Transcript Chromatin Assembly12

Chromatin Assembly
Lecture overview
Chromatin assembly
during replication
Nucleosome positioning
Epigenetic Inheritance
Chromatin assembly
during transcription
ac
DNA
methylation
from Horn and Peterson Science, 2002
ac ac
ac
ac
Nucleosome
Visualizing this primary structure
nucleosomal arrays
Partial micrococcal 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 nuclear extract
= 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 (HC)
Chromatin remodeling (CHR) activities
Enhanced assembly
Lusser et al., Nature Struc Mol Biol 2005
CHR
HC
Enhanced assembly
Lusser et al., Nature Struc Mol Biol 2005
CHR
HC
Chromatin (dis)assembly: when?
During
REPLICATION
TRANSCRIPTION
REPAIR
Chromatin (dis)assembly: when?
During
REPLICATION
TRANSCRIPTION
REPAIR
REPLICATION
MCM: unwinds
PCNA: sliding clamp, processivity
Avvakumov et al, Molec Cell, 2011
Leading and lagging strand synthesis
Vandenberg and Almouzni Nature Preview 2012
REPLICATION: key topics
Disassembly before fork
Reassembly after fork
need 2x histones: parental and newly synthesized!
how to copy pre-existing nucleosome position,
conformation, DNA and histone modifications?
REPLICATION: the players
1. Histone chaperones
bring histones to DNA
2. Histones and modifications
marked a newly synthesized
3. Chromatin remodeling complexes
position nucleosomes
Histone Chaperones (HC)
negatively charged protein
help assembly
prevent aggregation of histones and DNA
REPLICATION COUPLED (RC)
Chromatin Assembly
Histone chaperones
CAF1
H3/H4
H3/4 tetramer
import, assembly
ASF1
H3/H4
H3/H4 dimer
assembly, disassembly
import
Rtt106
H3/H4
assembly(yeast)
NAP1
H2A/H2B
(H3/H4)
import, assembly,
disassembly
FACT
H2A/H2B
(H3/H4)
assembly, disassembly
REPLICATION COUPLED (RC)
Chromatin Assembly
Histone chaperones
CAF1
H3/H4
H3/4 tetramer
import, assembly
ASF1
H3/H4
H3/H4 dimer
assembly, disassembly
import
Rtt106
H3/H4
assembly(yeast)
NAP1
H2A/H2B
import, assembly,
H3/H4 (with Vps75) dissassembly
FACT
H2A/H2B
assembly, disassembly
CAF-1 (chromatin assembly factor 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 (another HC)
p48 (Cac3)
binds histones
Roles
nucleosome deposition after replication
heterochromatin maintenance
Smallest CAF subunit bound to histone H4
Ransom et al., Cell, 2010
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
-
REPLICATION COUPLED (RC)
Chromatin Assembly
Histone chaperones
CAF1
H3/H4
H3/4 tetramer
import, assembly
ASF1
H3/H4
H3/H4 dimer
assembly, disassembly
import
Rtt106
H3/H4
assembly(yeast)
NAP1
H2A/H2B
import, assembly,
H3/H4 (with Vps75) dissassembly
FACT
H2A/H2B
assembly, disassembly
ASF1
binds H3H4 first, binds H3H4 heterodimer
brings to CAF1,
interacts with CAC2 subunit
is linked to replication:
interacts with MCM helicase (mammals)
or replication factor C (yeast)
binds to both newly synthesized and parental histones:
role in disassembly and assembly (Asf1-H3/H4-MCM
complex)
Mello and Almouzni, Current Opinion in Genetics & Development 2001.
Role of ASF1:
binds newly
synthesized H3 H4 dimer
(also parental)
presents H3/H4 dimer to CAF1
ASF1 can only bind H3/H4 dimer
Ransom et al., Cell, 2010
Histone chaperones
ASF1 H3/H4 dimer
CAF1 dimer plus
H3/H4 tetramer or 2 H3/H4 dimers
Why important?
