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′/B-1
B-1/ B-1
B′/B-1
B-1/B-1
3
F1
4
Epigenetics: explaining the unexpected
Altered phenotype e.g. coat color, gene expression
No change in underlying DNA sequence
Mitotically and/or meiotically heritable
Responsive to environment i.e. reversible
How to get many different sets of gene expression
from a single genome
5
Mosaic expression in a single individual, and
variation between isogenic individuals
Avy/Avy
x
a/a
6
Morgan et al, Nature Genetics 1999
Development of an entire animal from a single cell
7
Packing vs Access
DNA
Nucleosomes
ATCGGGATC
30 nm
fiber
Loop
domains
300X
More
Folding
Mitotic
chrom.
1000X
8
(Alberts, Molecular Biology of the Cell)
(Alberts, Molecular Biology of the Cell)
DNA methylation -- 5'CpG3'
cytosine
guanine
H
CH3
DNMT
N
H
O
N
H
N
N
C 1'
N
N
N
O
H
N
C 1'
H
Major groove of the double helix
9
DNA methylation in eukaryotes
DNA
methylation
—
—
+
—
(+)
+
+
DNA
methylation
binding
proteins
—
+
+
+
+
+
+
Imprinting
—
—
—
—
(+)
+
+
10
DNA methylation and cell memory
Self-complementary
CpG dinucleotides
DNA replication
Maintenance methylation
Holliday and Pugh 1975 Science
11
Riggs 1975 Cytogenet Cell Genet
Relative methylation at CpG sites
Establishment of DNA methylation
~ 70%
0
5
10
15
Days post conception
20
12
Yoder, Walsh & Bestor, 1997 Trends Genet
Relative methylation at CpG sites
Establishment of DNA methylation: imprinting
Autosomes
Inactive X & active X
CpG
islands
Paternal allele in
female germ cells
H19
Imprinted
gene
Maternal allele in
male germ cells
0
5
10
15
Days post conception
20
13
DNA methyl transferases
Regulatory domain
PCNA
Catalytic domain
RFT
Dnmt1
NLS
CXXC
BAH
Dnmt2
PWWP ATRX
Dnmt3a
PWWP ATRX
Dnmt3b
(Mammals)
14
Goll & Bestor 2005, AnnRevBiochem
DNA methyltransferase functions
Species
Activity
Loss-of-function Phenotype
Dnmt1
mouse
maintenance
E9.5 lethal, genome-wide loss MeCpG
Abnormal gene activation
(imprinting, X, TE)
Dnmt2
mouse
Cytoplasmic
tRNA
None
fish
“
Development defects retina, brain, liver
fly
“
None
S. pombe unknown
“
Dnmt3a
mouse
De novo
Death 4-8 wk, germ cell imprint failure,
no TE MeCpG
Dnmt3b
Mouse
Human
De novo
E14.5 lethal, no TE MeCpG
Immunodeficiency, centromeric instability,
Facial abnormalities (ICF) syndrome15
Rai et al. 2007 Genes Dev
DNA demethylation -- CpG islands
ON
OFF
? CpG methylation default, islands exclude global activity ?
16
How does DNA methylation inhibit transcription?
• Interference with transcription factor or other protein binding
• Biochemical experiments using
• artificial CpG methylation & transfection
• re-expression of X-linked genes after 5-azacytidine treatment
• e.g. imprinted expression of Igf2 gene in mice
Mat
Igf2
CTCF
CTCF
ICE
H19
E
H19
E
me
Pat
Igf2
ICE
17
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
Sin3A HDAC
MeCP2
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 proteins
19
Chromatin-based mechanisms
DNA
Nucleosomes: Fundamental repeating unit of chromatin
H1
147 bp
20
Histone modifications (≥ 56)
4
9
5
8
14
12
18
16
23
27
36
79
20
UB
UB
Allis, Jenuwein, Reinberg. Epigenetics. 2007
21
How many distinct combinations?
4
9
5
8
14
12
18
16
23
27
36
79
20
Is there a histone code?
22
Histone modifying enzymes
23
Chromatin remodelling – Polycomb and Trithorax
24
Chromatin remodelling – Polycomb and Trithorax
25
Epigenetic regulation of retrotransposons
26
Martens et al, EMBO2005
27
Kloc et al Current Biology 2008
DNA methylation and phenotype
• DNA methylation and mutation
• Genomic instability
• Cancer
• Complex disease
• Environmental effects on DNA methylation
29
Extra Reading
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
Assigned Reading
(Jin et al. GADD45A Does Not Promote DNA Demethylation
PLoS Genetics 2008)
See over page for suggestions for 2010
30
Chromatin state
has a profound
effect on biology
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
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