Epigenetic Mechanisms

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Epigenetic Mechanisms

• Victoria Lunyak

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What is Epigenetics?
Epigenetics can be defined as any inheritable
influence on gene activity that does not involve
a change in DNA sequence. Common mechanisms may
include but not limited to -DNA
methylation -Histone modification/histone
variants -Regulatory non-coding RNAs
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Two main directions in study of chromatin
inheritance

• First
• How are chromatin state specified?
• Second
• How are these states transmitted with high
  fidelity?

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DNA Methylation

• DNA methylation occurs at 5MC within CpG
  dinucleotides. 5MC constitutes lt1 of
  nucleotides.
• CpG frequency 5X less than expected, though to
  be due to increased mutation at CpGs.
• Transitions (C-T) occur at 10-40 fold higher than
  other dinucleotides.
• As many as 50 of point mutations in p53 gene in
  human tumors occur at CpG.
• CpGs are not randomly distributed in the genome.
• CpGs are methylated in repeats and 3 regions of
  genes, but not in CpG islands.
• There are estimated to be about 26,000-45,000 CpG
  islands, many which tend to be located in the
  promoter regions of
  housekeeping genes and some tissue specific
  genes.

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DNA Methylation
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DNA Methylation

• How does methylation occur?
• 1.De Novo methylation.
• What signals de novo methylation? Some evidence
  that repeats may alter chromatin structure and
  signal de novo methylation.
• 2. Maintenance Methylation- copies DNA so that
  methylation parttern on newly replicated DNA
  strand is identical to previous.
• 3. Demethylation of DNA- the existence of active
  demethylation is controversial.
• 4. Passive demethylation

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DNA Methylation
-Dnmt 1 thought to be primarily a maintenance
methylase. 10-40 fold preference for
hemimethylated DNA. Localizes to replication
foci. -Dnmt 3A and 3B are thought to be de novo
methylases. DNMT3 family contain similar
catalytic domain, but different regulatory
domains. Contains a PHD (plant homeodomain) found
in many chromatin associated proteins.
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DNA Methylation

• The Knock-out of any of the methyltransferase
  genes is lethal during development(Dnmt 1 or 3B)
  or shortly thereafter (3A about 4 weeks of age).
• -mutations in Dnmt3B cause ICF syndrome
  (Imunodeficiency, centromeric instability,facial
  anomalies).
• -ATRX is a chromatin remodeling protein.
  Mutations cause alfa thalassemia,mental
  retardation, are X-linked, and disrupt normal
  patterns of DNA methylation.
• -The function of DNMT2 and DNMT3L are largely
  unknown.

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DNA Methylation

• Dnmts associated with
• -Histone deacetylases (HDAC1/2).
• -tumor suppressors/transcriptional repressors
  (e.g. pRb, RP58).
• -Repressor oncogenes (PML-RAR).
• -methyl CpG binding proteins capable of binding
  methylated DNA (Mecp2/1, mbd2, mbd3) are in
  complex with chromatin modifying enzymes.

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Histone code hypothesis
Main directions of the research in this field

• Histone Modifications
• Histone Variants
• Enzymes providing histone modifications.
• Coordination of histone modifications
• Protein domains that bind the modified histones
• Nuclear architecture and histone code
• (Heterochromatin vs. euchromatin )

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Histone code hypothesis
Histone Modifications

• Different combinations of histone modifications,
  especially located near or within a genes
  promoter, may be VERY SPECIFIC to the
  transcriptional state of the gene

Peterson CL et al, Current Biology, 2004
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Histone code hypothesis
Enzymes providing histone modifications

Acetylation HATs - CBP,p300, GCN5, ATF2, Tip
60 Deacethylation HDACs- class I and
II Methyaltion Lysine SET-domain HMTase and
non-SET domain HMTase (Dot1) Arginine PRMT
family, CARM1 Demethylation LSD1 Ubiquitination
ubiquitin conjugase Rad6/ligase Bre1for
H2B De-Ubiquitination SAGA-associated Ubp10
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Histone code hypothesis
Enzymes providing histone methyaltion
Methylated histones first described in 1964,
Murray 1964 Biochemistry 3, 10-15
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Histone code hypothesis
Histone demethylase LSD-1
J. Wysocka,Cell (2005)
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Histone code hypothesis
Histone demethylase LSD-1
Shi et al., (2004).
Metzger et al. (2005)
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Histone code hypothesis
Nucleosomal histones and their variants
K. Sharma 2005,NATURE REVIEWS
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Histone code hypothesis
Connection between histone variants and
modifications
Example The modifications shown byChIP to be
enriched in active chromatin, includingacetylation
of K9, K14, K18 and K23 and methylation of K4
and K79, were found to be enriched two to
fivefold in H3.3 relative to H3.
Variant histones are assembled by specific
complexes
Example The SWR1(Snf2/Snf2-related ATPase)
complex appears to destabilize the
nucleosome,exchanging H2A.ZH2B for H2AH2B.
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Histone code hypothesis

• Something is writing this code.
• and
• Something is reading this code

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Histone code hypothesis
Protein domains that bind the modified histones
The bromodomain binds acetyl-lysine
Owen et al. 2000 EMBO J 19 6141-9
The chromodomain binds methyl-lysine
Brehmet al. 2004 BioEssays26 133-40
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Histone code hypothesis
Proteins that bind the modified histones
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Histone code hypothesis

• Coordination of histone modifications

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Histone code hypothesis
Nuclear architecture and histone code (
Heterochromatin vs. Euchromatin )
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Interaction between DNA methylation and Histone
acetylation/ methylation

• Rountree MR et al, Oncogene, 2001

Transcriptionally active DNA- unmethylated DNA
with acetylated histones Repressed (silent) DNA-
methylated DNA with deacetylated histones
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Non-coding RNA
Short interfering RNA (siRNA) Micro RNA (mir
RNA) Double-stranded RNA (ds RNA) Short
heterochromatic RNA (sh RNA) transcripts from
repeated sequences (ALU, LTR)
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Non-coding RNAMicroRNA biogenesis
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Non-coding RNA How Many Different MicroRNA genes
are There?
C. elegance 40,000 pairs of hairpins
35,697 had the minimal
conservation
to receive MiRscan score.
53 miRNAs were cloned
Drosophila melanogaster 436,000 pairs of
hairpins
118,000 structure with high score
124 miRNA genes
Human 800,000 pairs of hairpins
15,000 have a minimal conservation
to receive MIRscan score ( non-coding
regions) 255 miRNA genes
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Non-coding RNA How Many Different MicroRNA are
There?

• 41 of miRNA cloned from HeLa cells are variant
  of let-7

• 28 of human brain miRNAs are variants of mir-124

• 45 of miRNA cloned from human heart are mir-1
• which was initially cloned from Drosophila
  embryos

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Non-coding RNA How is MicroRNA Activity
Regulated?

• miRNAs have diverse temporal and quantitative
• expression profile
• miRNA genes are known to reside in local genomic
• clasters with possible operon-like organization

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Non-coding RNA MicroRNAs and Short Interfering
RNAs Might Use the Same RNA Processing Complex.
MicroRNA gene
Transposons , transgenes, viruses,
heterochromatic DNA
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Non-coding RNA What Do MicroRNA Do ?
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Interplay between different epigenetic strategies
Margueron ,Current Opinion in Genetics
Development (2005)