Silencing/ DNA methylation/Imprinting 1. Silencing mechanisms Sir2/ HP1/HP1 and DNA methylation budding yeast, fission yeast, mammals/plants 2. Insulators (boundary elements/ enhancer blocker Position effect variegation 3. DNA methylation de novo, maintenance , CpG islands functions methods of study 4. DNA demethylation plants mammals 4. Imprinting Silencing creates large domains of chromatin that are compacted and less accessible to DNA-binding proteins Silencers Silencing proteins Sir2 HP1 Polycomb group (PcG) proteins DNA Methylation noncoding RNAs Boundary elements S.c. S.p. A.th.D.m. Mamma Hypoacetyl. H3/H4 H3K9 me HP1 DNA methyl. - + + + + + - + + + +* + + + + - + - + Polycomb - - + + + Sir2 + + + + + * present but binds H3K27me notH3K9me Heterochromatin Condensed, deeply staining Regular nucleosome spacing; DNA mostly associated with histone core Gene poor Late replicating Localized at nuclear periphery Chromatin in silenced regions Tight nucleosome arrays (short linkers) Presence of certain histone modifications i.e. methylated H3K9 Presence of DNA methylation Bordered by boundary elements Presence of non-histone proteins HP1, Sirtuins, PcG, telosome HP1 localization Fanti and Pimpinelli, COGD, 2008 Purpose of heterochromatin protect repetitive DNA from recombination keep centromer/telomer intact (chromosome integrity) protect genome from transposons and other ‘selfish DNA’ prevent (not block) transcription Silencing: lessons from yeasts Saccharomyces cerevisiae or Saccharomyces pombe Telomers Centromers Silent mating type loci Mating type loci Saccharomyces cerevisiae or Saccharomyces pombe Haploid cells have one active mating type locus (MAT, mat1) Others are silenced in heterochromatin When needed a mating type switch can occur Yeast mating type loci S. cervisiae S. pombe S.c. S.p. A.th.D.m. Mamma Hypoacetyl. H3/H4 H3K9 me HP1 DNA methyl. - + + + + + - + + + +* + + + + - + - + Polycomb - - + + + Sir2 + + + + + * present but binds H3K27me notH3K9me silenced state initiated deacetylation of H3/H4 3 binds H4K16 (unacetylated) ultimerization of Sir proteins; spreading ontinuous presence of Sir proteins Molecular Cell, Vol. 8, 489–498, September, 2001 Common Themes in Mechanisms of Gene Silencing Danesh Moazed SIR silencing Function Sir2 NAD-dependent HDAC! Molecular Cell, Vol. 8, 489–498, September, 2001 Common Themes in Mechanisms of Gene Silencing Danesh Moazed Other roles of sirtuins Linked to caloric restriction/lifespan extension Resistance to Neurodegenerative disease Cancer (Tumor suppressor) Yeast mating type loci S. cervisiae S. pombe S.c. S.p. A.th.D.m. Mamma Hypoacetyl. H3/H4 H3K9me HP1 DNA methyl. - + + + + + - + + + +* + + + + - + - + Polycomb - - +$ + + Sir2 + + + + + * present but does not bind H3K9me $ PRC2 but not PRC1 Silencing S. pombe Clr1 DNA binding Clr3 HDAC Clr4 HMT Clr6 HDAC Swi6 HP1 Rik1 DNA binding (Hda1) (Suv3-9) (Rpd3) (WD40 repeats) Silencing S. pombe HP1 Grewal and Elgin, COGD, 2002. Silencing S. pombe Silent mating type loci (S. pombe) DNA: centromer-like region (cenH) boundary elements ( IR-L, IR-R) Order of events: 1. Deacetylation (Class I (Hda1 Clr3), Class II (Clr6)) key residue H3K9 2. Methylation H3K9 Clr4 3. Binding HP1 Swi6, Chp2 4. Establishment of Heterochromatin Silencing S. pombe Silent mating type loci (S. pombe) DNA: centromer-like region (cenH) boundary elements ( IR-L, IR-R) Order of events: 1. Deacetylation H3K9 2. Methylation H3K9 3. Binding HP1 4. Establishment of Heterochromatin/spreading Trigger: repetitive DNA, small noncoding RNAs recruiting factors, silencers A key silencing protein: HP1 (S. pombe Swi6, Chp2) Identified biochemically as nonhistone chromosomal protein Binds to centromers, telomers, silenced regions In mammals HP1 comes in different isoforms (HP, HP1, HP1) Not