Chapter 2
Histone Chaperones in the Assembly
and Disassembly of Chromatin
Briana K. Dennehey and Jessica Tyler
2.1
Introduction
Nucleosomes physically block access to the DNA, raising several questions (1) How
are new nucleosomes formed in vivo? (2) How are nucleosomes removed to facilitate DNA templated processes? and (3) How are histones restored to the DNA
following the completion of those processes? The intrinsic attraction between the
negatively charged DNA phosphate backbone and the positively charged lysine and
arginine-rich histone proteins is key to the formation of the nucleosome. Yet, their
intrinsic attraction is so high that mixing DNA and histones together in vitro results
in insoluble aggregates rather than nucleosomes. Consequently, additional factors
are required to allow interactions between histones and DNA to occur in a controlled
and ordered manner. These factors are collectively termed histone chaperones.
The term “Molecular Chaperone” was first applied to the biochemical activity
associated with a highly acidic 29 kDa protein purified from Xenopus eggs (Laskey
et al. 1978). “Thermostable assembly protein” (Mills et al. 1980), later renamed
“nucleoplasmin” (Laskey and Earnshaw 1980), not only prevented the in vitro
aggregation of histone proteins with DNA at physiological salt concentrations but
also promoted nucleosome assembly (Laskey et al. 1978). Nucleoplasmin was proposed to shield the positively charged histones from nonspecific ionic interactions
while promoting specific “correct” contacts (Laskey et al. 1978). Nucleoplasmin is
now understood to be a histone chaperone that stores maternal pools of H2A–H2B
in Xenopus eggs. However, the study of this founding member of the histone chaperone family led to the realization that histone chaperones facilitate nucleosome
assembly in vitro by preventing the aggregation of histones and DNA. In vivo histone chaperones bind to and guide histones in the cell both to prevent nonspecific
B.K. Dennehey • J. Tyler (*)
Department of Biochemistry and Molecular Biology, MD Anderson Cancer Center,
Houston, TX 77030, USA
e-mail: [email protected]
J.L. Workman and S.M. Abmayr (eds.), Fundamentals of Chromatin,
DOI 10.1007/978-1-4614-8624-4_2, © Springer Science+Business Media New York 2014
29
30
B.K. Dennehey and J. Tyler
interactions and to promote physiologically relevant interactions with other proteins
and/or DNA. It is now widely accepted that all free (nonnucleosomal) histones are
bound to histone chaperones in the cell (Osley 1991; Tagami et al. 2004; Campos
et al. 2010). Histone chaperones encompass a growing family of proteins that bind
stoichiometrically to histones and perform one or more of the following functions
(1) transport the newly synthesized histones from the cytoplasm to the nucleus,
(2) present the histones to histone-modifying enzymes for their posttranslational
modification, (3) store free histones in the cell, (4) deposit histones onto the DNA,
(5) remove histones from the nucleosome, and (6) remove histones from the DNA
when not specifically contained in nucleosomal DNA interactions. The ultimate
function of the histone chaperones is to achieve the assembly and disassembly of
chromatin both locally and globally, as required by the cell, working intimately with
ATP-dependent chromatin remodelers (See Chap. 3). In order to appreciate how
histone chaperones assemble and disassemble the nucleosome, we must first examine
the fundamental steps involved.
2.1.1
A Working Model for Stepwise Nucleosome
Assembly/Disassembly
In vitro, nonnucleosomal histones H2A–H2B and H3–H4 exist as heterodimers at
physiological ionic strength and pH, and although (H3–H4)2 tetramers can form,
they exist in dynamic equilibrium with H3–H4 dimers (Baxevanis et al. 1991; Banks
and Gloss 2003; Donham et al. 2011; Winkler et al. 2012). In vitro, nucleosome
reconstitution assays in combination with the molecular structure of the nucleosome
core particle (Luger et al. 1997), have led to the following model for nucleosome
assembly (Fig. 2.1). First, two dimers, or one tetramer, of H3–H4 is deposited onto
DNA to form the tetrasome, which includes almost one turn of the DNA around the
central (H3–H4)2 tetramer. This is followed by the addition of two flanking H2A–
H2B dimers and the wrapping of the remainder of the DNA around the histone
octamer. Similarly, nucleosome disassembly is a reversal of this process, initiating
with partial DNA unwrapping and loss of H2A–H2B dimers, followed by the loss
of the (H3–H4)2 tetramer from the DNA. Consequently, the exchange of any one
nucleosomal histone contained within the (H3–H4)2 tetramer core for a free histone
would conceivably require disassembly of the entire nucleosome (See Sect. 2.5). In the
cell, every step in the nucleosome assembly and disassembly process is coordinated by
histone chaperones.
