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Epigenetics: Modification of
DNA and chromatin to control
DNA structure and Gene
Expression
Genetic Variation is a Change in the DNA
Sequence

SNPs

Indels

Rearrangements
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Mendelian Inheritance
X
Epigenetics relies on DNA methylation

Epigenetics - heritable change in phenotype without a
change in DNA sequence
–
Organismal or cell generations

A mechanism involved in epigenetic phenotype regulation
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DNA methylation is a transcription (OFF) switch
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Histone methylation is a transcription (OFF) switch

Histone acetylation is a transcription (ON) switch

Will also involve other molecular mechanism
DNA methylation in different species
DNA
methylation
—
—
+
—
DNA
methylation
binding
proteins
—
+
+
Imprinting
—
—
—
(+)
+
+
+
+
+
+
—
(+)
+
+
A. Peaston TJL
DNA methylation -- 5'CpG3'
cytosine
guanine
H
CH3
DNMT
N
H
O
N
H
N
N
C1'
N
N
N
O
H
N
C1'
H
A. Peaston TJL
7
Establishment of DNA methylation
Relative methylation at CpG sites
~ 70%
0
5
10
15
Days post conception
20
Yoder, Walsh & Bestor, 1997 Trends Genet
8
Establishment of DNA methylation: imprinting
Relative methylation at CpG sites
Inactive X
Autosomes
& 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
Law & Jacobsen Nat Rev. Genet 2010
DNA demethylation
Passive
Active
e.g. zygotic paternal genome,
IL2 promoter
A. Peaston TJL
DNA demethylation -- CpG islands
ON
OFF
? CpG methylation default, islands exclude global activity ?
A. Peaston TJL
Law & Jacobsen Nat Rev. Genet 2010
DNA Demethylation and
5hydroxymethyl cytosine
5-methylcytosine oxidized to 5
hydroxymethylcytosine
Tet family (ten-eleven translocation)
Wu & Zhang 2011 Cell Cycle
How does DNA methylation inhibit transcription?
• Titratable transcriptional inhibition
• DNA-protein complex specific for methylated DNA
• MeCP2, MBD1-4, Kaiso
• Methyl-binding proteins recruit different co-repressor complexes
Sin3A HDAC
MeCP2
me
A. Peaston TJL
How does DNA methylation inhibit transcription?
• Biochemical experiments using
• artificial CpG methylation & transfection
• re-expression of X-linked genes after 5-azacytidine treatment
• Interference with transcription factor or other protein binding
• e.g. CTCF binding
CTCF
CTCF
Mat
Igf2
ICE
H19
E
H19
E
me
Pat
Igf2
ICE
A. Peaston TJL
Assessing DNA methylation
Avy/a
Morgan et al, Nature Genetics 1999
DNA methylation at the agouti locus
Avy allele
Hypothesis
LTR CpG methylated
IAP OFF
pseudoagouti
LTR CpG unmethylated
IAP ON
yellow
Methylation-sensitive restriction enzymes
Avy allele
B BamH1
M Msp1
H HpaII
Isoschizomers, C/CGG
A. Peaston TJL
Bisulfite sequencing
Locus-specific detailed single-molecule analysis
5MeCpG
bisulfite
DNA
PCR
CpG
CG
UG
CG
TG
Frommer et al, PNAS 1992
Bisulfite Sequencing
Sperm
95%
Oocytes
98%
Zygotes
90%
Blastocyst
62%
Lane et al, Genesis 2003
Higher throughput

Pyrosequencing of clones (You still have to clone)

SAGE
Higher throughput
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Next Generation Sequencing

High throughput, non-biological cloning
And permutations.....
Murrell, Rakyan & Beck, Hum Mol Genet 2005
Chromatin and DNA methylation
Neurospora and Arabidopsis – methylation depends on H3K9me
Plants – RNAi targets chromatin modification, gene silencing & DNAme
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
The nucleosome core. Site of action. Tails that
get modified are evident in this image.
http://atlasgeneticsoncology.org/Deep/Images/ChromatinFig2Engl.gi
f
Histone modifying enzymes
29
Chromatin remodelling – Polycomb and Trithorax
30
31
Kloc et al Current Biology 2008
Epigenetic changes and phenotype

DNA methylation and mutation
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Genomic instability

Cancer

Complex disease

Environmental effects on DNA methylation
A. Peaston TJL
Epigenetics/Epigenomics

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Modification of histones
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Mostly Acetylation (+) and Methylation (-)
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Histone acetyl transferases
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Histone deacetylases
Modification of DNA
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In eukaryotes, mostly 5-methylcytosine (-)
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Dnmt (DNA methyl transferases)
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Removal?
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A sixth base; 5-hydroxymethylcytosine
Epigenetics/Epigenomics
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Methylated DNA recruits HDACs
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

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Increased condensation of chromatin decreased expression
Methylated chromatin may recruit Dnmts
Transcription factors may recruit Dnmts,
HDACs or HMTs or HATs depending on function
Signals can propagate along the chromosome
using bi-functional enzymes (chromodomain
and HMT activity)