chromatin assembly nucleosomal dna primary chromatin structure beads-on-a-string

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Chromatin Assembly Nucleosomal DNA Primary chromatin structure beads-on-a-string Slide 2 Nucleosome from Horn and Peterson Science, 2002 ac DNA methylation Slide 3 Visualizing this primary structure nucleosomal arrays Partial nuclease digestion generates mono, di, trinucleosomes Slide 4 How is chromatin assembly achieved? Slide 5 Irregular assembly Dynamics of ATP-dependent chromatin assembly by ACF Dmitry V. Fyodorov & James T. Kadonaga NATURE | VOL 418 | 22 AUGUST 2002 | Assembly in vitro Histones + DNA + salt assemble into aggregates Histones + DNA + negatively charged proteins = mononucleosomes and poor nucleosmal array (irregularly spaced; smear) Slide 6 Dynamics of ATP-dependent chromatin assembly by ACF Dmitry V. Fyodorov & James T. Kadonaga NATURE | VOL 418 | 22 AUGUST 2002 | in vitro Xenopus/Drosophila extracts =chromatin (regularly spaced) Slide 7 What is in the extract? Histone chaperones Slide 8 negatively charged proteins help assembly; prevent aggregation NAP1 H2A/H2BNuclear Import, nucleosome disruption, histone variant incorporation nucleoplasmin H2A/H2BStorage, sperm histone exchange N1/N2 H3/H4Storage Slide 9 Slide 10 CAF-1 Chromatin assembly factor 1 More histone chaperones Slide 11 Replication-coupled chromatin assembly factor CAF-1 (identified in yeast; Bruce Stillman lab) brings H3/H4 onto newly synthesized DNA CAF-1 3 subunits:p150 (Cac1) binds PCNA p60(Cac2) binds ASF1 p48(Cac3) binds histones histone binding in many complexes Roles nucleosome deposition after replication heterochromatin maintenance Slide 12 Ransom et al., Cell, 2010 Smallest CAF subunit bound to histone H4 Slide 13 Replication Slide 14 Cell, Vol. 96, 575585, February 19, 1999, Replication-Dependent Marking of DNA by PCNA Facilitates CAF-1-Coupled Inheritance of Chromatin Kei-ichi Shibahara and Bruce Stillman Slide 15 Cac2 interacts with PCNA when Cac1 is present PCNA connects DNA replication to epigenetic inheritance in yeast Zhiguo Zhang, Kei-ichi Shibahara & Bruce Stillman NATURE | VOL 408 | 9 NOVEMBER 2000 PCNA --- Slide 16 Role of PCNA in chromatin assembly? 1. Adenine deficient yeast are red 2. Move Adenine biosynthesis gene into heterochromatin inactive; red colonies 3. Mutagenize strain 4. improper heterochromatin assembly in PCNA mutants causes Adenine biosynthesis gene reactivation; more whitish colonies Activation of Ade in telomeric heterochromatin in PCNA mutants PCNA connects DNA replication to epigenetic inheritance in yeast Zhiguo Zhang, Kei-ichi Shibahara* & Bruce Stillman* NATURE | VOL 408 | 9 NOVEMBER 2000 | Ade - Ade + PCNA wtPCNA mutant Slide 17 PCNA coordinates: DNA synthesis (processivity) Chromatin assembly Repair Methylation Epigenetic Inheritance Cell-cycle control Slide 18 Parental nucleosome segregation (hatched) De novo nucleosome assembly (unhatched) Slide 19 New nucleosome formation after replication 1 H3/H4 tetramer OR 2 H3/H4 dimers bind DNA 2 dimers H2A/H2B bind H3/H4 tetramer DNA first very reactive with histones, then matures Nucleosome deposition is essential if repressed, DNA will be duplicated but cell cycle arrest in G 2 Histone synthesis occurs at G 1 /S Slide 20 Newly synthesized histones have acetylated tails cytoplasmic (B-type) HAT: HAT1 HAT: histone acetyltransferase pattern of acetylation differs from acetylation involved in gene regulation Modifications on H4: H4K5ac, H4K12ac modifications lost after incorporation (HDAC, histone deacetylase) Slide 21 Corpet and Almouzni TICB 2008 H3K56ac H3K9/14ac GCN5 Rtt109 Slide 22 These modifications are important reduced viability in yeast if mutated Role:Nuclear import Binding/Deposition by histone chaperone Chromatin maturation Mark the histone as newly synthesized for chromatin assembly Modifications are rapidly removed after assembly Slide 23 Journal of Cell Science 113, 2647-2658 (2000) CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repair Patricia Ridgway and Genevive Almouzni or tetramer Slide 24 ASF1 acts together with CAF1, interacts with CAC2 subunit interacts with HAT1 complex interacts with MCM helicase Another histone chaperone Slide 25 Trujillo and Osley Nature Structural & Molecular Biology 2008 Slide 26 The ins and outs of nucleosome assembly Jill A Mello* and Genevive Almouzni Current Opinion in Genetics & Development 2001, 11:136141 Slide 27 Role of ASF1: generates H3 H4 dimer prevents tetramer formation breaks up tetramers presents H3/H4 dimer to CAF1 also role in H3K56ac by Rtt109 Slide 28 Ransom et al., Cell, 2010 Slide 29 