1. Polycomb Group PcG Regulators Identified in Drosophila genetically Mutations in PcG proteins cause ectopic expression of homeotic genes ANT-C and BX-C (Antennapedia and Bithorax Complex)

2. Homeotic mutations transcriptional regulation defects Mutant: T3 haltere into T2 wing

3. PcG Three complexes PRC2 PRC1 PhoRC

5. PRC2 E(z):HMT H3K27me enhancer of zeste Su(z)12:Zn finger protein suppressor of zeste; E(z) cofactor ESC/EED: WD40 protein interaction domains specificity of HMT; E(z) cofactor extra sex combs P55: RbApAB46/48/NURF55 (CAF subunit; sometimes complexed with HDACs) -------------------------------------------------------- PCL: polycomb like; PhD and Tudor domains Stimulates enzyme activity, role in recruitment?

6. Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

7. PRC2 unique roles Required for X-inactivation, imprinting Stem cell maintenance EZH2 (human E(z)) important marker for tumor metastasis)

9. PRC2 is required for Xi in the extra-embryonic lineage and in the embryo puts on H3K27me mark occurs right after Xist coating Xist is necessary and sufficient for PRC2 recruitment

10. PRC2 recruitment by long nc RNAs XIST (X-inactivation) Kcnq1ot1 (Imprinting) Hotair (Hox gene expression)

12. Pc: polycomb has N-terminal chromodomain binds me H3K27 PH:polyhomeotic Zn- finger, Q-repeats Psc: posterior sex combs (human: Bmi1) Ring finger protein interaction domain acts also as suppressor of telomeric position effect dRING: Ubiquitin ligase H2A K119ub PRC1

13. Simon and Kingston, Nature Reviews Mol Cell Biol, 2009

14. Lund and Lohuizen COCB16 239-246 (2004) Two step process? PRC1 is also recruited independently of H3K27me3 PRC1 occupies large domains, stable repression

15. PRC1 functions Inhibits transcription Blocks SWI/SNF chromatin remdoleing Compacts chromatin arrays

16. Second complex: Ubiquitylation of H2A? PRC1-like Simon and Kingston, 2009

18. PhoRC Pho: DNA binding protein SFMBT:can bind to certain methylated lysines on H3

19. YY1

20. Mammals (and plants) Many PRC2 and PRC1 complexes Organism complexity, response to the environment Simon and Kingston, 2009

21. PRE PcG Response Element cis-acting DNA elements that PcG proteins bind to in Drosophila Many PREs in homeotic gene regulatory regions PREs are comprised of many factor biding sites

22. PREs have many binding sites for certain TFs

23. PREs Genomewide studies: PRC1 binding largely overlaps with Pho binding Less (but still considerable) overlap with GAF and DSP1 Binding site mutations: Pho and GAF contribute.

24. Drosophila PRC1 PRC2

25. PcG recruitment Noncoding RNAs can recruit PRC2 PhoRC binds PRE & can recruit PRC2 result: H3K27me H3K27me can recruit PRC1 PhoRC binds PRE & can recruit PRC1 PREs are devoid of nucleosomes PhoRC can wrap DNA around itself in presence of PRC1

26. Simon and Kingston, 2009

29. HOX gene regulation Normal expression

30. PREs have many binding sites for certain TFs

31. Fab-7 Enhancer blocker between iab-6 and iab-7 regulatory regions Blocks enhancers and binding site PcG! Deletion causes homeotic phenotypes Is regulated, allows is tissue/stage specific enhancer blocker

32. Molecular Cell, Vol. 8, 1145–1151, November, 2001, Developmentally regulated enhancer blockers

33. HOX genes turned on in certain regions PcG maintain this pattern (memory) Also required for proper spatial regulation later in development

34. How does the silencing memory work? PcG required continuously (adult) maintained through cell cycle most PcG leaves chromatin during mitosis some remains = mark? H3K27 = mark? PRE = mark? Also associated with histone deacetylation other (H3K9) histone methylation DNA methylation memory

35. PhoRc Noncoding RNA

36. Mechanism for PcG repression Generates large H3K27me domains Compacts chromatin Prevents PolII elongation Form PcG bodies (subnuclear silencing compartments) Recruits HDACs, DNMTs Inhibits SWI/SNF activity

37. Science 308 (2005)

38. mammals

39. Drosophila

40. Block of transcription elongation? Simon and Kingston, 2009

41. http://www.igh.cnrs.fr/equip/cavalli/Figure0.jpeg

42. Supressor screen in polycomb mutants Look for wild-type looking flies suppress ectopic HOX gene expression Identified trithorax group (TrxG) proteins

43. Simon and Kingston, 2009

44. TrxG Brahma: SWI/SNF ATPase Moira Osa Ash1, 2 Trithorax (TRX)/MLL Identified as suppressors of PcG mutations

45. TrxG TRX: H3K4 KMT ASH1: H3K4 KMT, also H3K36 H3K4-(me)3: TrxG binding inhibits PcG binding inhibits HP1 binding

46. PREs have many binding sites for certain TFs

47. Often PcG and TrxG bind the same element PRE Continuous inactivation required for PcG silencing Transcription through PRE causes loss of silencing

48. Homeotic Gene Expression Transcription initiation Induced by maternal gene products and segment identity gene products in 3.5 hr embryo Transcription maintenance 5-7 hr embryo: activators are gone TrxG maintenance of activated state of homeotic genes PcG maintenance of repressed state of homeotic genes

49. Mammals Some PREs recently identified TrxG and PcG opposing roles conserved Also in plants

50. PcG (and TrxG) Not just transcriptional memory more dynamic gets reset Cell fate not as fixed as was previously assumed

51. Noncoding RNA

53. Not this simple

54. Opposing roles of TrxG and PcG Reversibility of marks UTX JMJD3 part of TrxG complex LSD1 and JARID associate with PcG complex

55. Bivalent domains

57. Embryonic stem cells PcG proteins occupies promoters of differentiation genes prevents differentiation

59. Associated with poised polymerase

60. Some concerns Contradictory data Marks not shown to be present at the same locus: population effect? In some cases no evidence for poised PolII.

61. Going further Both TrXG and PcG stable epigenetic memory Both can be reversed; needs multiple steps/cues Generally TrxG and PcG have a dynamic role Lund and Lohuizen COCB16 239-246 (2004)

62. PcG silencing reversal (needs several steps) Resetting by transcription Signaling repression of PcG (WNT/TGFB/HH) Posttranslational modifications PcG (BMI1-P dissociates) Recruitment of TrxG proteins MLL plus H3K27 demethylase chromatin remodeling