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.

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: >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

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; 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.

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