Another histone chaperone Rtt106 interacts with CAF1 complex involved in H3/H4 assembly during replication high affinity for H3K56ac (as does CAF1) Slide 30 Modified from Fillingham and Greenblatt, Cell 2008 Slide 31 Ransom et al., Cell, 2010 H2A/H2B chaperone: FACT? Slide 32 BOTH long arrays AND properly spaced nucleosomes require other enzymes: ATP-dependent chromatin remodelers Chromatin assembly Chromatin array formation ATPasecomplex ISWI ACF/ChRAC CHD1-INO80 Slide 33 ISWI: chromatin remodeling ATPase roles in chromatin assembly together with NAP1 or Asf1 linker length, formation of long nucleosomal arrays transcriptional elongation silencing chromosome organization Slide 34 CHD1: chromatin remodeling ATPase roles in chromatin assembly together with NAP1 histone variant assembly Slide 35 Enhanced assembly Lusser et al., Nature Struc Mol Biol 2005 Slide 36 Different periodicity Lusser et al., Nature Struc Mol Biol 2005 Slide 37 Clapier and Cairns, Annu. Rev. Biochem., 2009 Slide 38 INO80: chromatin remodeling ATPase roles in replication stress Slide 39 Morrison and Shen, Nature reviews Mol Cell Biol, 2009 Slide 40 Open questions H3/H4 assembled as tetramer or as dimer? H2A/H2B chaperone? how are the parental histones incorporated? how are the chromatin marks copied? Epigenetic memory Slide 41 Epigenetic Memory Slide 42 Slide 43 Hypothesis 1: H3H4 dimers are incorporated can copy modifications, variants intramolecular Hypothesis 2A: H3 H4 tetramers are incorporated repressive marks are default activating marks incorporated during RI assembly variants Slide 44 Copy modification by recruiting the enzyme that makes the modification to this same modification (Intermolecular) Hypothesis 2 Slide 45 DNA methylation (DNMT1) interacts with PCNA KMT1C (H3K9me) interacts with DNMT1 KMT1C also interacts with CAF1 HP1 interacts with CAF1 Late replicating HETEROCHROMATIN Characterized by DNA methylation, HeK9me, and presence of HP1 Slide 46 Heterochromatin: new possibilities for the inheritance of structure Shiv IS Grewal and Sarah CR Elgin Current Opinion in Genetics & Development 2002, 12:178187 HS-2: heterochromatin 39C-X: euchromatin Slide 47 Chromatin Assembly During Replication Transcription Repair Slide 48 Chromatin assembly comes in two flavors: 1.Replication coupled (RC) 2.Replication independent (RI) Slide 49 CAF1H3/H4assembly, disassembly ASF1H3/H4H3/H4 dimer assembly, disassembly FACTH2A/H2Bassembly, disassembly ISWINAnucleosomal arrays, periodicity Heterochromatin replic. CHD1NAnucleosomal arrays INO80NAreplication stress Chromatin Assembly (RC) Slide 50 HirAH3.3/H4deposition during transcription; ASF1H3.3/H4 interact with transcription elongation factor FACT FACTH2A/H2B disassembly and reassembly during transcription elongation CHD1H3.3interacts with HirA SWRH2A.Z/H2Bdeposition during transcription INO80H2A.X/H2Bdeposition during DNA repair Chromatin Assembly (RI, replacement) Slide 51 Slide 52 = CAC3 =Htz1 =CENP-A Slide 53 DNA repair Slide 54 Transcription Slide 55 Dis-assembly and assembly Slide 56 Slide 57 Histone turnover in genes Consequence of nucleosome dis-assembly during transcription elongation FACT elongation factor Slide 58 Transcription elongation Slide 59 Histone turnover at promoters/genes Consequence of disassembly/assembly transcription nucleosome eviction chromatin remodeling histone modifications Slide 60 Histone turnover at promoters/genes Consequence of disassembly/assembly transcription nucleosome eviction chromatin remodeling histone modifications Slide 61 Slide 62 promoter 5 end gene H3.3 incorporation during transcription Drosophila Slide 63 Histone turnover at promoters/genes Consequence of disassembly/assembly transcription nucleosome eviction chromatin remodeling histone modifications Slide 64 High histone turnover at promoters is high = red nucleosomes Slide 65 Slide 66 Glossary PCNAsliding clampPOLII processivity MCMhelicaseDNA unwinding/replication NAP1histone chaperoneH2A/H2B nuclear import N1/N2histone chaperoneH3/H4 (storage) Nucleoplasminhistone chaperoneH2A/H2B (storage) Rtt106histone chaperoneH3K56ac/H4 RC assembly CAF1histone chaperoneH3/H4 RC assembly ASF1histone chaperoneH3/H4 RC and H3.3/H4 RI assembly HirAhistone chaperoneH3.3/H4 RI assembly HAT1cytoplasmic KATacetylates newly synthesized H4 Rtt109KATacetylates H3K56 ISWIchromatin remodelingassembly, spacing ATPase CHD1chromatin remodelingassembly, H3.3 with HirA ATPase ACScomplexes containingassembly ISWI INO80chromatin remodelingreplication stress, repair (H2AX) ATPase/complex SWRchromatin remodelingH2AZ exchange

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