Download CAF-1

Chromatin Assembly
Nucleosomal DNA
Primary chromatin structure
beads-on-a-string
ac
DNA
methylation
from Horn and Peterson Science, 2002
ac ac
ac
ac
Nucleosome
Visualizing this primary structure
nucleosomal arrays
Partial nuclease digestion generates mono, di, trinucleosomes…
How is chromatin assembly
achieved?
Assembly
Irregular assembly
in vitro
Histones + DNA + salt
assemble into aggregates
Histones + DNA
+ negatively charged proteins
= mononucleosomes and
poor nucleosmal array
(irregularly spaced; “smear”)
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)
Dynamics of ATP-dependent chromatin assembly by ACF
Dmitry V. Fyodorov & James T. Kadonaga
NATURE | VOL 418 | 22 AUGUST 2002 |
What is in the extract?
Histone chaperones
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
More histone chaperones
CAF-1
Chromatin assembly factor 1
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)
p60 (Cac2)
p48 (Cac3)
binds PCNA
binds ASF1
binds histones
in many complexes
Roles
nucleosome deposition after replication
heterochromatin maintenance
Smallest CAF subunit bound to histone H4
Ransom et al., Cell, 2010
Replication
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
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
-
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
Ade -
PCNA mutant
Ade +
Activation of Ade in telomeric heterochromatin in PCNA mutants
PCNA coordinates:
DNA synthesis (processivity)
Chromatin assembly
Repair
Methylation
Epigenetic Inheritance
Cell-cycle control
Parental nucleosome segregation (hatched)
De novo nucleosome assembly (unhatched)
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
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)
GCN5
H3K9/14ac
Rtt109 H3K56ac
Corpet and Almouzni TICB 2008
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
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
Another histone chaperone
ASF1
acts together with CAF1,
interacts with CAC2 subunit
interacts with HAT1 complex
interacts with MCM helicase
Trujillo and Osley Nature Structural & Molecular Biology 2008
The ins and outs of nucleosome assembly
Jill A Mello* and Geneviève Almouzni †
Current Opinion in Genetics & Development 2001, 11:136–141
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
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
H2A/H2B chaperone: FACT?
Ransom et al., Cell, 2010
Chromatin assembly
Chromatin array formation
BOTH long arrays AND properly spaced nucleosomes
require other enzymes:
ATP-dependent chromatin remodelers
ATPase
ISWI
complex
ACF/ChRAC
CHD1
-
INO80
INO80
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
CHD1: chromatin remodeling ATPase
roles in
chromatin assembly
histone 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 incorporated
can “copy” modifications, variants
intramolecular
Hypothesis 2A:
H3 H4 tetramers are incorporated
repressive marks are default
activating marks incorporated
during RI assembly variants
Hypothesis 2
Copy modification by
recruiting the enzyme that
makes the modification
to this same modification
(Intermolecular)
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
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
Chromatin Assembly
During Replication
Transcription
Repair
Chromatin assembly comes in two flavors:
1. Replication coupled (RC)
2. Replication independent (RI)
Chromatin Assembly (RC)
CAF1
H3/H4
assembly, disassembly
ASF1
H3/H4
H3/H4 dimer
assembly, disassembly
FACT
H2A/H2B
assembly, disassembly
ISWI
NA
CHD1
NA
nucleosomal arrays,
periodicity
Heterochromatin replic.
nucleosomal arrays
INO80
NA
replication stress
Chromatin Assembly (RI, replacement)
HirA
H3.3/H4
deposition during
transcription;
interact with transcription
elongation factor FACT
ASF1
H3.3/H4
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
=CENP-A
= CAC3
=Htz1
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
Drosophila
H3.3 incorporation during transcription
promoter
promoter
5’ end gene
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
Glossary
PCNA
MCM
NAP1
N1/N2
Nucleoplasmin
Rtt106
CAF1
ASF1
HirA
HAT1
Rtt109
ISWI
CHD1
ACS
INO80
SWR
sliding clamp
helicase
histone chaperone
histone chaperone
histone chaperone
histone chaperone
histone chaperone
histone chaperone
POLII processivity
DNA unwinding/replication
H2A/H2B nuclear import
H3/H4 (storage)
H2A/H2B (storage)
H3K56ac/H4 RC assembly
H3/H4 RC assembly
H3/H4 RC and H3.3/H4 RI
assembly
histone chaperone
H3.3/H4 RI assembly
cytoplasmic KAT acetylates newly synthesized H4
KAT
acetylates H3K56
chromatin remodeling
assembly, spacing
ATPase
chromatin remodeling
assembly, H3.3 with HirA
ATPase
complexes containing
assembly
ISWI
chromatin remodeling
replication stress, repair (H2AX)
ATPase/complex
chromatin remodeling
H2AZ exchange