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Chromatin Assembly

Chromatin Assembly. Nucleosomal DNA Primary chromatin structure beads-on-a-string. ac. ac. ac. ac. DNA methylation. ac. Nucleosome. from Horn and Peterson Science , 2002. Visualizing this primary structure nucleosomal arrays.

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Chromatin Assembly

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  1. Chromatin Assembly Nucleosomal DNA Primary chromatin structure beads-on-a-string

  2. ac ac ac ac DNA methylation ac Nucleosome from Horn and Peterson Science, 2002

  3. Visualizing this primary structure nucleosomal arrays Partial nuclease digestion generates mono, di, trinucleosomes…

  4. How is chromatin assembly achieved?

  5. Assembly in vitro Histones + DNA + salt assemble into aggregates Histones + DNA + negatively charged proteins = mononucleosomes and poor nucleosmal array (irregularly spaced; “smear”) Irregular assembly Dynamics of ATP-dependent chromatin assembly by ACF Dmitry V. Fyodorov & James T. Kadonaga NATURE | VOL 418 | 22 AUGUST 2002 |

  6. in vitro Xenopus/Drosophila extracts =chromatin (±regularly spaced) Dynamics of ATP-dependent chromatin assembly by ACF Dmitry V. Fyodorov & James T. Kadonaga NATURE | VOL 418 | 22 AUGUST 2002 |

  7. What is in the extract? Histone chaperones

  8. Histone chaperones negatively charged proteins help assembly; prevent aggregation NAP1 H2A/H2B Nuclear Import, nucleosome disruption, histone variant incorporation nucleoplasmin H2A/H2B Storage, sperm histone exchange N1/N2 H3/H4 Storage

  9. More histone chaperones CAF-1 Chromatin assembly factor 1

  10. 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 histoneshistone binding in many complexes Roles nucleosome deposition after replication heterochromatin maintenance

  11. Smallest CAF subunit bound to histone H4 Ransom et al., Cell, 2010

  12. Replication

  13. Cell, Vol. 96, 575–585, February 19, 1999, Replication-Dependent Marking of DNA by PCNA Facilitates CAF-1-Coupled Inheritance of Chromatin Kei-ichi Shibahara and Bruce Stillman

  14. - - - Cac2 interacts with PCNA when Cac1 is present PCNA PCNA connects DNA replication to epigenetic inheritance in yeast Zhiguo Zhang, Kei-ichi Shibahara & Bruce Stillman NATURE | VOL 408 | 9 NOVEMBER 2000

  15. 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 PCNA connects DNA replication to epigenetic inheritance in yeast Zhiguo Zhang, Kei-ichi Shibahara* & Bruce Stillman* NATURE | VOL 408 | 9 NOVEMBER 2000 | PCNA wt PCNA mutant Ade + Ade - Activation of Ade in telomeric heterochromatin in PCNA mutants

  16. PCNA coordinates: DNA synthesis (processivity) Chromatin assembly Repair Methylation Epigenetic Inheritance Cell-cycle control

  17. Parental nucleosome segregation (hatched) De novo nucleosome assembly (unhatched)

  18. 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 G2 Histone synthesis occurs at G1/S

  19. 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)

  20. GCN5 H3K9/14ac Rtt109 H3K56ac Corpet and Almouzni TICB 2008

  21. 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

  22. 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 Geneviève Almouzni or tetramer

  23. Another histone chaperone ASF1 acts together with CAF1, interacts with CAC2 subunit interacts with HAT1 complex interacts with MCM helicase

  24. Trujillo and Osley Nature Structural & Molecular Biology 2008

  25. The ins and outs of nucleosome assembly Jill A Mello* and Geneviève Almouzni † Current Opinion in Genetics & Development 2001, 11:136–141

  26. 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

  27. Ransom et al., Cell, 2010

  28. Another histone chaperone Rtt106 interacts with CAF1 complex involved in H3/H4 assembly during replication high affinity for H3K56ac (as does CAF1)

  29. Modified from Fillingham and Greenblatt, Cell 2008

  30. H2A/H2B chaperone: FACT? Ransom et al., Cell, 2010

  31. Chromatin assembly Chromatin array formation BOTH long arrays AND properly spaced nucleosomes require other enzymes: ATP-dependent chromatin remodelers ATPase complex ISWI ACF/ChRAC CHD1 - INO80 INO80

  32. 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

  33. CHD1: chromatin remodeling ATPase roles in chromatin assembly together with NAP1 histone variant assembly

  34. Enhanced assembly Lusser et al., Nature Struc Mol Biol 2005

  35. Different periodicity Lusser et al., Nature Struc Mol Biol 2005

  36. Clapier and Cairns, Annu. Rev. Biochem., 2009

  37. INO80: chromatin remodeling ATPase roles in replication stress

  38. Morrison and Shen, Nature reviews Mol Cell Biol, 2009

  39. 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

  40. Epigenetic Memory

  41. 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

  42. Hypothesis 2 Copy modification by recruiting the enzyme that makes the modification to this same modification (Intermolecular)

  43. HETEROCHROMATIN Characterized by DNA methylation, HeK9me, and presence of HP1 DNA methylation (DNMT1) interacts with PCNA KMT1C (H3K9me) interacts with DNMT1 KMT1C also interacts with CAF1 HP1 interacts with CAF1 Late replicating

  44. HS-2: heterochromatin 39C-X: euchromatin Heterochromatin: new possibilities for the inheritance of structure Shiv IS Grewal and Sarah CR Elgin Current Opinion in Genetics & Development 2002, 12:178–187

  45. Chromatin Assembly During Replication Transcription Repair

  46. Chromatin assembly comes in two flavors: Replication coupled (RC) Replication independent (RI)

  47. Chromatin Assembly (RC) CAF1 H3/H4 assembly, disassembly ASF1 H3/H4 H3/H4 dimer assembly, disassembly FACT H2A/H2B assembly, disassembly ISWI NA nucleosomal arrays, periodicity Heterochromatin replic. CHD1 NA nucleosomal arrays INO80 NA replication stress

  48. Chromatin Assembly (RI, replacement) HirA H3.3/H4 deposition during transcription; ASF1 H3.3/H4 interact with transcription elongation factor FACT FACT H2A/H2B disassembly and reassembly during transcription elongation CHD1 H3.3 interacts with HirA SWR H2A.Z/H2B deposition during transcription INO80 H2A.X/H2B deposition during DNA repair

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