epigenetic regulation of gene expression anne peaston phd research scientist the jackson laboratory...
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Epigenetic regulation of gene expression
Anne Peaston PhD
Research Scientist
The Jackson Laboratory
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Definition of the problem
Mechanisms DNA methylation
chromatin-based
non-coding RNA
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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
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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
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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
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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
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DNA methylation and cell memory
Holliday and Pugh 1975 ScienceRiggs 1975 Cytogenet Cell Genet
DNA replication
Maintenance methylation
Self-complementaryCpG dinucleotides
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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
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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)
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DNA demethylation -- CpG islands
ON
OFF
? CpG methylation default, islands exclude global activity ?
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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
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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
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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
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4 9 14 18 23 27 36 79
5 8 12 16 20
UB
UB
Allis, Jenuwein, Reinberg. Epigenetics. 2007
Histone modifications (≥ 56)
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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
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Chromatin remodelling – Polycomb and Trithorax
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Chromatin remodelling – Polycomb and Trithorax
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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
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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