2.1.2
Overview of Histone Chaperones
Different histone chaperones exist to perform the variety of functional roles listed
above, i.e., escorting the histones from the cytoplasm, histone presentation for histone modification, storage of histone pools, and nucleosome assembly/disassembly.
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
H2A-H2B
(H3-H4)2
Tetrasome
Histone
chaperone
31
H2A-H2B
Hexasome
Histone
chaperone
Nucleosome
core particle
Histone
chaperone
or
Histone
chaperone
Histone
chaperone
Histone
chaperone
2 X H3-H4
H2A-H2B
H2A-H2B
Fig. 2.1 Schematic of the stepwise assembly and disassembly of the nucleosome core particle.
Green arrows represent steps in chromatin assembly and the red arrows indicate steps in chromatin disassembly. On the right is shown the nucleosome core particle, derived from the X-ray crystal
structure of Luger and others (Luger et al. 1997) including the DNA (grey—DNA backbone and
cyan—bases), and histones H3 (blue), H4 (green), H2A (yellow), and H2B (red). The unstructured
tails of the histones that were not visible in the nucleosome core particle structure have been added,
and the rods indicate the alpha helices of the histones. The remaining images are models derived
from the nucleosome core particle structure. Whether or not it is a tetramer of histones (H3–H4)2
or two dimers of H3–H4 that are deposited onto, or removed from, the DNA depends on which
specific histone chaperone and which H3 variant is being utilized. We are extremely grateful to
Jean-Marc Victor, Hua Wong, and Julien Mozziconacci for generating the models
To add to the complexity, there are histone chaperones specific for H2A–H2B
dimers, H3–H4 dimers, (H3–H4)2 tetramers, and for certain histone variants. There
are histone chaperones that assemble chromatin specifically during DNA synthesis
(termed replication dependent), while others function specifically during times
when the DNA is not being synthesized, such as during transcription (termed replication independent). This information is summarized in Table 2.1.
Histone chaperones are structurally diverse sharing few similarities in sequence,
making them difficult to identify in silico. Many histone chaperones contain patches
of acidic residues that presumably help stabilize interactions with positively charged
histones. As the structures of increasing numbers of chaperones are solved, a diversity of histone chaperone forms has emerged, underscoring the distinct roles of
each. Below, we discuss how structural biology, biochemistry, and cell biology have
led to our understanding of how histone chaperones guide the histones along their
intrepid journey, which begins at the site of protein synthesis in the cytoplasm and
ends at the DNA.
H3–H4 (obligate
dimer)
H3.1–H4
CAF-1 complex
(Sc: Cac1, Cac2, Cac3/Msi1)
(Dm: p180, p105, p55)
(Hs: p150, p60, RbAp48)
H1
Transcriptional silencing
Promotion of Rtt109 mediated H3
K56 acetylation
Replication-dependent chromatin
assembly
UV induced NER repair
Chromatin assembly after DSB
repair, and heterochromatin
formation
Cytoplasmic escort of H1 with
Hsp90
Deposition of H1
Cytoplasmic escort of H3–H4
Replication-dependent chromatin
assembly
Replication-independent chromatin
assembly
Chromatin assembly after DSB repair
Chromatin disassembly before
replication
Chromatin disassembly during
transcription
Cytoplasmic escort of H3–H4
Promotes HAT1 mediated H4K5,
K12 diacetylation
H3–H4 dimerization platform with
Hsp90
Sink for excess H3–H4
H3–H4
H3.1–H4
Function
Cargo
Asf1 (Hs, Mm, Xl, Sc, Dm),
CIA1 (Sp)
NASP (Hs, Mm), Hif1(Sc)
N1/N2 (Xl)
Chaperone
H3–H4 chaperones
RbAp46 (Hs)
Table 2.1 Histone chaperones, and their cargoes, their chromatin assembly/disassembly related functions
Adkins et al. (2004, 2007), Korber et al. (2006),
Schwabish and Struhl (2006), Gkikopoulos
et al. (2009), Takahata et al. (2009)
Le et al. (1997), Singer et al. (1998)
Recht et al. (2006), Han et al. (2007), Tsubota
et al. (2007)
Smith and Stillman (1989), Tagami et al. (2004)
Gaillard et al. (1996)
Kim and Haber (2009)
Quivy et al. (2008)
Finn et al. (2008)
Campos et al. (2010), Alvarez et al. (2011)
Tyler et al. (1999), Groth et al. (2005),
Sanematsu et al. (2006)
Schermer et al. (2005), Rufiange et al. (2007);
see also Galvani et al. (2008)
Chen et al. (2008), Kim and Haber (2009)
Groth et al. (2007), Jasencakova et al. (2010)
Cook et al. (2011), Finn et al. (2012) and
references therein
Alekseev et al. (2003, 2005)
Campos et al. (2010)
Campos et al. (2010), Alvarez et al. (2011)
Select references for function
32
B.K. Dennehey and J. Tyler
Prevention of CenH3 degradation
(Scm3)
Receptor for CenH3 (Scm3)
Deposition of CenH3
Deposition of CenH3
Helps with deposition of CenH3?