H3/H4 assembled first, than H2A H2B
or tetramer
Ridgway and Almouzni, Journal of Cell Science (2000)
H2A H2B
H2A/H2B bind the ‘peripheral’ core DNA
40 bp on each side
assembled last, disassembled first
REPLICATION COUPLED (RC)
Chromatin Assembly
Histone chaperones
CAF1
H3/H4
H3/4 tetramer
assembly, import
ASF1
H3/H4
H3/H4 dimer
assembly, disassembly
import
Rtt106
H3/H4
assembly(yeast)
NAP1
H2A/H2B
H3/H4
import, assembly,
dissassembly
FACT
H2A/H2B
H3/H4
assembly, disassembly
H2A H2B chaperones
FACT: binds H2A/H2B
interacts with DNA polymerase and localizes
to replication origins
promotes replication initiation in vivo
role in dissassembly, also assembly
Can also bind H3/H4 on a different surface
H2A H2B chaperones
NAP1
can assemble chromatin in vitro
a second possible H2A/H2B chaperone during
replication
IMPORTANT FOR H2A/H2B IMPORT
Summary thus far
FACT
NAP1
FACT
NAP1
From Alabert and Groth Nature Reviews Cell and Molecular Biology 2012
REPLICATION
The players:
1. Histone chaperones
2. Histones and modifications
3. Chromatin remodeling complexes
Histone deposition
250 bp before and after the replication fork are naked DNA
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 is coupled to replication
occurs in G1/S
Leading and lagging strand synthesis
Vandenberg and Almouzni Nature Preview 2012
Feser et al. Molec Cell, 2010
Histone deposition: modifications
Newly synthesized histones have acetylated tails
pattern of acetylation differs from acetylation involved in gene
regulation
H3 and H4 tails are required for Assembly
Histone synthesis: modifications
Enzymes
Acetylation
H3K56ac (more important in yeast)
H4K5ac, H4K12ac (more important in mammals)
Histone synthesis: modifications
mark as ‘newly synthesized’
H4 tail ac: nuclear import, CAF1 interaction
H3K56ac: in histone core, loosens DNA interaction,
nucleosome assembly, CAF1interaction
H3K56ac: stronger CAF1 interaction
Li et al., Cell 2008
Histone deposition summary
Newly synthesized histone modifications are important
reduced viability in yeast if mutated
Role:
Nuclear import
Binding/Deposition by histone chaperone
Octamer assembly; Chromatin maturation
Mark the histone as newly synthesized for chromatin
assembly
Modifications are rapidly removed after assembly,
chromatin continues to mature
REPLICATION
The players:
1. Histone chaperones
2. Histones and modifications
3. Chromatin remodeling complexes
ATP-dependent activities
ISWI
NA
nucleosomal arrays,
periodicity
Heterochromatin
replication
CHD1
NA
nucleosomal arrays
INO80
NA
alleviate replication stress
Chromatin assembly
Chromatin array formation
BOTH long arrays AND properly spaced nucleosomes
require:
ATP-dependent chromatin remodelers
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
Iswi
INO80: chromatin remodeling ATPase
role in replication stress
Morrison and Shen, Nature reviews Mol Cell Biol, 2009
What positions nucleosomes?
Clapier and Cairns, Annu. Rev. Biochem., 2009
What positions nucleosomes?
Does DNA sequence matter?
Do micrococcal nuclease (MNase)
digestion genomewide, look for enriched
sequences
nucleosome
The nucleosome free region
Red: unfavorable for nucleosome positioning
AT rich stretch >5 or 10bp
Do micrococcal nuclease (MNase)
digestion genomewide, look for enriched
sequences
Anti positioning sequences
1. yeast, C. elegans: A-rich nucleosome depleted region
2. mammal:s CpG islands?
Bendabilibity
1. AT, TT, AA 10 bp periodicity
DNA is a poor predictor
many algorithms developed
no more than 50% accurate
attempts to reconstitute nucleosome positioning
genomewide not very successful
requirement for ATP-dependent chromatin remodelers
NFR
Not surprisingly
sequence is not
sufficient for
nucleosome
positioning
Zhang et. al, Science 2010
Other protein could contribute to
positioning
Barrier could be
transcription factor and PolII
well positioned (container) nucleosome
Allis review
What positions nucleosomes?
DNA (in some cases)
Trans factors
chromatin remodeling ATPases
and histone chaperones
transcription factors
general transcriptional machinery
Lecture overview
Chromatin assembly
during replication
Nucleosome positioning
Epigenetic Inheritance
Chromatin assembly
during transcription
Chromatin assembly is
Required for Epigenetic Memory
How are existing modifications copied?
