Epigenetics In the 1940’s (Waddington et al.) The sum of the genes and their products and how they...

Epigenetics

In the 1940’s (Waddington et al.)• The sum of the genes and their products and how they define a phenotype (dividing kidney and skin cells are identical in their DNA but give rise to cells of different phenotype).

Today• Changes in gene function that are mitotically and/or meiotically heritable and that do not entail a change in DNA sequence.

What does this mean?

•• Production of genetically normal mosaic micefrom malignant teratocarcinoma cells (Mintzand Illmense 1975).•• Some carcinogens (tumor promoters, i.e. TPA) are not genotoxic.

Therefore, modification of gene expression, function due to developmental changes orexogenous factors could lead to cancer.

Evidence for Epigenetic Mechanisms of Carcinogenesis

Importance of Epigenetics

1. DNA methylation2. Imprinting3. X-chromosome inactivation4 Development/Reprogramming of somatic nucleus5. Cancer6. Rett syndrome1. DNA methylation7. Non-coding RNAs & heterochromatin

Anders H. Lund et al. Genes Dev. 2004; 18: 2315-2335

trx-GPc-G

Homeotic gene expression

Cloned Members of the Pc-G and trx-G

(PRE/TRE)

Pc M33/MPc1, MPc2, MPc3ph Rae28/Mph1Psc Mel18, Bmi-1Scm Scmh1, Scmh2Pcl M96Pho YY1Su(z)2 Mel18, Bmi-1dRING Ring1a, Ring1bE(z) Enx-1, -2/Ezh-1, -2esc eedAsx Asxl1, Asxl2crm

trx Mll, Mll2trl brm Brm, Brg1mo Baf155, Baf170osa Baf250snr1 Baf47/Snf5zash-1 Ash1lash-2 Ash2llidkislawc

PRC1

PRC2

SWI/SNF

Ying-Yang Theory

A natural homoestasis occurs between protein

isoforms with and without SET domains. This

balance is perturbed in human cancer development

by either the loss of the SET domain isoform or a busion gene lacking the SET domain.

Disease Link to Chromatin (ref.)

Myeloid leukemias Translocation of the MOZ HAT (Smith, 1998)

Promyelocytic leukemias Fusion of a HDAC to transcription factors

(Grignani, 1998; Lin, 1998)

Acute Myeloid leukemia CBP-HRX fusion (Cairns, 1996)

Rubinstein-Taybi Syndrome CBP point mutations (Petrij, 1995)

Colorectal/gastric p300 missense mutations (Muraoka, 1996)

carcinomas

Breast cancer Amplification/Overexpression of

AIB1/HAT (Sobulo, 1997; Anzick, 1997)

Prostate cancer Overexpression of EZH2 (Varambally, 2002)

Characteristics of Hetero- and Eu-chromatin

Euchromatin Heterochromatin(constitutive)

Staining Dispersed Condensed (speckles)

DNA sequence Predominantly unique Repetitive & gene poor & gene rich

Replication Early to Late S-Phase Late S-Phase

Recombination Abundant Limited

Chromatin Irregular nucleosomes Regular nucleosomesAccessible to nucleases Less accessible to nucleasesHypersensitive Sites Few Hypersensitive Sites

Modifications H3-K4 methylation H3-K9 methylation Low CpG methylation More CpG methylation

DNA Methylation• In mammals, ~1% of DNA bases methylated on carbon-5 of cytosine pyrimidine ring (5-methylcytosine).• Most frequent at 5’-CpG-3’ dinucleotides (~70% of all CpGs).• In general, CpGs are under-represented (suppressed)

CpG islands• Regions (500-1 kb) of higher G+C than genome average.• Relatively devoid of methylation.• ~ 60% mammalian RNA Pol II promoters found in CpG islands.

DNA Methylation and Gene Expression

•• Cytosine residues in 5’CpG are often postsynthetically methylated•• CpG methylation is involved in long-term silencing of certain gene during development•• The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression.

DNA Methylation and Cancer•• Hypomethylation

•• Human tumors normally exhibit decreased overall genome methylation

•• Cancer Res. 48:1159-1161, 1988; 64:4238-4243,2001•• Adv. Cancer Res. 54:1023,1990•• Gene. Cytogenet. 97:83-89,1987

•• Hypomethylation occurs relatively early in carcinogenesis

•• New Engl J./med. 319:525-532,1988.•• Cancer Res. 48:1159-1161, 1988•• Gut 39:434-438, 1996.

A significant reduction in 5- methylcytosinecontent in adenoma and adenocarcinomacompared with normal, paired colonicmucosa, whereas no difference existedbetween benign adenoma and malignantadenocarcinoma.

Feinberg A.P. et al. Cancer Res.48:1159-1161, 1988.

•• Hypermethylation•• Tumor Suppressor genes hypermethylated in human cancerCancer Res. 61:3225-3229,2001.

A Gene Hypermethylation Profile of Human CancerM. Esteller et al. Cancer Res.61:3225-3229, 2001.

Analyzed promoter hypermethylation changes in 12genes from 600 primary tumor samplesrepresenting 15 major tumors.