H2A–H2B dimer removal/
displacement
CenH3–H4
CenH3–H4
CenH3, H3
CenH3–H4
H2A–H2B,
H3–H4
CAL1 (Dm)
Sim3 (Sp) (NASP-like)
RbAp48 (Hs), p55 (Dm)
H2A–H2B chaperones
FACT complex
Incorporation of H3.3 during
nuclear receptor mediated
transcription
Deposition of CenH3
Incorporation of H3.3 at telomeres
H3.3–H4 (obligate
dimer)
H3.3–H4
Scm3 (Sc, Sp), HJURP (Hs),
DEK (Hs, Mm) dDEK (Dm)
Replication-independent chromatin
assembly
Transcriptional silencing/repression
H3–H4
Vps75 (Sc)
Replication-dependent chromatin
assembly
Chromatin assembly after RNA Pol
II passage
Promotion of Rtt109 stability and
Rtt109 mediated H3K9, K27
acetylation
Function
H3.3–H4
H3–H4
Rtt106 (Sc), Mug183 (Sp)
H3 variant chaperones
HIR complex
(Sc: Hir1, Hir2, Hir3 and
Hpc2)
(Sp: Hip1, Hip3, Hip4 and
Slm9)
(Hs: HIRA, Ubinuclein-1,
and Cabin-1)
DAXX (Hs, Mm), DLP (Dm)
Cargo
Chaperone
Select references for function
Orphanides et al. (1999), Kireeva et al. (2002),
Belotserkovskaya et al. (2003)
(continued)
Stoler et al. (2007), Dunleavy et al. (2009),
Shuaib et al. (2010), Barnhart et al. (2011)
Hewawasam et al. (2010), Ranjitkar et al.
(2010)
Pidoux et al. (2009)
Erhardt et al. (2008)
Dunleavy et al. (2007)
Furuyama et al. (2006)
Drane et al. (2010), Goldberg et al. (2010),
Lewis et al. (2010), Wong et al. (2010)
Sawatsubashi et al. (2010)
Tagami et al. (2004)
see for example Vishnoi et al. (2011)
Silva et al. (2012)
Silva et al. (2012)
Li et al. (2008), Zunder et al. (2012)
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
33
H1
H3–H4
H2A–H2B
H3–H4
CenH3–H4
H2A–H2B
CenH3–H4
H2A–H2B,
H3–H4,
Cargo
Removal of nonnucleosomal H2A–
H2B from DNA
Removal of linker histone H1
Unclear
Chromatin disassembly
Chromatin reassembly after RNA
Pol II passage
Centromere assembly/maintenance?
Chromatin assembly after
transcription
Heterochromatic silencing
Histone storage and deposition
Nucleosome assembly
Centrosome assembly/maintenance?
Cytoplasmic escort of H2A–H2B
Function
Kepert et al. (2005)
Abe et al. (2011), McCullough et al. (2011)
Kaplan et al. (2003), Mason and Struhl (2003),
Nakayama et al. (2007), Jamai et al. (2009)
Okada et al. (2009), Choi et al. (2012)
Winston et al. (1984), Kaplan et al. (2003),
Adkins and Tyler (2006), Cheung et al. (2008)
Kiely et al. (2011)
Finn et al. (2012) and references therein
Okuwaki et al. (2001)
Barnhart et al. (2011)
Chang et al. (1997), Mosammaparast et al.
(2001, 2002a, b)
Andrews et al. (2010)
Select references for function
H2A.Z–H2B
Trafficking and deposition of H2A.Z
Luk et al. (2007)
H3–H4
rDNA silencing
Kuzuhara and Horikoshi (2004)
H2A.Z
Unclear
Luk et al. (2007)
Chaperones capable of interacting with (H3–H4)2 tetramers are: Nap1 (Andrews et al. 2008; Bowman et al. 2011), Vps75 (Park et al. 2008; Bowman et al.
2011), NASP (Wang et al. 2012), FACT (Belotserkovskaya et al. 2003), CAF-1 (Liu et al. 2012; Winkler et al. 2012), Rtt106 (Fazly et al. 2012; Su et al. 2012),
and Spt6 (Bortvin and Winston 1996). Chaperones that bind exclusively to H3–H4 dimers are Asf1 (English et al. 2005, 2006) and DAXX (Elsasser et al. 2012)
Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl,
Xenopus laevis; DSB, double-strand break; NER, nucleotide excision repair
H2A variant chaperones
Chz1 (Sc)
Fkbp39 (Sp), Fpr3/Fpr4 (Sc)
Nap1 (Hs, Mm, Dm, Xl, Ce
Sc, Sp)
Nucleoplasmin/
nucleophosmin
Spt6 (Dm, Sp, Sc), SUPT6H
(Hs, Mm)
Spt16, SSRP1 (Hs, Mm)
Spt16, Pob3, Nhp6 (Sc)
Chaperone
Table 2.1 (continued)
34
B.K. Dennehey and J. Tyler
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
2.2
35
Histone Transport from the Cytoplasm to the Nucleus
Before histones can be assembled into chromatin, they must first transit from the
cytoplasm to the nucleus. The transport of H2A–H2B is likely mediated by the histone chaperone Nap1 (Fig. 2.2). Nap1 is primarily a cytosolic protein (Kellogg et al.