How is nucleosome positioning copied?
Parental AND newly synthesized
histone octamers
Parental nucleosome segregation (hatched)
De novo nucleosome assembly (unhatched)
Some Regulators manage to stay on the
mitotic chromatin
Replication timing helps concentrate relevant
chromatin factors
Example: heterochromatin is late replicating.
Many factors required for heterochromatin are at
the replication fork at this time
DNA methylation, HeK9me3, and presence of HP1
Some modifications are regenerated by
combined readers/writers
Copy modification by
recruiting the enzyme that
makes the modification
to this same modification
(Intermolecular)
Newly synthesized chromatin matures
Epigenetic Memory
Many questions remain
How to copy nucleosome positioning?
Is copying inter- or intra- nucleosomal: H3H4 dimer versus
tetramer
Note most chromatin regulators are not associated with
chromatin during replication
Why should we care about replication?
1. Is the key time or decision point for epigenetic inheritance
versus loss/reversal of this information
2. The mechanisms discussed thus far are relevant for many
other processes that occur in the context
of chromatin.
Lecture overview
Chromatin assembly
during replication
Nucleosome positioning
Epigenetic Inheritance
Chromatin assembly
during transcription
Chromatin Assembly
During Replication
Transcription
(Repair)
Chromatin assembly during transcription
Replication independent (RI)
Linked to incorporation of histone variants
‘memory’ of transcriptional activity
Chromatin Assembly (RI)
HirA
H3.3/H4
ASF1
H3.3/H4
FACT (Spt6)
H2A/H2B
disassembly and
reassembly during
transcription
initiation and elongation
NAP1
H2A/H2B/
H2A.Z/H2B
H2A.Z /H2B
promoters
Chz1
deposition during
transcription initiation and
elongation
interact with transcription
elongation factor FACT
Chromatin Assembly (RI)
CHD1
H3.3
interacts with HirA
SWR
H2A.Z/H2B
deposition during
transcription
INO80
H2A/H2A.Z
exchange
HIRa versus CAF1
Histone turnover in genes
Consequence of nucleosome disassembly during
transcription elongation
FACT elongation factor
Transcription elongation
H3.3 is incorporated during transcription
Drosophila
H3.3 is incorporated during transcription
promoter
promoter
5’ end gene
H2A.Z is incorporated flanking the
nucleosome depleted region
Biochem. Cell Biol vol 84 2006
Histone turnover in during transcription
Genes: Consequence of disassembly/assembly
during transcription
Promoters: nucleosome eviction or exchange
have different properties
alter nucleosome stability
can serve as transcriptional state memory
Lecture summary
Chromatin assembly
Who are the players. How is it accomplished
Why does it matter?
during replication
during transcription
Nucleosome positioning
What is it? How is it determined?
Why is it important?
Replication
Why is it a critical process for Epigenetics
Glossary
PCNA
MCM
FACT
NAP1
sliding clamp
POLII processivity
helicase
DNA unwinding/replication
histone chaperone
H2A/H2B RC and RI assembly
histone chaperone
H2A/H2B nuclear import
H3/H4 assembly RC, H2AZ/H2B RI
Rtt106
histone chaperone
H3/H4 RC assembly
CAF1
histone chaperone
H3/H4 RC assembly
ASF1
histone chaperone
H3/H4 RC and H3.3/H4 RI
assembly
HirA
histone chaperone
H3.3/H4 RI assembly
HAT1
cytoplasmic KAT acetylates newly synthesized H4
Rtt109
HAT
acetylates H3K56
ISWI
chromatin remodeling
assembly, spacing
ATPase
CHD1
chromatin remodeling
assembly, H3.3 with HirA
ATPase
INO80
chromatin remodeling
replication stress, H2A exchange
ATPase/complex
SWR
chromatin remodeling
H2AZ exchange
Nucleosome positioning nucleosome free region, anti positioning sequence,
dinucleotides, barrier proteins that bind the DNA
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
Zhang et al., Nature 2000
PCNA wt
Ade -
PCNA mutant
Ade +
Activation of Ade in telomeric heterochromatin in PCNA mutants
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
Zhang et al., Nature 2000
CAF1: required for heterochromatin assembly
PCNA wt
Ade -
PCNA mutant
Ade +
Activation of Ade in telomeric heterochromatin in PCNA mutants
Useful terminology