all are repressive HP1 structure H1 Chromodomain: binds H3K9me Hinge region: binds RNA Chromoshadow domain: dimerization, interaction with other proteins such as Suv39 HP1 structure and binding Vermaak and Malik, Ann. Rev. Genet., 2009 HP1 chromodomain binds H3K9me2/3 Jacobs and Khorasanizadeh, Science 2001 HP1 Binds to: H3K9 me2/3 (chromodomain) su(var)3-9 KMT (chromoshadow domain; spreading) mammals: Suv39h1, SETDB1,G9a/GLP itself (chromoshadow domain; spreading) DMT3a, DMT1 (DNA methyltransferases) HDACs CAF1: replication linked recruitment lamin B (nuclear periphery) RNA (hinge region; recruitments, stabilize binding) Su(var)3-9 Is HMT: methylates H3K9 (di/tri methylation) has set domain and chromodomain (both required for Su(var)3-9 binding to chromatin) Binds HP1 HP1 heterochromatin: recruits other factors DNA methylation Grewal and Jia Nature Reviews Genetics 2007 Reversal of Heterochromatin formation H3K9me demethylase H3S10ph Both cause HP1 removal Methyl/phospho switch Hirota et al. Nature 2005 Genes within heterochromatin Uniquely regulated Require heterochromatin for proper expression Require HP1 for proper expression Heterochromatin spreads: prevented by boundary Gaszner and Felsenfeld Nature Reviews Genetics 2006 Defects in boundary Silencing of adjascent genes: transgene silencing translocation PEV (position effect variegation) PEV in Drosophila PEV in Drosophila Drosophila white gene (responsible for red eye color) is located in heterochromatin in the PEV flies PEV strain PEV strain HP1 mutant http://www.biology.wustl.edu/faculty/elgin/hetchrom.html PEV Gaszner and Felsenfeld Nature Reviews Genetics 2006 PEV in yeast Lunyak COCB, 2008 PEV Variegation due to imprecise establishment or inheritance of the silenced state of euchromatic gene Dissecting Heterochromatin Biology via genetic identification of modifiers of PEV Great system because is part on/part off so can easily identify: enhancers of PEV (more silenced) suppressors of PEV (less silenced) Types of PEV modifiers identified Boundary: a type of insulator that prevents the spreading of heterochromatin Valenzuela and Kamakaka , Annu. Rev. Genet. 2006 Delete boundary: H3K9me3 and HP1 (Swi5) spread! Noma et al. Science, 2001 How is boundary made? 1. Region of high transcriptional activity, low nucleosome density, high acetylation Counteract heterochromatin 2. Region with RNA secondary structure Can be active/ inactive Second type of insulator: enhancer blocker Valenzuela and Kamakaka , Annu. Rev. Genet. 2006 Blocks transcription if BETWEEN enhancer and promoter Gaszner and Felsenfeld Nature Reviews Genetics 2006 DNA Methylation and Imprinting DNA Methylation • Found in: – Prokaryotes: E. coli – Eukaryotes: Some Fungi Plants Vertebrates not found in: S. cerevisiae, C.elegans or Drosophila DNA Methylation http://www.med.ufl.edu/biochem/keithr/fig1pt1.html Eukaryotic DNA methylation • Mostly methylated cytosines at CpG* • Plants are also methylated at CHG or CHH 60-90% of CpGs are methylated at cytosine * CpA and CpT in ESCs DNA Methylation function • Generally a repressive mark • Reduced DNA-binding of many proteins • Condensed chromatin structure • Binding site for methyl binding proteins General roles imprinting X inactivation differentiation regulation of gene expression aging and cancer genome stabilty defense against transposons, viruses counteracts recombination of repetitive DNA chromosome segregation Experimental Techniques- Bisulfite Mutagenesis and Sequencing Slide from Marisa Bartolomei Experimental Techniques- Bisulfite Mutagenesis and Sequencing -Each row represents a different strand of DNA -Each circle represents a CpG -Filled in circles-methylated CpG -Open circles- unmethylated CpG Engel, 2004, Nat. Genet. Experimental Techniques- Methylation