Epigenetics

1
Epigenetics
2
Nakao M, 2001
3

• Dr Alan Wolffe (1999) Epigenetics is heritable
  changes in gene expression that occur without a
  change in DNA sequence
• Epigenetics is ingenious system to selectively
  utilize genome information, through activating or
  inactivating functional genes.
• Identified epigenetic processes involved in human
  disease
• DNA methylation
• imprinting
• histone modifications
• Each of these processes influences chromatin
  structure and
• Thus regulates gene expression and DNA
  methylation, replication, recombination and
  repair.

4
(No Transcript)
5
(No Transcript)
6
Ac -acetylated histones mC-methylated
Cytosine HDAC -histone deacetylases Pol II- RNA
polymerase II GTF- general transcription
factors HAT -histone acetyltransferases MBD
-methylated DNA binding domain
Nakao M, 2001
7
1. System of DNA methylation
CpG islands gt200 bp stretches of DNA that have
a significantly higher concentration of 5-CpG3
dinucleotides than the bulk of the
genome Cytosine resudue in complementary
3-GpC-5 that makes a basepair, is also
methylated symmetrically, and these two methyl
groups show a three-dimentional structure
prominent in the major groove of the
dsDNA 50-60 of human genes have CpG islands in
front of and covering core promotor and
transcription start site 70-80 of CpGs in the
genome is methylated CpG islands in front of
genes are mostly unmethylated exceptions
imprinted genes and X-linked genes CpG island
are divided into several classes (1) methylated
on both alleles in all tissues located in high CG
isochores (2) differentially methylated and
located in low CG (lt0.5) isochores genomic
methylation pattern is stable and
heritable genome-wide methylation patterns are
reprogrammed in mammalian germ cells and in
pre-implantation embryos
8

• Mammalian methyltransferases
• DNMT1 - maintenance DNA methyltransferase
• methylates hemi-methylated DNA providing
  methylation pattern to the newly replicated
  daugther strand, based on parent strand
• represses transcription in complex with histone
  deacetylases
• 2. DNMT3a, DNMT3b - de novo methylases
• add a methyl group to unmethylated CpG base
  pairs, resulting in creation of a new
  hemi-methylated and then fully methylated CpG
• de novo methylation is implicated in cell growth
  and differentation,
• and in altered methylation in tumorigenesis.
• DNMT3b - mutated (common splice variant) in
  patients with ICF syndrome (immunodeficiency in
  association with centromere instability of
  chromosome 1, 9, 16, and facial anomalies)
  hypomethylation of pericentromeric satellite
  sequences
• Methyl-CpG binding proteins MeCP2, MBD1-4

9
Methylated DNA is replicated later than actively
transcribed DNA Monoallelically expressed genes
(imprinted) have coordinated replication timing
along human chromosomes
Replicated (active) genes
Non-replicated (silenced) genes
FISH analysis with imprinted gene pairs selected
from one chromosome
Ensminger and Chess, 2004
10
2. Histone modifications
The amino termini of histones contain a diversity
of posttranslational modifications. The most
promonent of them are acetylation and methylation
of Lysine (K) residues in the highly concerved H3
and H4
174 bp of
Histone tails
Histone fold domain
ACETYLATION
Methyl modifications
TRANSCRIPTION
Acetyl modifications
Grewal and Moazed, 2003
11
Many of trans-acting factors required for HT
assembly are either enzymes that directly modify
histones or factors binding to histones
e.g. SIR (silent information regulator) genes in
yeast - Sir 2 is a NAD-dependent histone
deacetylase Sir34 bind to deacetylated histone
tails in mammals, Drosophila and yeast
methylation of H3 lysine 9 correlates with
heterochromatin assembly. This residue is
methylated by concerved methyltransferase SUV39H1
in human, Su(var)3-9 in drosophila and Clr4 in
fission yeast Swi6 (yeast) and HP1 (human,
Drosophila) bind to Lys 9 methylated H3 tails
Histone methylation/HP1-binding cycle is an
ancient mechanism for propagating epigenetic
states. CpG methylation/histone deacetylase
binding cycle in evidently added later.
12
How are heterochromatin complexes targeted to a
specific chromosomal domain? Evidence suggests a
role for repetitive DNA elements and non-coding
RNAs in regional targeting of HT complexes.
S. cerevisiae
S. pombe
Small HT RNAs
Grewal and Moazed, 2003
13
RNA interference (RNAi) pathway

• Required for HT formation and H3 Lys9 methylation
  in S. pombe
• Argonaute (ago1), member of PAZ/Piwi family
• Dicer (dcr1), RNaseIII-like protein
• RNA-dependent RNA polymerase (rdp1)
• 2. Centromeric repeat sequences that are
  transcribed at low levels and produce ds RNA are
  sufficient to recruit HT at an ectopic site
• 3. Small HT RNAs provide specificity for
  targeting histone modifying activities and
  epigenetic modification of the genome through
  homology recognition
• 4. The role of RNAi in epigenetic gene silencing
  appears to be concerved among diverse species

