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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)
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Homeotic mutations transcriptional regulation defects Mutant: T3 haltere into T2 wing
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PcG Three complexes PRC2 PRC1 PhoRC
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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?
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Simon and Kingston, Nature Reviews Mol Cell Biol, 2009
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PRC2 unique roles Required for X-inactivation, imprinting Stem cell maintenance EZH2 (human E(z)) important marker for tumor metastasis)
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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
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PRC2 recruitment by long nc RNAs XIST (X-inactivation) Kcnq1ot1 (Imprinting) Hotair (Hox gene expression)
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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
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Simon and Kingston, Nature Reviews Mol Cell Biol, 2009
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Lund and Lohuizen COCB16 239-246 (2004) Two step process? PRC1 is also recruited independently of H3K27me3 PRC1 occupies large domains, stable repression
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PRC1 functions Inhibits transcription Blocks SWI/SNF chromatin remdoleing Compacts chromatin arrays
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Second complex: Ubiquitylation of H2A? PRC1-like Simon and Kingston, 2009
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PhoRC Pho: DNA binding protein SFMBT:can bind to certain methylated lysines on H3
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YY1
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Mammals (and plants) Many PRC2 and PRC1 complexes Organism complexity, response to the environment Simon and Kingston, 2009
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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
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PREs have many binding sites for certain TFs
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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.
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Drosophila PRC1 PRC2
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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
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Simon and Kingston, 2009
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HOX gene regulation Normal expression
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PREs have many binding sites for certain TFs
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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
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Molecular Cell, Vol. 8, 1145–1151, November, 2001, Developmentally regulated enhancer blockers
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HOX genes turned on in certain regions PcG maintain this pattern (memory) Also required for proper spatial regulation later in development
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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
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PhoRc Noncoding RNA
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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
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Science 308 (2005)
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mammals
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Drosophila
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Block of transcription elongation? Simon and Kingston, 2009
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http://www.igh.cnrs.fr/equip/cavalli/Figure0.jpeg
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Supressor screen in polycomb mutants Look for wild-type looking flies suppress ectopic HOX gene expression Identified trithorax group (TrxG) proteins
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Simon and Kingston, 2009
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TrxG Brahma: SWI/SNF ATPase Moira Osa Ash1, 2 Trithorax (TRX)/MLL Identified as suppressors of PcG mutations
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TrxG TRX: H3K4 KMT ASH1: H3K4 KMT, also H3K36 H3K4-(me)3: TrxG binding inhibits PcG binding inhibits HP1 binding
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PREs have many binding sites for certain TFs
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Often PcG and TrxG bind the same element PRE Continuous inactivation required for PcG silencing Transcription through PRE causes loss of silencing
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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
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Mammals Some PREs recently identified TrxG and PcG opposing roles conserved Also in plants
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PcG (and TrxG) Not just transcriptional memory more dynamic gets reset Cell fate not as fixed as was previously assumed
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Noncoding RNA
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Not this simple
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Opposing roles of TrxG and PcG Reversibility of marks UTX JMJD3 part of TrxG complex LSD1 and JARID associate with PcG complex
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Bivalent domains
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Embryonic stem cells PcG proteins occupies promoters of differentiation genes prevents differentiation
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Associated with poised polymerase
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Some concerns Contradictory data Marks not shown to be present at the same locus: population effect? In some cases no evidence for poised PolII.
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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)
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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
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