sensitive Restriction Enzyme Other techniques Miho, Nature Reviews, 2008 De Novo vs. Maintenance DNA methylation Maintenance Methylation Chen, Current topics in developmental biology, 2004 Maintenance Methyltransferases methylate hemi-methylated DNA after replication copy existing pattern onto new strand Dnmt1 mammals MET1 Arabidopsis CTM Arabidopsis generally ubiquitously expressed Dnmt1 is essential (mutants are embryonic lethal)Li, Cell, 1992 Bestero Bestor, Hum. Molec. Genet., 2000 Slide from Doris Wagner DNA Methyltransferases DRM2 Arabidopsis De novo methylation DRM Arabidopsis DNA methylation throughout development Imprints paternal maternal Modified from Reik, Theriogenology, 2003 DNA Demethylation Active vs. Passive Active demethylation Enzyme Demethylates DNA independent of Replication Passive Demethylation DNA methylation is not maintained through replication. Methylation marks get “diluted” out Active and Passive DNA demethylation in preimplantation mouse embryo 1 cell Paternal= Active Maternal=Passive Passive 2 cell- 4 cell Mayer, Nature, 2000 Lesson from Plants Law and Jacobsen, Nature Review Genetics 2010 Demethylation in Mammals Carey et al. Drug discovery today, 2011 DNA Methylation and transcription 1. Promoter regions CpG islands (CGIs): non-methylated CpG poor promoters: can be methylated, repressive CGIs that are methylated do exist: long term silencing imprinted loci; also seen in cancer CpG Islands (CGI) Definition: presence of normal (genome average) level of CpGs in promoters G +C observed/ expected CpG* CGI 65% close to 1 bulk DNA 40% <1 *Num of CpG/(Num of C × Num of G) × Total number of nucleotides in the sequence Kept unmethylated by: transcription factor binding actively demethylated DNMT cannot methylate well (NDR, H2A.Z, H3K4me2/3) DNA Methylation and transcription 2. Gene bodies A. DNA methylation is required for Efficient transcription • Prevents access to cryptic transcription start sites found in gene bodies B. Is important for Splicing • DNA methylation and nucleosomes predominantly found in exons also H3K9me3 • DNMT3 substrate is nucleosomal DNA • DNAme and H3K9me3 recruit one another DNA Methylation and transcription 3. Enhancers variable methylation level found = diffferentially methylated regions • prevent TF binding (E2F, NF-KB, AP2) • Recruit Methyl binding proteins • TF binding may alter DNAme (CTCF) cause and effect relationship not very clear Methyl-CpG-Binding proteins Bogdanovic, Chromosoma, 2009 Methyl-CpG-binding proteins MeCP2: first identified, has MBD (methyl binding domain), binds mSin3a (HDAC) MBD1: interacts with SETDB1 (H3K9 HMT) MBD2 : in MeCP1 complex: 1: heterochromatin, binds HP1, Suv39, p150 (CAF1) 2: also in NuRD (HDAC) MBD3: NuRD (HDAC) complex component MBD4: DNA glycosylase; Cme-T repair KAISO: no MBD, binds methylated DNA via Zn-finger Important for amphibian development DNA Methylation patterns and epigenetic memory Bird, GenesDev, 2002 DNA Hydroxymethylation In mouse cerebellum, hematopoesis, and ESCs 10% of all meC in mammals Promoters and gene bodies pluripotency role in bivalent domains in ESCs differentiation of hematopoeitic cells TET enzymes; bind to CGIs one pathway for demethylation for example of the paternal genome (hmC by TET1) followed by passive loss or deamination/mismatch repair or other pathways DNA Methylation The key EPIGENETIC mark for meiotic or transgenerational inheritance! Of critical importance in carcinogenesis Genomic Imprinting Both parental genomes are necessary! Genomic Imprinting The unequal expression of the maternal and paternal alleles of a gene. ~100 imprinted genes clustered throughout the genome Why Imprinting? Genetic conflict hypothesis Paternal allele: aggressive in obtaining maternal resources for particular offspring Maternal