1995) that shuttles in and out of the nucleus (Ito et al. 1996; Mosammaparast et al.
2002a). In HeLa cells, Nap1 binds to newly synthesized H2A–H2B from cytosolic
extracts (Chang et al. 1997), and in budding yeast Nap1 binds to H2A–H2B and
Kap114 (a karyopherin). The association of Nap1 with Kap114 enhances the ability
of Kap114 to interact with the nuclear localization signal (NLS) of H2A–H2B facilitating its transport into the nucleus (Mosammaparast et al. 2002a). Yet, in the absence
of Nap1, H2A–H2B are still transported to the nucleus and karyopherins other than
Kap114 can function in their import (Mosammaparast et al. 2001). Nap1 also associates with the linker histone H1 in Xenopus egg extracts (Shintomi et al. 2005), but
evidence for Nap1 function in the cytosolic transport of linker histones is lacking.
In Humans:
Cytoplasm
K9me1
K9me1
H3
K9me1
K14Ac
K9me1
K14Ac
HSC70
Protein
synthesis
of histones
K9me1
K14Ac
K5Ac
K12Ac
Asf1a
HSP90 NASP
PAR
Nucleus
K14Ac
K5Ac
K12Ac
Asf1a
Importin4
NASP
Histones handed
off to downstream
histone chaperones
that deposit them
onto the DNA
NASP
H4
K9me1
HSP70
HSP90
HAT1
PAR
K5Ac
Asf1b
K12Ac
K14Ac
K9me1
K9me1
Asf1b
K5Ac
K12Ac
K5Ac
K12Ac
Importin4
H2B H2A
?
Nap1
Protein
synthesis
of histones
NASP
In Yeast:
Nap1
H1
NASP
?
Cytoplasm
Nucleus
GCN5
Rtt109
K56Ac
H3 H4
K5Ac
K12Ac
Hat1,2
Hif1
Hif1
Asf1
K5Ac
K12Ac
K27Ac
K9Ac
K5Ac
K12Ac
Asf1
Kap123
Histones handed
off to downstream
histone chaperones
that deposit them
onto the DNA
Protein
synthesis
of histones
H2B H2A
Nap1
H1
Kap114
Nap1
Nap1
Fig. 2.2 The histone’s journey from the cytoplasm to the nucleus. The histones are depicted and
colored as in Fig. 2.1. The red and green dots indicate the positions of methylation (red) or acetylation (green) that occur on the histones during their journey described in the text. The yellow ovals
depict specific histone acetyl transferase enzymes. The orange oval shapes depict nuclear importers
and the remaining shapes depict specific histone chaperones as described in the text. With the exception of the histone chaperone Asf1, the region of the histone shown bound to the histone chaperone
has not been experimentally proven and should be considered to be arbitrary. Question marks indicate predicted functions for histone chaperones that have not yet been unequivocally proven
36
B.K. Dennehey and J. Tyler
Although the relationship between Nap1 and H2A–H2B transport has been
known for over a decade, only recently has the trafficking of H3–H4 begun to be
clarified. Two studies have examined the passage of these histones from the cytoplasm to the nucleus by isolating epitope tagged H3–H4 from different cellular
compartments and examining their binding partners and posttranslational modifications (Campos et al. 2010; Alvarez et al. 2011). From these studies, a highly ordered
program of sequential histone chaperone interactions and histone posttranslational
modifications has been established (Fig. 2.2). In HeLa cytosolic extracts, newly
synthesized histone H3 monomers associate with the general chaperone HSC70.
These histones are poly(ADP-ribosyl)ated and are also monomethylated at H3K9
(H3K9me1). Similarly, histone H4 monomers are also poly(ADP-ribosyl)ated and
associate with the general chaperones HSP90/HSP70. Given that the poly(ADPribosyl)ation is removed when H3 and H4 assemble into an H3–H4 dimer, it has
been speculated that the poly(ADP-ribosyl)ation may help keep H3 and H4 folded
in the absence of their histone binding partner (Alvarez et al. 2011). By the time that
the H3–H4 dimer forms, H3 is acetylated at K14 (H3K14ac), and H3–H4 are associated with HSP90 and/or the histone chaperone NASP (nuclear autoantigenic
sperm protein). Once associated with NASP, the RbAp46–HAT1 complex diacetylates H4 at K5 and K12. However, it should be noted that although both H3.3–H4
and H3.1–H4 copurify with the RbAp46–HAT1 complex (Alvarez et al. 2011;
Zhang et al. 2012), H4 is differentially modified within these complexes with acetylation of H4 in H3.1–H4 complexes favored over H3.3–H4 complexes (Zhang et al.
