Epigenetic regulation of gene expression

Anne Peaston PhD

Research Scientist

The Jackson Laboratory

anne.peaston@jax.org

1

Definition of the problem

Mechanisms DNA methylation

chromatin-based

non-coding RNA

2

B-1/B-1

B′/ B′ B-1/ B-1

B′/B-1 B-1/ B-1

B′/B-13

F1

4

Mitotically and/or meiotically heritable

Altered phenotype e.g. coat color, gene expression

No change in underlying DNA sequence

Epigenetics: explaining the unexpected

How to get many different sets of gene expression from a single genome

Responsive to environment i.e. reversible

5

Morgan et al, Nature Genetics 1999

Mosaic expression in a single individual, and variation between isogenic individuals

Avy/Avy x a/a6

Development of an entire animal from a single cell

7

Packing vs Access

(Alberts, Molecular Biology of the Cell)

30 nm fiber

Loopdomains

MoreFolding

Mitoticchrom.

(Alberts, Molecular Biology of the Cell)

Nucleo-somes

ATCGGGATC 300X 1000X

DNA

8

DNA methylation -- 5'CpG3'

H

H

H

N

O

N

H

N

N

N

C1'

N

OC1'

N

N

H

cytosine guanine

CH3

DNMT

Major groove of the double helix 9

S. cerevisiae

S. pombe

N. crassa

C. elegans

Drosophila

Vertebrates

Arabidopsis

DNAmethylationbinding proteins

— + + ++ + +

DNAmethylation

— +— — (+) + +

Imprinting ———— (+) + +

DNA methylation in eukaryotes

10

DNA methylation and cell memory

Holliday and Pugh 1975 ScienceRiggs 1975 Cytogenet Cell Genet

DNA replication

Maintenance methylation

Self-complementaryCpG dinucleotides

11

0 5 10 15 20

Days post conception

Rel

ativ

e m

eth

ylat

ion

at

Cp

G s

ites

Establishment of DNA methylation

~ 70%

Yoder, Walsh & Bestor, 1997 Trends Genet 12

Establishment of DNA methylation: imprinting

0 5 10 15 20Days post conception

Rel

ativ

e m

eth

ylat

ion

at

Cp

G s

ites

H19ImprintedgeneMaternal allele in

male germ cells

Paternal allele in female germ cells

Inactive XAutosomes& active X

CpG islands

13

DNA methyl transferases

Dnmt1

Regulatory domain Catalytic domain

PCNA

NLS

RFT

CXXC BAH

Dnmt2

Dnmt3a

PWWP ATRX

Dnmt3b

PWWP ATRX

(Mammals) Goll & Bestor 2005, AnnRevBiochem14

DNA methyltransferase functions

Dnmt2 mouse CytoplasmictRNA

None

fish “ Development defects retina, brain, liver

“S. pombe unknown

fly “ None

Dnmt3b MouseHuman

E14.5 lethal, no TE MeCpGImmunodeficiency, centromeric instability,Facial abnormalities (ICF) syndrome

De novo

Rai et al. 2007 Genes Dev

Dnmt3a mouse De novo Death 4-8 wk, germ cell imprint failure,no TE MeCpG

Species Activity Loss-of-function Phenotype

Dnmt1 mouse maintenance E9.5 lethal, genome-wide loss MeCpGAbnormal gene activation (imprinting, X, TE)

15

DNA demethylation -- CpG islands

ON

OFF

? CpG methylation default, islands exclude global activity ?

16

How does DNA methylation inhibit transcription?

• Biochemical experiments using • artificial CpG methylation & transfection• re-expression of X-linked genes after 5-azacytidine treatment

Mat Igf2 ICE

CTCF

EH19

CTCF

Pat Igf2 ICE EH19

me

• Interference with transcription factor or other protein binding

• e.g. imprinted expression of Igf2 gene in mice

17

MeCP2

How does DNA methylation inhibit transcription?

• MethylCpG-binding proteins recruit different co-repressor complexes • Titratable transcriptional inhibition

• DNA-protein complex specific for methylated DNA

• MeCP2, MBD1-4, Kaiso

me

Sin3A HDAC

HKMT

Me

18

Chromatin and DNA methylation

Neurospora and Arabidopsis – methylation depends on H3K9me

Plants/fungi – RNAi targets chromatin modification, gene silencing & meC

Mouse reduced CpG methylation with :

deficiency of H3K9 methyltransferases

deficiency of EZH2 (H3K27 methyltransferase)

increased CpG methylation with :

antisense transcription through promoters

unsettled role of RNAi

Many species – link to SWI/SNF-like remodeling proteins19

Chromatin-based mechanisms

Nucleosomes: Fundamental repeating unit of chromatin

147 bp

DNA

H1

20

4 9 14 18 23 27 36 79

5 8 12 16 20

UB

UB

Allis, Jenuwein, Reinberg. Epigenetics. 2007

Histone modifications (≥ 56)

21

How many distinct combinations?

4 9 14 18 23 27 36 79

5 8 12 16 20

Is there a histone code?22

Histone modifying enzymes

23

24

Chromatin remodelling – Polycomb and Trithorax

25

Chromatin remodelling – Polycomb and Trithorax

26

Epigenetic regulation of retrotransposons

Martens et al, EMBO2005

Kloc et al Current Biology 200827

DNA methylation and phenotype

• DNA methylation and mutation

• Genomic instability

• Cancer

• Complex disease

• Environmental effects on DNA methylation

29

Multiple reviews – scholar google, pub med, other.

Epigenetics. 2007. Eds Allis, Jenuwein, Reinberg. Cold Spring Harbor Press

Cold Spring Harbor Symposia on Quantitative Biology - Epigenetics

Extra Reading

Assigned Reading

(Jin et al. GADD45A Does Not Promote DNA Demethylation PLoS Genetics 2008)

See over page for suggestions for 2010 30

http://www.sciencemag.org.ezproxy.jax.org/cgi/reprint/323/5912/373.pdf

http://www.sciencemag.org.ezproxy.jax.org/cgi/rapidpdf/science.1181495v1.pdf

Chromatin state has a profound effect on biology

Assigned Reading for 2010 possibilities (from 2009)

An Epigenetic Role for maternally inherited piRNAs in Transposon Silencing. Brennecke et al 2008 Science 322:1387

A mouse speciation gene encodes a meiotic histone H3 methyltransferase. Ondrej Mihola et al 2009 Science 323:373 (commentary p350) . See also next article on gene for Drosophila hybrid sterility

Chromatin-associated periodicity in genetic variation downstream of transcriptional start sites. Sasaki et al 2009 Science 323:401

Science 323:220-222 commentary on stem/pluripotent maintenance/resistance to differentiation via chromatin modifications/histone ubiquitination