
• ALTERED EXPRESSION OF NON-CODING RNAs, e.g. microRNAs
EPIGENETIC GENE-SPECIFIC ALTERATIONS IN CANCER
E P I G E N O M E E P I G E N O M E
NORMAL CELL CANCER CELL
CpG island promoter region
DNA level
Chromatin level
XCpG island promoter region
DNA level
Chromatin level
Hinshelwood and Clark, J. Mol. Med., 2008
DNA METHYLATION CHANGES DURING SKIN CARCINOGENESIS
NS MCA3D PB MSCP6 PDV PAM212 MSCB119
MSC11A5
HaCa4 CarB CarC
BRCA1 U U U U U U U U U U U
MLH1 U U U U U U U U U U U
MGMT U M M M M M M M M M M
CDH1 U U U U U U U M M M M
Snail U M M M M M M U U U U
MLT1 U M M M M M M M M M M
Abbreviations: NS - normal skin; M - methylated; U - unmethylated
Status of global DNA methylation
CpG island methylation status of selected genes
Fraga et al., 2004
EPIGENETIC CHANGES IN CARCINOGENESIS
CAN EPIGENETIC ALTERATIONS BE USED FOR CARCINOGENICITY ASSESSMENT?
CARCINOGENICITY TESTING – MODERN ERA CHALLENGES
1%
≥ 65,000 chemicals have been manufactured for commercial use in industrialized countries
Eaton and Gallagher, 1997≥ 700 chemicals tested by NTP and RF in two-year bioassays
50% are carcinogens 50% are non-genotoxiccarcinogens
•Most compounds presented to regulatory agencies today are non-genotoxic carcinogens•No reliable rapid test for non-genotoxic chemicals•Liver is a sensitive organ to non-genotoxic chemicals
CARCINOGEN-DNA ADDUCTS AS MARKERS OF EXPOSURE AND CANCER RISK
Clonal selection and expression of initiated cells
Mutator phenotype cells
ACQUISITION OF ADDITIONAL RANDOM MUTATIONS
Initiated cells
Cancer cells
En
do
gen
ou
sEn
do
gen
ou
s
En
vir
on
men
tal
En
vir
on
men
tal
Normal cells
Benzo[a]pyrene
BPDE-DNA ADDUCTS
Benzo[a]pyrene-diol-epoxide (BPDE)
EXPOSURE
REACTIVE METABOLITES
CARCINOGEN-DNA ADDUCTS
CANCER
1,3-BUTADIENE
Humans: known human carcinogen “Group 1” (IARC, 2008)
Rodents: potent rodent carcinogen
Mice: lymphoma, hemangiosarcoma, lung, liverRat: pancreas, mammary gland
Mice and humans share common sites of tumor formation
METABOLIC ACTIVATION OF BD TO REACT ELECTROPHILES AND FORMATION OF DNA ADDUCTS
Goggin M et al., Cancer Res 2009
THB-Gua-BD ADDUCTS IN HEPATIC DNA FROM MICE EXPOSED TO 1,3-BUTADIENE
0
50
100
150
200
250*
**
Am
ount
of T
HB
-Gua
addu
cts/
107
ntds
Total Racemic Meso
* **
EFFECTS OF 1,3-BUTADIENE EXPOSURE ON EPIGENETIC ALTERATIONS IN THE LIVERS
0
50
100
150
200
250
[3 H]d
CTP
inco
rpor
atio
n, %
DNA methylation
**
Control 1,3-butadiene 1,3-butadiene0 ppm 6.2 5ppm 625 ppm
0.0
0.5
1.0
1.5
Fold
cha
nge
*
Control 1,3-butadiene 1,3-butadiene0 ppm 6.25 ppm 625 ppm
0
20
40
60
80
100
120
140
**
*
Cha
nge
from
con
trol,
%
H3K9me3 H4K20me3 H3K27me3
0 ppm 6.25 ppm
H3K9me3
H4K20me3
H3K27me3
Loading
625 ppm
Control 1,3-butadiene
LINE-1 methylation
Histone methylation
TAMOXIFEN
• ICI46,474 (TAMOXIFEN) discovered in 1960s by A.L. Walpole, J.K. Harper and D. Richardson
• First targeted agent for the treatment of breast cancer
• Approved by the U.S. Food and Drug Administration for the reduction of breast cancer risk in pre- and postmenopausal women in 1998
ICI46,474(TAMOXIFEN)
• Risk factor for endometrial cancer in women (IARC, 2011)
• Potent genotoxic liver carcinogen in rats
• Induces NASH in humans, rats, and mice
GENOTOXIC ALTERATIONS INDUCED BY TAMOXIFEN EXPOSURE IN RAT LIVER
Hepatic tamoxifen–DNA adduct levels in rats fed 420 p.p.m. tamoxifen for 6, 12, 18 or 24 weeks
Weeks of feeding dG-TAM dG-DesMeTAM Total tamoxifen–DNA adducts
