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

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Chromatin Assembly

Nucleosomal DNA Primary chromatin structure

beads-on-a-string

Nucleosomefrom Horn and Peterson Science, 2002

acacac

ac

acDNA

methylation

Visualizing this primary structure

nucleosomal arrays

Partial nuclease digestion generates mono, di, trinucleosomes…

How is chromatin assembly achieved?

Irregular assembly

Dynamics of ATP-dependent chromatin assembly by ACFDmitry V. Fyodorov & James T. KadonagaNATURE | VOL 418 | 22 AUGUST 2002 |

Assembly

in vitroHistones + DNA + saltassemble into aggregates

Histones + DNA + negatively charged proteins= mononucleosomes andpoor nucleosmal array (irregularly spaced; “smear”)

Dynamics of ATP-dependent chromatin assembly by ACFDmitry V. Fyodorov & James T. KadonagaNATURE | VOL 418 | 22 AUGUST 2002 |

in vitroXenopus/Drosophila extracts=chromatin (±regularly spaced)

What is in the extract?

Histone chaperones

Histone chaperonesnegatively charged proteinshelp assembly; prevent aggregation

NAP1 H2A/H2B Nuclear Import, nucleosomedisruption, histone variant incorporation

nucleoplasmin H2A/H2B Storage, sperm histone exchange

N1/N2 H3/H4 Storage

CAF-1

Chromatin assembly factor 1

More histone chaperones

Replication-coupled chromatin assembly factorCAF-1 (identified in yeast; Bruce Stillman lab)

brings H3/H4 onto newly synthesized DNA

CAF-13 subunits: p150 (Cac1) binds PCNA

p60 (Cac2) binds ASF1p48 (Cac3) binds histones histone binding

in many complexesRoles

nucleosome deposition after replicationheterochromatin maintenance

Ransom et al., Cell, 2010

Smallest CAF subunit bound to histone H4

Replication

Cell, Vol. 96, 575–585, February 19, 1999, Replication-Dependent Marking of DNA by PCNAFacilitates CAF-1-Coupled Inheritance of ChromatinKei-ichi Shibahara and Bruce Stillman

Cac2 interacts with PCNA when Cac1 is present

PCNA connects DNA replication to epigenetic inheritance in yeastZhiguo Zhang, Kei-ichi Shibahara & Bruce StillmanNATURE | VOL 408 | 9 NOVEMBER 2000

PCNA

- - -

Role of PCNA in chromatin assembly?1. Adenine deficient yeast are red2. Move Adenine biosynthesis gene into heterochromatin ± inactive; red colonies3. Mutagenize strain4. 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 yeastZhiguo Zhang, Kei-ichi Shibahara* & Bruce Stillman*NATURE | VOL 408 | 9 NOVEMBER 2000 |

Ade - Ade +

PCNA wt PCNA mutant

PCNA coordinates: DNA synthesis (processivity)Chromatin assemblyRepairMethylation Epigenetic InheritanceCell-cycle control

Parental nucleosome segregation (hatched)

De novo nucleosome assembly (unhatched)

New nucleosome formationafter replication1 H3/H4 tetramer OR 2 H3/H4 dimers bind DNA2 dimers H2A/H2B bind H3/H4 tetramer

DNA first very reactive with histones, then matures

Nucleosome deposition is essentialif repressed, DNA will be duplicated but cell cycle arrest in G2

Histone synthesis occurs at G1/S

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)

Corpet and Almouzni TICB 2008

H3K56ac

H3K9/14acGCN5

Rtt109

These modifications are important reduced viability in yeast if mutated

Role: Nuclear importBinding/Deposition by histone chaperoneChromatin maturation

Mark the histone as newly synthesized for chromatinassembly

Modifications are rapidly removed after assembly

Journal of Cell Science 113, 2647-2658 (2000)CAF-1 and the inheritance of chromatin states: at the crossroads of DNA replication and repairPatricia Ridgway and Geneviève Almouzni

or tetramer

ASF1

acts together with CAF1, interacts with CAC2 subunit

interacts with HAT1 complex

interacts with MCM helicase

Another histone chaperone

Trujillo and Osley Nature Structural & Molecular Biology 2008

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

Role of ASF1:

generates H3 H4 dimer

prevents tetramer formationbreaks up tetramers

presents H3/H4 dimer to CAF1

also role in H3K56ac by Rtt109

Ransom et al., Cell, 2010

Another histone chaperone

Rtt106

interacts with CAF1 complex

involved in H3/H4 assembly during replication

high affinity for H3K56ac (as does CAF1)

Modified from Fillingham and Greenblatt, Cell 2008

Ransom et al., Cell, 2010

H2A/H2B chaperone: FACT?