2012). Nonetheless, the next histone chaperone to receive histones H3–H4 along
their journey to the nucleus is Asf1.
The universal H3–H4 dimer chaperone Asf1 is a highly conserved protein in
eukaryotes. Asf1 is essential in organisms ranging from fission yeast to humans, but
is not essential in budding yeast. Asf1 was originally identified as a gene that when
overexpressed in budding yeast led to a reduction in transcriptional silencing
(Le et al. 1997; Singer et al. 1998). Cocrystals of the conserved N-terminal core of
Asf1 complexed with a dimer of H3–H4 show that Asf1 physically binds to and
blocks the H3–H3 tetramerization interface (English et al. 2006; Natsume et al.
2007). Consequently, it is likely that H3–H4 exist as dimers, rather than tetramers,
until and during their delivery to Asf1 (Fig. 2.2). In mammals, but not yeast or
Drosophila, Asf1 exists in two isoforms, Asf1a and Asf1b. The dually modified
H3(K9me1, K14ac) and H4(K5ac, K12ac) dimer is transferred to Asf1a, whereas
the singly modified H3(K9me1) doubly modified H4(K5ac, K12ac) dimer associates with Asf1b (Alvarez et al. 2011). The H3–H4 dimers associated with Asf1 next
form complexes with Importin-4, a karyopherin, presumably to facilitate import
into the nucleus (Campos et al. 2010; Alvarez et al. 2011). The latter part of this
pathway is conserved in budding yeast, with Hif1 serving in the role of NASP and
with Hat1 and Hat2 diacetylating H4 at K5 and K12 prior to transfer of histones to
Asf1 (Campos et al. 2010). Nuclear entry occurs aided by Kap123, which is
associated with acetylated cytosolic histones, and to a lesser extent Kap121
(Mosammaparast et al. 2002b). In addition to the escort of H3–H4, NASP and
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
37
HSP90 are also implicated in escorting the linker histone H1 to the nucleus
(Alekseev et al. 2003, 2005). The chaperoning of H1 by NASP is mutually exclusive to H3–H4 association with NASP (Wang et al. 2012).
2.3
Replication-Dependent Chromatin Assembly
The most basic function of a cell is its own division. Prior to division, a cell must
replicate its genome, and to maintain cellular identity, its epigenome must be either
faithfully replicated or reestablished.
Histone synthesis is tightly linked to DNA replication in order to meet the cellular
requirement for chromatin assembly onto the two daughter DNA strands. These newly
synthesized histones can be differentiated from preexisting histones by their unique
pattern of deposition-specific, posttranslational modifications (Fig. 2.2), which can be
distinguished immediately after replication, but over time, are lost as the histones
resume the modification pattern of the parental chromatin and the epigenomic information is reestablished (Scharf et al. 2009) (Fig. 2.3). During replication, parental histones
are segregated to both nascent DNA duplexes, where parental (H3–H4)2 tetramers are
generally transferred intact, whereas H2A–H2B dimers reassort freely with new and
parental H2A–H2B dimers and (H3–H4)2 tetramers (Senshu et al. 1978; Jackson and
Chalkley 1981; Jackson 1988) (Fig. 2.3).
2.3.1
Histone Eviction in Front of the Replication Fork
In the first step of replication, origins of replication are recognized by ORC (origin
recognition complex), which is then joined by the replication helicase MCM2–7
(minichromosome maintenance complex 2–7) (Diffley 2011). It is from these
licensed sites that DNA synthesis is initiated during S-phase. Sogo and colleagues
used an SV-40 mini-chromosome replication system and psoralen cross-linking
wherein psoralen intercalates between bases and cross-links adjacent thymidines in
open, but not nucleosomal DNA, thereby identifying DNA that had been wrapped
around a histone octamer core. They found that nucleosomes are disrupted approximately 300 bp in front of the advancing replication fork (Gasser et al. 1996), but the
precise mechanism behind this disruption is still not known. It is likely that histone
chaperones aid this process, but it is not yet clear whether histone chaperones are
actively involved in histone removal from the parental DNA duplex, or merely
provide temporary lodging for histones displaced by the advancing replication
machinery. Although experimentally, loss of many individual histone chaperones
(i.e., NASP, Asf1, CAF-1) inhibits replication, this is likely due to a negative feedback loop that inhibits DNA replication when delivery of histones to the DNA is
compromised. By contrast, the evidence for a direct involvement of the histone
38
B.K. Dennehey and J. Tyler
Rtt109
Asf1 Ac
H3-H4 heterotetramer
?