6 265 ± 13 318 ± 16 583 ± 21
12 493 ± 28 769 ± 43 1262 ± 66
18 631 ± 22 1020 ± 36 1651 ± 57
24 639 ± 56 1073 ± 87 1712 ± 143The tamoxifen–DNA adduct levels are expressed as adducts/108 nt (n=5, mean ± SEM)
EPIGENETIC ALTERATIONS IN LIVER DURING TAMOXIFEN-INDUCED RAT HEPATOCARCINOGENESIS
100150200
50
Control TAM Control TAMM
12 weeks 24 weeks
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
6 weeks 12 weeks 18 weeks 24 weeks
ControlTAM
[3H
]dC
TP in
corp
orat
ion/
µg D
NA
, x10
3
**
* *
Time of treatment
Control TAM
12 weeks
18 weeks
6 weeks
24 weeks
0
20
40
60
80
100
120
140
160
180
200
ControlTAM
6 weeks 12 weeks 18 weeks 24 weeksTime of treatment
Leve
l of m
odifi
catio
n, D
LU x
103
** * *
Global DNA hypomethylation
LINE-1 hypomethylation
H4K20me3
Tryndyak et al., 2007 Tryndyak et al., 2006
GENETIC AND EPIGENETIC CHANGES INDUCED BY CARCINOGENIC AGENTS
AGENT GenotoxicityEpigenetic alterations
DNA hypo-methylation
DNA hyper-methylation
Histone modifications microRNA References
Tobacco smoke + N/A + + + Belinsky, 2005; Vassière et al., 2009
1,3-Butadiene + + N/A + N/A Koturbash et al., 2011
2-AAF + - + + + Bagnyukova et al., 2008
Tamoxifen + + N/A + + Tryndyak et al., 2006; Pogribny et al., 2007
Phenobarbital - + + + N/A Bachman et al., 2006
Arsenic - + + + N/A Zhao et al., 1997; Ren et al., 2011
Lipotrope diets - + + + +Wainfan and Poirier,
1992; Christman, 1995; Pogribny et al, 2004
AGENT GenotoxicityEpigenetic alterations
DNA hypo-methylation
DNA hyper-methylation
Histone modifications microRNA References
Tobacco smoke + N/A + + + Belinsky, 2005; Vassière et al., 2009
1,3-Butadiene + + N/A + N/A Koturbash et al., 2011
2-AAF + - + + + Bagnyukova et al., 2008
Tamoxifen + + N/A + + Tryndyak et al., 2006; Pogribny et al., 2007
Phenobarbital - + + + N/A Bachman et al., 2006
Arsenic - + + + N/A Zhao et al., 1997; Ren et al., 2011
Lipotrope diets - + + + +Wainfan and Poirier,
1992; Christman, 1995; Pogribny et al, 2004
GENETIC AND EPIGENETIC MARKERS OF CARCINOGENIC EXPOSURE
EPIGENETIC MODEL OF CARCINOGENESIS
Carcinogen
Alteration of cellular epigenome• Global DNA and repetitive DNA elements hypomethylation • Gene-specific hypermethylation • Gene-specific hypomethylation• Aberrant histone modifications• Altered expression of miRNAs
Epigenetically altered cells
Epigenetically reprogrammed cells
Cancer cells
ADVANTAGES OF EPIGENETIC BIOMARKERS FOR CANCER RISK ASSESSMENT
• EARLY APPEARANCE OF CARCINOGEN-INDUCED EPIGENETIC ALTERATIONS
• STABILITY OF EPIGENETIC CHANGES
• INHERITANCE OF CARCINOGEN-INDUCED EPIGENETIC CHANGES THROUGH TRANSMISSION FROM ONE CELL GENERATION TO ANOTHER
• DETECTION AT RELATIVELY LOW COSTS
• SPECIFICITY FOR GENOTOXIC AND NON-GENOTOXIC CARCINOGENS
• GREATER NUMBER OF EPIGENETIC CHANGES OVER GENETIC ALTERATIONS AFTER EXPOSURE
CONCLUSIONS
EPIGENETIC ALTERATIONS are a major component and driving force in development of pathological states associated with exposures to harmful personal lifestyle factors and environmental physical and chemical agents. Understanding and manipulating the epigenome holds enormous promise for preventing these pathological states. Specifically, epigenetics holds substantial potential for developing biological markers to predict which exposures would put exposed individuals at risk and, more importantly, which individuals will be more susceptible to developing a disease.