BOTH long arrays AND properly spaced nucleosomes

require other enzymes:

ATP-dependent chromatin remodelers

Chromatin assemblyChromatin array formation

ATPase complexISWI ACF/ChRAC

CHD1 -

INO80 INO80

ISWI: chromatin remodeling ATPase

roles in chromatin assembly together with NAP1 or Asf1linker length, formation of long nucleosomal arrays

transcriptional elongationsilencingchromosome organization

CHD1: chromatin remodeling ATPase

roles in chromatin assembly together with NAP1histone variant assembly

Enhanced assembly

Lusser et al., Nature Struc Mol Biol 2005

Different periodicity

Lusser et al., Nature Struc Mol Biol 2005

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

INO80: chromatin remodeling ATPase

roles in replication stress

Morrison and Shen, Nature reviews Mol Cell Biol, 2009

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

Epigenetic Memory

Hypothesis 1: H3H4 dimers are incorporatedcan “copy” modifications, variantsintramolecular

Hypothesis 2A: H3 H4 tetramers are incorporatedrepressive marks are defaultactivating marks incorporated during RI assembly variants

Copy modification by recruiting the enzyme that makes the modificationto this same modification(Intermolecular)

Hypothesis 2

DNA methylation (DNMT1) interacts with PCNA

KMT1C (H3K9me) interacts with DNMT1KMT1C also interacts with CAF1

HP1 interacts with CAF1

Late replicating

HETEROCHROMATINCharacterized by DNA methylation, HeK9me, and presence of HP1

Heterochromatin: new possibilities for the inheritance of structureShiv IS Grewal and Sarah CR ElginCurrent Opinion in Genetics & Development 2002, 12:178–187

HS-2: heterochromatin

39C-X: euchromatin

Chromatin Assembly

During Replication

Transcription

Repair

Chromatin assembly comes in two flavors:

1. Replication coupled (RC)

2. Replication independent (RI)

CAF1 H3/H4 assembly, disassembly

ASF1 H3/H4 H3/H4 dimerassembly, disassembly

FACT H2A/H2B assembly, disassembly

ISWI NA nucleosomal arrays, periodicityHeterochromatin replic.

CHD1 NA nucleosomal arrays

INO80 NA replication stress

Chromatin Assembly (RC)

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

Chromatin Assembly (RI, replacement)

= CAC3

=Htz1

=CENP-A

DNA repair

Transcription

Dis-assembly and assembly

Histone turnover in genes

Consequence of nucleosome dis-assembly during transcription elongation

FACT elongation factor

Transcription elongation

Histone turnover at promoters/genes

Consequence of

disassembly/assembly

transcription

nucleosome eviction

chromatin remodeling

histone modifications

Histone turnover at promoters/genes

Consequence of

disassembly/assembly

transcription

nucleosome eviction

chromatin remodeling

histone modifications

promoter

promoter 5’ end gene

H3.3 incorporation during transcription

Drosophila

Histone turnover at promoters/genes

Consequence of

disassembly/assembly

transcription

nucleosome eviction

chromatin remodeling

histone modifications

High histone turnover at promoters is high = red nucleosomes

GlossaryPCNA sliding clamp POLII processivityMCM helicase DNA unwinding/replicationNAP1 histone chaperone H2A/H2B nuclear importN1/N2 histone chaperone H3/H4 (storage)Nucleoplasmin histone chaperone H2A/H2B (storage)Rtt106 histone chaperone H3K56ac/H4 RC assemblyCAF1 histone chaperone H3/H4 RC assemblyASF1 histone chaperone H3/H4 RC and H3.3/H4 RI

assemblyHirA histone chaperone H3.3/H4 RI assemblyHAT1 cytoplasmic KATacetylates newly synthesized H4Rtt109 KAT acetylates H3K56ISWI chromatin remodeling assembly, spacing

ATPaseCHD1 chromatin remodeling assembly, H3.3 with HirA

ATPaseACS complexes containing assembly

ISWIINO80 chromatin remodeling replication stress, repair (H2AX)

ATPase/complexSWR chromatin remodeling H2AZ exchange