FACT
ne
sto rs
Hi difie
o
m
Ac
H2A-H2B dimer
Rtt106
Ac
CAF-1
FACT
MCM
Replication
machinery
Sequence
specific DNA
binding factor
PCNA
Asf1
NASP
Chromatin
disassembly
Parental H3-H4 heterodimer
Parental H2A-H2B heterodimer
New H3-H4 heterodimer
New H2A-H2B heterodimer
H1
?
Disassembly of
non-nucleosomal
H2A-H2B
?
Nap1
Chromatin
reassembly
H
mo iston
dif e
ier
s
Incorporation of the parental
pattern of histone modifications
onto the new histones
Fig. 2.3 Replication-dependent chromatin disassembly and assembly. Schematic showing
chromatin disassembly ahead of the replication fork and stepwise chromatin reassembly behind
the DNA replication fork. Ac refers to the acetylation of H3K56 by the fungal-specific Rtt109 HAT
enzyme that promotes the interaction of the histones with CAF-1 and Rtt106 in yeast. Question
marks indicate predicted functions for histone chaperones that have not yet been unequivocally
proven. The model shown is a compilation of information derived from studies in both yeast and
mammalian cells as specifically described in the text
chaperone FACT (facilitates chromatin transcription) in H2A–H2B removal during
DNA replication is more convincing.
FACT is composed of two subunits, SPT16 (suppressor of Ty) and SSRP1
(structure specific recognition protein) in humans, and Spt16 and Pob3 (polymerase
one binding) in yeast. FACT is important for replication in Xenopus egg extracts
(Okuhara et al. 1999) and in human cell lines, where it copurifies with MCM
helicase components and enhances MCM helicase activity in vitro (Tan et al. 2006).
In budding yeast, FACT copurifies with the DNA replication factor RPA
(VanDemark et al. 2006), the MCM helicase (Gambus et al. 2006), and DNA
polymerase-α (Wittmeyer and Formosa 1997) that are all required for initiation of
replication from origins and for lagging strand synthesis (Kunkel 2011). Consistent
with a physical requirement for histone removal from the DNA to allow movement
of the replication machinery, Pob3 mutants are replication defective (Schlesinger
and Formosa 2000) and Spt16 is localized to replication origins in G1 and early S
phases (Han et al. 2010). Given that FACT can displace a single H2A–H2B dimer
from a nucleosome during transcription (Orphanides et al. 1999; Kireeva et al. 2002;
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
39
Belotserkovskaya et al. 2003), it follows that FACT might do the same during
replication. Indeed, Spt16 binds to H2A–H2B with an affinity higher than that of H2A–
H2B for DNA and can effectively compete H2A–H2B from DNA (Winkler et al. 2011).
In vivo FACT has been suggested to be important for both replication initiation
and elongation. FACT facilitates MCM-helicase DNA unwinding, and disruption of
the MCM–FACT interaction in HeLa cells leads to a lag in replication initiation
(Tan et al. 2006). Further, chromatin single fiber analyses have shown that SSRP1 is
required for efficient elongation following initiation (Abe et al. 2011). To assess
whether inefficient elongation resulted from defects in chromatin assembly or
disassembly, micrococcal nuclease (MNase) analyses were performed on newly
replicated, BrdU labeled, DNA. Depletion of SSRP1 did not alter MNase sensitivity,
whereas depletion of the p150 subunit of CAF-1, a key H3–H4 histone chaperone in
replication-dependent chromatin assembly (see Sect. 2.3.2), increased MNase
sensitivity, consistent with FACT being unimportant for chromatin assembly during
replication (Abe et al. 2011). Additional evidence for FACT in chromatin disassembly during DNA replication comes from allele-specific suppression studies in yeast.
H2A–H2B mutations that result in looser association of H2A–H2B with the
(H3–H4)2 tetramer overcome the replication defect caused by FACT deficiency
(McCullough et al. 2011).
Following the removal of H2A–H2B, the (H3–H4)2 tetramer must be removed to
allow passage of the replication machinery. The histone chaperone involved in
disassembly of H3–H4 during DNA replication is currently unknown, although
Asf1 appears to serve as a repository for the dislodged parental histones, as discerned
by their pattern of histone modifications. Specifically, when replication is inhibited,
but helicase activity continues, the displaced parental histones accumulate in Asf1–
MCM–H3–H4 complexes (Groth et al. 2007; Jasencakova et al. 2010). However,
Asf1 itself is unlikely to disassemble the H3–H4 from the DNA because in vitro
studies have indicated that Asf1 can neither remove (H3–H4)2 tetramers from DNA
within a tetrasome (Donham et al. 2011) nor disassemble (H3–H4)2 tetramers from
DNA in the presence of ATP and the chromatin remodeler RSC (Lorch et al. 2006).
