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P oly c omb G roup PcG Regulators

P oly c omb G roup 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). Homeotic mutations transcriptional regulation defects. Mutant: T3 haltere into T2 wing.

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P oly c omb G roup PcG Regulators

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  1. Polycomb Group PcG Regulators Identified in Drosophilagenetically 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

  4. 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?

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

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

  7. 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

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

  9. PRC1 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

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

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

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

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

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

  15. YY1

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

  17. 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

  18. PREs have many binding sites for certain TFs

  19. 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.

  20. Drosophila PRC1 PRC2 PRC1

  21. 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

  22. Simon and Kingston, 2009

  23. HOX gene regulation Normal expression

  24. PREs have many binding sites for certain TFs

  25. 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

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

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

  28. 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

  29. PhoRc Noncoding RNA

  30. 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

  31. Science 308 (2005)

  32. mammals

  33. Drosophila

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

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

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

  37. Simon and Kingston, 2009

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

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

  40. PREs have many binding sites for certain TFs

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

  42. 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 TrxGmaintenance of activated state of homeotic genes PcG maintenance of repressed state of homeotic genes

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

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

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