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Silencing/ DNA methylation/Imprinting

Silencing/ DNA methylation/Imprinting. Silencing mechanisms Sir2/ HP1/HP1 and DNA methylation budding yeast, fission yeast, mammals/plants Insulators (boundary elements/ enhancer blocker Position effect variegation 3. DNA methylation de novo, maintenance , CpG islands functions

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Silencing/ DNA methylation/Imprinting

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  1. Silencing/ DNA methylation/Imprinting • Silencing mechanisms • Sir2/ HP1/HP1 and DNA methylation • budding yeast, fission yeast, mammals/plants • Insulators (boundary elements/ enhancer blocker • Position effect variegation • 3. DNA methylation • de novo, maintenance , CpG islands • functions • methods of study • DNA demethylation • plants • mammals • 4. Imprinting

  2. Silencing creates large domains of chromatin that are compacted and less accessible to DNA-binding proteins

  3. Silencers Silencing proteins Sir2 HP1 Polycomb group (PcG) proteins DNA Methylation noncoding RNAs Boundary elements

  4. S.c. S.p. A.th.D.m. Mammals Hypoacetyl. + + + + + H3/H4 H3K9 me - + + + + HP1 - + +* + + DNA methyl. - - + - + Polycomb - - + + + Sir2 + + + + + * present but binds H3K27me notH3K9me

  5. Heterochromatin Condensed, deeply staining Regular nucleosome spacing; DNA mostly associated with histone core Gene poor Late replicating Localized at nuclear periphery

  6. 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

  7. HP1 localization Fanti and Pimpinelli, COGD, 2008

  8. 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

  9. Silencing: lessons from yeasts Saccharomyces cerevisiae or Saccharomyces pombe Telomers Centromers Silent mating type loci

  10. 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

  11. Yeast mating type loci S. cervisiae S. pombe

  12. S.c. S.p. A.th.D.m. Mammals Hypoacetyl. + + + + + H3/H4 H3K9 me - + + + + HP1 - + +* + + DNA methyl. - - + - + Polycomb - - + + + Sir2 + + + + + * present but binds H3K27me notH3K9me

  13. silenced state initiated deacetylation of H3/H4 Sir3 binds H4K16 (unacetylated) Multimerization of Sir proteins; spreading Molecular Cell, Vol. 8, 489–498, September, 2001 Common Themes in Mechanisms of Gene Silencing Danesh Moazed Continuous presence of Sir proteins

  14. SIR Function Sir2 NAD-dependent HDAC! silencing Molecular Cell, Vol. 8, 489–498, September, 2001 Common Themes in Mechanisms of Gene Silencing Danesh Moazed

  15. Other roles of sirtuins Linked to caloric restriction/lifespan extension Resistance to Neurodegenerative disease Cancer (Tumor suppressor)

  16. Yeast mating type loci S. cervisiae S. pombe

  17. S.c. S.p. A.th.D.m. Mammals Hypoacetyl. + + + + + H3/H4 H3K9me - + + + + HP1 - + +* + + DNA methyl. - - + - + Polycomb - - +$ + + Sir2 + + + + + * present but does not bind H3K9me $ PRC2 but not PRC1

  18. Silencing S. pombe Clr1 DNA binding Clr3 HDAC (Hda1) Clr4 HMT (Suv3-9) Clr6 HDAC (Rpd3) Swi6 HP1 Rik1 DNA binding (WD40 repeats)

  19. Silencing S. pombe HP1 Grewal and Elgin, COGD, 2002.

  20. 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

  21. 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

  22. 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

  23. HP1 structure H1 Chromodomain: binds H3K9me Hinge region: binds RNA Chromoshadow domain: dimerization, interaction with other proteins such as Suv39

  24. HP1 structure and binding Vermaak and Malik, Ann. Rev. Genet., 2009

  25. HP1 chromodomain binds H3K9me2/3 Jacobs and Khorasanizadeh, Science 2001

  26. 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)

  27. 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

  28. HP1 heterochromatin: recruits other factors DNA methylation Grewal and Jia Nature Reviews Genetics 2007

  29. Reversal of Heterochromatin formation H3K9me demethylase H3S10ph Both cause HP1 removal

  30. Methyl/phospho switch Hirota et al. Nature 2005

  31. Genes within heterochromatin Uniquely regulated Require heterochromatin for proper expression Require HP1 for proper expression

  32. Heterochromatin spreads: prevented by boundary Gaszner and Felsenfeld Nature Reviews Genetics 2006

  33. Defects in boundary Silencing of adjascent genes: transgene silencing translocation PEV (position effect variegation)

  34. PEV in Drosophila

  35. PEV in Drosophila Drosophila white gene (responsible for red eye color) is located in heterochromatin in the PEV flies PEV strain HP1 mutant PEV strain http://www.biology.wustl.edu/faculty/elgin/hetchrom.html

  36. PEV Gaszner and Felsenfeld Nature Reviews Genetics 2006

  37. PEV in yeast Lunyak COCB, 2008

  38. 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)

  39. Types of PEV modifiers identified

  40. Boundary: a type of insulator that prevents the spreading of heterochromatin Valenzuela and Kamakaka , Annu. Rev. Genet. 2006

  41. Delete boundary: H3K9me3 and HP1 (Swi5) spread! Noma et al. Science, 2001

  42. How is boundary made? Region of high transcriptional activity, low nucleosome density, high acetylation Counteract heterochromatin 2. Region with RNA secondary structure Can be active/ inactive

  43. Second type of insulator: enhancer blocker Valenzuela and Kamakaka , Annu. Rev. Genet. 2006

  44. Blocks transcription if BETWEEN enhancer and promoter Gaszner and Felsenfeld Nature Reviews Genetics 2006

  45. DNA Methylation and Imprinting

  46. DNA Methylation • Found in: • Prokaryotes: E. coli • Eukaryotes: Some Fungi Plants Vertebrates not found in: S. cerevisiae, C.elegansor Drosophila

  47. DNA Methylation http://www.med.ufl.edu/biochem/keithr/fig1pt1.html

  48. 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

  49. DNA Methylation function • Generally a repressive mark • Reduced DNA-binding of many proteins • Condensed chromatin structure • Binding site for methyl binding proteins

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