The inability of Asf1 to disassemble chromatin in vitro is consistent with the fact
that Asf1 binds to the H3–H3 dimerization interface that is inaccessible to Asf1 in
a nucleosomal context (English et al. 2006; Natsume et al. 2007). As such, it is still
unclear which histone chaperones mediate the removal of H3–H4 dimers or tetramers from DNA during either DNA replication-dependent or replication-independent
chromatin disassembly.
2.3.2
Histone Deposition Behind the Replication Fork
Nucleosomes, or nucleosome-like particles, are rapidly reassembled (McKnight
and Miller 1977) on both leading and lagging DNA (Cusick et al. 1984; Sogo et al.
1986) approximately 100–300 bp behind the replication fork (Herman et al. 1981;
Sogo et al. 1986). Methods utilizing nuclease digestion, salt extraction, and pulse
40
B.K. Dennehey and J. Tyler
labeling followed by nuclease digestion of both formaldehyde cross-linked samples
and native samples indicate that fully nuclease-resistant, properly positioned nucleosome particles do not form immediately following replication (Seale 1975, 1976;
Schlaeger and Knippers 1979; Smith et al. 1984) an idea that has recently resurfaced
(Torigoe et al. 2011). The current understanding is that histone chaperones are
important for the rapid histone deposition step, followed by proper positioning of
the octamer, and DNA wrapping resulting in a mature nucleosome. Additional steps
include removal of the deposition-specific pattern of histone modifications after
chromatin assembly and their replacement with the local parental pattern of histone
modifications, a process mediated by the recruitment of histone modification
enzymes by sequence-specific DNA-binding factors (Fig. 2.3).
2.3.2.1
H3–H4 Deposition onto Newly Replicated DNA
The identity of the histone chaperones involved in deposition of the parental
(H3–H4)2 tetramers onto the newly replicated DNA is currently unknown. In contrast, much is known about how newly synthesized histones are assembled onto
newly replicated DNA. In mammals, histone H3.1 (and H3.2) is expressed during S
phase and used for replication-dependent chromatin assembly, as opposed to the H3
variant H3.3 that is expressed throughout the cell cycle and is used for replicationindependent chromatin assembly. Budding yeast has a single histone H3 variant
(apart from the centromeric H3, see Sect. 2.4) that most closely resembles metazoan
H3.3. Newly synthesized dimers of H3.1–H4, after being imported into the nucleus
(Fig. 2.2), are delivered to sites of DNA synthesis. The available data, discussed
below, indicate that Asf1 transfers H3.1–H4 dimers to the histone chaperones
CAF-1 (chromatin assembly factor 1) and Rtt106 (in yeast) at sites of DNA replication. These downstream chaperones then assemble the (H3.1–H4)2 tetramer and
deposit it onto the newly replicated DNA (Fig. 2.3).
CAF-1 was originally identified and characterized as a factor that could assemble
nucleosomes onto newly replicated DNA using cytosolic extracts and a T-antigen
bound SV-40 minichromosome (Smith and Stillman 1989). The largest subunit of
CAF-1 (p150) interacts with PCNA (Shibahara and Stillman 1999; Moggs et al.
2000) thus positioning CAF-1 near replication sites and providing the opportunity
for CAF-1 to deposit histones onto newly replicated DNA. In addition to this physical connection to replication, there is also a functional connection: loss of CAF-1 in
mammalian cells inhibits nucleosome reassembly (Smith and Stillman 1989;
Nabatiyan and Krude 2004; Takami et al. 2007). Asf1–H3–H4 complexes stimulate
CAF-1-mediated chromatin assembly onto replicating DNA both in vitro (Tyler
et al. 1999) and in vivo (Groth et al. 2005; Sanematsu et al. 2006), but not in cases
where there are large pools of stored histones, as found in Xenopus egg extracts
(Ray-Gallet et al. 2007). The transfer of the histones from Asf1 to CAF-1 is likely
to occur in the immediate vicinity of the replication fork given that Asf1 localizes to
replication foci in Drosophila S2 cells (Schulz and Tyler 2006) and is physically
linked to sites of replication in mammalian cells by its association with the
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
41
mammalian MCM helicase complex via histones H3–H4 (Groth et al. 2007;
Jasencakova et al. 2010). In yeast, binding of Asf1 to Rfc1 (replication factor C,
which loads PCNA onto DNA) is sufficient to recruit Asf1 to the newly replicated
DNA (Franco et al. 2005).