P16INK4a, P15INK4b － Cell cycle regulationBRCA1, hMLH1, MGMT － DNA repairTIMP3, CDH1, DAPK － Cell adherence, metastasisGSTP1 － Metabolic enzymeP14ARF, P73 － P53 networkAPC － β - catenin function

Principle of Bisulfite Modification

Ggg gcg gac cgc

Ggg gug gau ugu

Ggg gcmg gacm cmgcm

Ggg gcmg gacm cmgcm

Bisulfite modification

Bisulfite modification

Unmethylated DNA

Methylated DNA

A unique profile of promoterhypermethylation may exist for humancancers where some gene changes are

shared and others are cancer-type specific:

•• P16 INK4a － Many tumor types•• APC － Liver, pancreas, stomach

•• P14 ARF － Colon, stomach•• GSTP － Liver, breast, prostate

•• MGMT － Colon, brain•• P15 INK4b － Leukemia

Simultaneous inactivation ofmultiple tumor suppressor/promoter

pathways may also occur:

Colon : P16 INK4a, hMLH1, TIMP3Breast : P16 INK4a, BRCA1, CDH1Lung : P16 INK4a, MGMT, DAPK

Sir2 (S.c.) =Clr4/Suv39 (S.p.,human)Sir3/4 (S.c.) =Swi6/HP1 (S.p.,human)

trx-GPc-G

Homeotic gene expression

Cloned Members of the Pc-G and trx-G

(PRE/TRE)

Pc M33/MPc1, MPc2, MPc3ph Rae28/Mph1Psc Mel18, Bmi-1Scm Scmh1, Scmh2Pcl M96Pho YY1Su(z)2 Mel18, Bmi-1dRING Ring1a, Ring1bE(z) Enx-1, -2/Ezh-1, -2esc eedAsx Asxl1, Asxl2crm

trx Mll, Mll2trl brm Brm, Brg1mo Baf155, Baf170osa Baf250snr1 Baf47/Snf5zash-1 Ash1lash-2 Ash2llidkislawc

PRC1

PRC2

SWI/SNF

Copyright ©1999 by the National Academy of Sciences

Toyota, Minoru et al. (1999) Proc. Natl. Acad. Sci. USA 96, 8681-8686

H19

IGF2*

H19*

IGF2

Paternal Imprinting

Maternal Imprinting

The SWI/SNF Complex

• ATP-dependent chromatin remodeling complex

• Yeast mating type switching (SWI) and sucrose non-

fermenting (SNF)

• Expression of 6% of yeast genes require SWI/SNF function

• 9-12 subunits - 2MDa

• Each subunit is required for function of the entire complex

• Evolutionarily conserved: C. elegans, yeast, drosophila, rat,

mouse, human

Variations in Human SWI/SNF Complexes

Actina

BRG1/BAF

BRG1

BAF155

BAF170BAF57

BAF53a

BAF250a

BAF60a

BAF47

Actin

PBAF

BRG1

BAF155

BAF170BAF57

BAF53a BAF180a & b

BAF60a

BAF47

Actin

BRM/BAF

BRM

BAF155

BAF170BAF57

BAF53aBAF250a

BAF60a

BAF47

Actin

BRG1

BAF155

BAF170BAF57

BAF53a

BAF250b

BAF60a & b

BAF47

EBAF70

EBAF140EBAF100

Actin

ENL

EBAFb

BRG1

BAF155

BAF170BAF57

BAF53a

BAF250a

BAF60a & b

BAF47

EBAF70

EBAF140EBAF100

Actin

ENL

EBAFa

BRG1

BAF250

BAF170

BAF60BAF57

BAF53

BAF155

hSNF5

c-Myc p53

SWI/SNF-Mediated Chromatin Remodeling

c-Myc p53

BRG1

BAF250

BAF170

BAF60BAF57

BAF53

BAF155

hSNF5


c-Myc p53

BRG1

BAF250

BAF170

BAF60BAF57

BAF53

BAF155

hSNF5


ATP

ADP+Pi

c-Myc p53

BRG1

BAF250

BAF170

BAF60BAF57

BAF53

BAF155

hSNF5


Evidence for BRG1’s Role in Human Tumor Development

- Required for RB-mediated growth arrest

- Mutations found in 10% of human tumor cell lines

- Expression lost in ~50% of NSCLC tumor cell lines, ~20% of adenocarcinoma cell lines & 10% of primary tumors.

- BRG1+/- mice develop adenocarcinomas

- LOH in region surrounding BRG1 locus (19p13.2)

How does loss of BRG1/BRM promote tumor development?

1. Abrogation of Rb mediated growth arrest?

2. Alterated DNA methylation patterns?

3. Aberrant cytoskeletal organization/signaling?

Summary

• Chromatin structure plays a central role many cellularprocesses.• Relevant to most aspects of gene expression as well aschromosome stability, DNA replication, recombination and repair.• Chromatin regulated at local and global levels.• Misregulation of chromatin or DNA methylation states leads to cancer.• Chromatin is highly dynamic both in terms of structure &chemical composition.• CpG methylation and heterochromatin formation partition the genome.

Epigenetics In the 1940’s (Waddington et al.) The sum of the genes and their products and how they...

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