Epigenetics: Modification of DNA and chromatin to control DNA structure and Gene Expression

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Epigenetics Modification of DNA and chromatin
to control DNA structure and Gene Expression
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Genetic Variation is a Change in the DNA Sequence

• SNPs
• Indels
• Rearrangements
• Mendelian Inheritance

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X
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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
• DNA methylation is a transcription (OFF) switch
• Histone methylation is a transcription (OFF)
  switch
• Histone acetylation is a transcription (ON)
  switch
• Will also involve other molecular mechanism

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DNA methylation in different species
S. cerevisiae
Arabidopsis
Vertebrates
Drosophila
C. elegans
S. pombe
N. crassa
DNA methylation

()?





DNA methylation binding proteins







()?


Imprinting




A. Peaston TJL
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DNA methylation -- 5'CpG3'
A. Peaston TJL
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Yoder, Walsh Bestor, 1997 Trends Genet
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Establishment of DNA methylation imprinting
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DNA methylation and cell memory
Self-complementary CpG dinucleotides
DNA replication
Holliday and Pugh 1975 Science Riggs 1975
Cytogenet Cell Genet
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Law Jacobsen Nat Rev. Genet 2010
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DNA demethylation
Passive
Active e.g. zygotic paternal genome, IL2
promoter
A. Peaston TJL
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DNA demethylation -- CpG islands
? CpG methylation default, islands exclude
global activity ?
A. Peaston TJL
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Law Jacobsen Nat Rev. Genet 2010
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DNA Demethylation and 5hydroxymethyl cytosine
5-methylcytosine oxidized to 5
hydroxymethylcytosine Tet family (ten-eleven
translocation)
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Wu Zhang 2011 Cell Cycle
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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

HDAC
Sin3A
HKMT
MeCP2
Me
me
A. Peaston TJL
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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
• Interference with transcription factor or other
  protein binding
• e.g. CTCF binding

A. Peaston TJL
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Assessing DNA methylation
Avy/a
Morgan et al, Nature Genetics 1999
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DNA methylation at the agouti locus
Avy allele
Hypothesis
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Methylation-sensitive restriction enzymes
Avy allele
A. Peaston TJL
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Bisulfite sequencing
Locus-specific detailed single-molecule analysis
Frommer et al, PNAS 1992
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Bisulfite Sequencing
Lane et al, Genesis 2003
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Higher throughput

• Pyrosequencing of clones (You still have to
  clone)
• SAGE

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Higher throughput

• Next Generation Sequencing
• High throughput, non-biological cloning

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And permutations.....
Murrell, Rakyan Beck, Hum Mol Genet 2005
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Chromatin and DNA methylation
Neurospora and Arabidopsis methylation depends
on H3K9me Plants RNAi targets chromatin
modification, gene silencing DNAme Mouse reduce
d 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
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• The nucleosome core. Site of action. Tails that
  get modified are evident in this image.

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http//atlasgeneticsoncology.org/Deep/Images/Chrom
atinFig2Engl.gif
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Histone modifying enzymes
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Chromatin remodelling Polycomb and Trithorax
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Kloc et al Current Biology 2008
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Epigenetic changes and phenotype

• DNA methylation and mutation
• Genomic instability
• Cancer
• Complex disease
• Environmental effects on DNA methylation

A. Peaston TJL
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Epigenetics/Epigenomics

• Modification of histones
• Mostly Acetylation () and Methylation (-)
• Histone acetyl transferases
• Histone deacetylases
• Modification of DNA
• In eukaryotes, mostly 5-methylcytosine (-)
• Dnmt (DNA methyl transferases)
• Removal?
• A sixth base 5-hydroxymethylcytosine

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Epigenetics/Epigenomics

• Methylated DNA recruits HDACs
• 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)

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