14
2.
1.
3.
RISC- RNA induced silencing complex
Grewal and Moazed, 2003
15
Model for formation of silenced chromatin
domains E-histone-modifying Enzyme SF- silencing
factor BE- boundary element Deacetylation
and methylation of H3 Lys9 are followed by
deacetylation of H3 Lys 14 and create a binding
site for Swi6 silencing factor
H3 Lys 9 acetylation H3 Lys4 methylation STOP
heterochromatin
16
Ac -acetylated histones H3 Lys9 CpG-Me
-methylated Cytosine HDAC -histone
deacetylases DNMT -DNA methyltransferase
HMT-histone methyltransferase MBD -methylated
DNA binding domain HDAC deacetylates lysine
residues as the prerequisite for methylation HP1
protein recognizes MeK9, binds also HMT and
heterchromatin can spread
17
3. Chromatin remodeling
The positioning of histones along DNA is mediated
by ATP-dependent nucleosome - remodeling
complexes that use the energy of ATP hydrolysis
to noncovalently reposition histone octamers and
generate nucleosome free or dense chromatin.
18
Genes
Mechanims where involved
Diseases
SIOD - Schimle immuno-osseous dysplasia COFS -
cerebro-oculo-facio-skeletal syndrome CBS -
Cockayne syndrome type B RTS - Rubinstein Taybi
syndrome
Huang et al., 2003
19
Chromatin remodelling disorders 1.ATRX,
SNF2-family helicase (a-thalassemia X-linked
mental retardation) mutations Causes several
mental retardation disorders, facial, skeletal,
an urigenital abnormalities, a -thalassemia and
microcephaly ATRX protein resides predominantly
in repetitive DNA, ribosomal gene clusters,
pericentromeric heterochromatin. In ATRX cells,
the ribosomal DNA repeats are hypomethylated. 3.
SMARCAL1 (SWI/SNF-related matrix-associated,
actin-dependent regulator of chromatin, subfamily
A-like protein 1) Schimke immuno-osseous
dysplasia characterized by T-cell
immunodeficiency, renal failure, hypothyroidism,
bone-marrow failure etc. SMARCAL1 probably
regulates a subset of genes necessary for
cellular proliferation.
20
2. ERCC6 gene (excision repair cross-complementing
rodent repair deficiency, compelentation group
6) (a) COFS (cerebro-oculo-facio-skeletal)
syndrome failure of multiple systems and
premature death (b) Cockayne syndrome
UV-sensitivity, dwarfism, skeletal abnormalities,
mental retardation etc. Both cellular phenotypes
include increased sensitivity to oxydative and
UV-induced DNA-damage and failure to recover RNA
synthesis after UV irradiation ERCC6 plays key
role in transcription coupled DNA repair,
presumably opens the chromatin allowing access of
the DNA repair apparatus to the DNA
21
MeCP - Methyl-CpG binding protein IGF2 -
insulin-like growth factor 2
Epigenetics and human disease
CBP - CREB binding protein, co-activator of
transcription Mi2 - nucleosome remodelling
histone deacetylase
Nakao M, 2001
22
How is the heterochromatic state
inherited? During DNA replication, histones H3
and H4 are randomly distributed to sister
chromatides. modified parental histones and
assambled heterochromatin proteins (Swi6/HP1 or
Sir 3) can serve as molecular bookmarks to
imprint the parental histone-modification pattern
onto newly assambled nucleosomes.
23
4. Cancer epigenetics
Feinberg and Vogelstein (1983) loss of DNA
methylation in cancer cells compared to normal
tissues
Feinberg and Tycko, 2004
24
Hypomethylation and gene activity 1.
Hypomethylation can lead to gene activation (e.g.
HRAS, which is normally expressed only in
testis) Overexpression of cyclin D2 in gastric
carcinoma MN/CA9 in renal-cell carcinoma S100A4
metastasis associated gene in colon-cancer HPV16
in cervical cancer 2. A cellular methylator
phenotype has been linked to mismatch repair
(Lengauer et al) Hypermethylation of the
mismatch-repair gene MLH1 is commonly found in
mismatch-repair-defective tumors
25
3. Hypomethylation in cancer is related to
chromosomal instability Frequent unbalanced
chromosomal translocations with breakpoints in
pericentromeric satellite sequences (otherwise
highly methylated) 4. Hypomethylation is a
mechanism of drug, toxin and viral effects in
cancer MDR1, multidrug resistance gene
correlates with increased expression and drug
resistance in acute myelogenous leukemia Cadmium
inhibits DNA methyltransferase activity and leads
to acute hypomethylation, which is followed by
hypermethylation of dna after chronic exposure to
this epigenic carcinogen Arsenic induces Ras
hypomethylation in mice cervical cancer latency
is caused by hypermethylation of HPV16 genome
26
Hypermethylation and cancer
Promotor CpG hypermethylation of tumor supressor
genes Retinoblastoma gene RB Cyclin-dependent
kinase inhibitor (INK4A,p16, CDKN2A) Mismatch
repair gene MLH1 Von Hippel-Lindau (VHL) tumour
supressor E-cadherin Is the INITIAL SILENCING
HYPERMETHYLATION? Or is HYPERPEMTHYLATION a
consequence? Probably it is part of programmed
silencing, but is not per se responsible for
inactivation of a gene
27
Alternative models for CpG methylation in cancer
28
Loss of imprinting in cancer
germline
sporadic
BWS is fetal overgrowth disorder due to
deregulation of imprinted genes at 11p15
paternally expressed IGF2, KCnQ1OT1 maternally
expressed H19, CDKN1C, KNCQ1
Wilms tumour hypermethylation of H19 due to LOI
of IGF2 leading to biallelic expression and
twofold increase in doses