Epigenetics

Nakao M, 2001

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:

1. DNA methylation2. imprinting3. histone modifications

Each of these processes influences chromatin structure andThus regulates gene expression and DNA methylation, replication, recombination and repair.

Nakao M, 2001

Ac -acetylated histones; mC-methylated CytosineHDAC -histone deacetylases: Pol II- RNA polymerase II GTF- general transcription factorsHAT -histone acetyltransferases;MBD -methylated DNA binding domain

1. System of DNA methylation

*CpG islands: >200 bp stretches of DNA that have a significantly higher concentration of 5’-CpG;3’ 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 (<0.5) isochores*genomic methylation pattern is stable and heritable*genome-wide methylation patterns are reprogrammed in mammalian germ cells and in pre-implantation embryos

Mammalian methyltransferases:

1.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

*Methylated DNA is replicated later than actively transcribed DNA

*Monoallelically expressed genes (imprinted) have coordinated replication timing along human chromosomes

Non-replicated (silenced) genes

Replicated (active) genes

Ensminger and Chess, 2004

FISH analysis with imprinted gene pairs selected from one chromosome

Grewal and Moazed, 2003

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 fold domain

Histone tails

Acetyl modifications

Methylmodifications

ACETYLATION

TRANSCRIPTION

2. Histone modifications

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; Sir3&4 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.

Grewal and Moazed, 2003

Small HT RNAs

S. cerevisiae S. pombe

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.

RNA interference (RNAi) pathway

1.Required for HT formation and H3 Lys9 methylation in S. pombe :Argonaute (ago1), member of PAZ/Piwi familyDicer (dcr1), RNaseIII-like proteinRNA-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

1. 2.

3.

Grewal and Moazed, 2003

RISC- RNA induced silencing complex

Model for formation of silenced chromatin domainsE-histone-modifyingEnzymeSF- silencing factorBE- 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

Ac -acetylated histones H3 Lys9CpG-Me -methylated Cytosine

HDAC -histone deacetylasesDNMT -DNA methyltransferase HMT-histone methyltransferaseMBD -methylated DNA binding domain

HDAC deacetylates lysine residues as the prerequisite for methylation

HP1 protein recognizes MeK9, binds also HMT and heterchromatin can spread

3. Chromatin remodelingThe 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.

Huang et al., 2003

Genes

Mechanimswhereinvolved

Diseases

SIOD - Schimle immuno-osseous dysplasiaCOFS - cerebro-oculo-facio-skeletal syndromeCBS - Cockayne syndrome type BRTS - Rubinstein Taybi syndrome

Chromatin remodelling disorders:

1.ATRX, SNF2-family helicase (-thalassemia X-linked mental retardation) mutations:Causes several mental retardation disorders, facial, skeletal, an urigenital abnormalities, -thalassemia and microcephalyATRX 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.

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

Epigenetics and human disease

Nakao M, 2001

CBP - CREB binding protein, co-activator of transcriptionMi2 - nucleosome remodelling histone deacetylase

MeCP - Methyl-CpG binding proteinIGF2 - insulin-like growth factor 2

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.

4. Cancer epigenetics

Feinberg and Tycko, 2004

Feinberg and Vogelstein (1983): loss of DNA methylation in cancer cells compared to normal tissues

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

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

Hypermethylation and cancer

Promotor CpG hypermethylation of tumor supressor genes:

Retinoblastoma gene RBCyclin-dependent kinase inhibitor (INK4A,p16, CDKN2A)Mismatch repair gene MLH1Von Hippel-Lindau (VHL) tumour supressorE-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

Alternative models for CpGmethylation in cancer

Loss of imprinting in cancer

BWS is fetal overgrowth disorder due to deregulation of imprinted genes at 11p15: paternally expressed IGF2, KCnQ1OT1 & maternally expressed H19, CDKN1C, KNCQ1

sporadic

Wilms tumour: hypermethylation of H19 due to LOI of IGF2 leading to biallelic expression and twofold increase in doses

germline