allele: equal allocation of resources to all offspring Many paternally expressed genes encode growth factors, Many maternally expressed genes inhibit growth factors trophoblast hypothesis Avoid depletion of maternal resources (after spontaneous oocyte activation) Make process fertilization dependent DNA Methylation • • • • Differential methylation during gametogenesis Reversible Stably inherited Repression of transcription ICR: Imprinting Control Region DMR: Differentially methylated region Differential Methylation ICR/DMR Hypothetical exampleEstablishment and maintenance of DNA methylation imprint sperm ~100% methylation at this specific imprinted locus ~50% methylation at this specific imprinted locus ~100% methylation at this specific imprinted locus Fertilization egg ~0% methylation at this specific imprinted locus Somatic cells of offspring Germline of male offspring DNA methylation throughout development Imprints paternal maternal Modified from Reik, Theriogenology, 2003 Differential DNA methylation established during gametogenesis Li, Nature, 2002 Techniques- Allele specific Expression F1 Hybrid! M. castaneous C57BL/6 X F1 Hybrid Examples of imprinted loci H19/Igf2 locusInsulator Model Wan, Advances in Genetics, 2008 ICR= Imprinting control region DMR/DMD= Differentially methylated region (domain) If CTCF is bound = enhancer blocker Igf2= Fetal Mitogen H19- ncRNA of unknown function Igf2r/Air locus- long ncRNA mediated imprinting Ideraabdullah, Mut. Res., 2008 Igf2r= female scavenger receptor for Igf2 Slide from Marisa Bartolomei ncRNA mediated vs. Insulator mediated • ncRNA mediated imprinting seems to be a more widespread mechanism of imprinting • Insulator mediated imprinting is more ancient (found similar mechanism in marsupials) Features of Imprinted loci • The imprinting mechanism acts in cis • Imprinted genes are clustered and are controlled by a single imprinting control region (ICR) • The ICR acquires an imprint in one gamete (often DNA methylation) • Imprinted gene clusters contain at least 1 long ncRNA Imprinted loci conserved in humans and associated with disease Ideraabdullah, Mut. Res. 2008 Example diseases: Angelman Syndrome, Prader-Willi Syndrome, Beckwith-Wiedemann Syndrome, Silver-Russell Syndrome Causes: Genetic mutation in expressed gene (i.e. deletion) Genetic mutations at the ICR that result in epigenetic defect Epimutations, with no genetic change Uniparental Disomy DNA methylation throughout development The requirement is to erase essentially all methylation in the PGC is thought to be linked to the low number of transgenerational inheritance events in mammals Pressure less strong on plants, imprinting mainly occurs in extra-embryonic tissue Imprints paternal maternal Silencing/ DNA methylation/Imprinting 1. Silencing mechanisms Sir2/ HP1/HP1 and DNA methylation budding yeast, fission yeast, mammals/plants 2. Insulators (boundary elements/ enhancer blocker Position effect variegation 3. DNA methylation de novo, maintenance , CpG islands functions methods of study 4. DNA demethylation plants mammals 4. Imprinting Glossary I Sir2, Sir1T NAD-dependent HDAC Swi6, HP1 H3k9me binding, heterochromatin Clr4, Su(var)3-9 KMT, generates H3K9me PEV position effect variegation due to juxtaposition of euchromatic gene and heterochromatin Boundary insulators that prevents heterochromatin spreading Enhancer blocker insulator that prevents enhancer from acting on the wrong promoter Glossary II DNMT1 (CMT, MET1) maintenance DNA methyltransferases DMNT3 (DRM2) DNA demethylation de-novo DNA methyltransferases plants: glycosylases plus base excision repair mammals: deamination plus mismatch repair hmcC TET pathway ICR/DMD Imprinting control region/ differentially methylated domain CHH and CA or CT always de novo methylation!