The hand-off of histones from Asf1 to the downstream histone chaperones
depends on their physical interaction. Asf1, when bound to H3–H4, maintains an
open binding surface that provides an interface for the mutually exclusive (Malay
et al. 2008) docking of two distinct H3–H4 chaperones, CAF-1 and HIRA (Tagami
et al. 2004; Tang et al. 2006; Malay et al. 2008). Asf1 delivers new H3.1–H4 dimers
to the CAF-1 complex for replication-dependent chromatin assembly, and new
H3.3–H4 dimers to the HIRA complex for replication-independent chromatin
assembly (Tagami et al. 2004). How these two downstream chaperones recognize
histone proteins that vary by only five amino acids has not been fully determined,
but may depend on posttranslational histone modifications. For example, the HAT1–
RbAp46 complex differentially acetylates H3.1–H4 and H3.3–H4, favoring H3.1–H4
dimers (Zhang et al. 2012), suggesting that this modification might be important for
replication and/or recognition by CAF-1. Conversely, the phosphorylation of H4S47
by PAK2 increases the binding affinity of HIRA for H3.3–H4 and reduces the
affinity of CAF-1 for H3.1–H4 in mammalian systems (Kang et al. 2011).
In S. cerevisiae, most, if not all newly synthesized H3 is acetylated in the nucleus
on lysine 56 (H3K56ac) (Masumoto et al. 2005). Asf1, in conjunction with the histone acetyltransferase Rtt109, is required for the acetylation of H3K56 (Recht et al.
2006; Han et al. 2007; Tsubota et al. 2007). H3 K56Ac, in yeast, appears to drive
replication-dependent chromatin assembly as this modification increases the histone
binding affinities for CAF-1 and Rtt106. In fact, H3K56ac is required for a detectable interaction of H3–H4 with Rtt106 in vivo (Zunder et al. 2012) and H3K56ac
leads to an enhanced binding affinity of CAF-1 for H3–H4 (Li et al. 2008; Nair et al.
2011; Winkler et al. 2012). Gcn5-mediated acetylation of lysines on the N terminus
of H3, including K27ac, also increases the binding affinity for yeast CAF-1 (Fig. 2.2)
(Burgess et al. 2010). Therefore, the deposition-specific histone acetylations on H3
appear to promote chromatin assembly by enhancing their interaction with the
histone chaperones that will deposit them onto the newly synthesized DNA. Within
the nucleosome, H3K56ac has been proposed to loosen the nucleosome–DNA interaction at the DNA entry and exit sites, allowing for the binding of one or more
chromatin remodeling factors (Xu et al. 2005). As such, it is possible that H3K56ac
aids in proper nucleosome positioning following deposition onto the DNA. H3K56ac
is rapidly removed after histone H3–H4 incorporation onto the newly replicated
DNA (Masumoto et al. 2005; Celic et al. 2006), potentially stabilizing the position
of the nucleosome once established. Although H3K56ac is prevalent in both S. cerevisiae and D. melanogaster, in mammalian cells K56ac is found on less than 1 % of
the histone H3 population (Das et al. 2009), suggesting that this modification is
either unlikely to be important for replication-dependent chromatin assembly in
mammals or is highly dynamic.
In yeast, both CAF-1 and the fungal-specific chaperone Rtt106 receive newly synthesized histones from Asf1 (Fig. 2.3). Rtt106 has a poorly defined role in replication;
42
B.K. Dennehey and J. Tyler
however, in the absence of CAF-1 function, Rtt106 becomes important for resistance
to the DNA damaging agent camptothecin, a topoisomerase inhibitor, suggesting that
it has a role redundant with CAF-1 during replication (Li et al. 2008). Indeed, Rtt106
has been found at both early and late replication origins as assessed by ChIP (Zunder
et al. 2012). Additionally, mutations in Rtt106 (in a CAF-1-deficient background)
that interfere with Rtt106–histone interactions, also lead to camptothecin sensitivity
(Su et al. 2012; Zunder et al. 2012).
The (H3–H4)2 tetramer appears to be assembled from two H3–H4 dimers on the
CAF-1/Rtt106 histone chaperones, before its incorporation onto the DNA. Indeed,
a single CAF-1 complex can bind (H3–H4)2 tetramers in vitro and tetramers can
form on CAF-1 (Liu et al. 2012; Winkler et al. 2012). Further, CAF-1 can be immunoprecipitated with (H3–H4)2 tetramers from yeast extracts (Winkler et al. 2012)
and can deposit (H3–H4)2 onto DNA in vitro (Liu et al. 2012). Like CAF-1, Rtt106
can bind to (H3–H4)2 tetramers (Fazly et al. 2012; Su et al. 2012). Mechanistically,
how the (H3–H4)2 tetramer is transferred from either CAF-1 or Rtt106 to the newly
replicated DNA is not yet clear, but may be driven by the high affinity of the
(H3–H4)2 tetramer for DNA (Andrews et al. 2010; Winkler et al. 2012).
2.3.2.2
H2A–H2B and H1 Deposition onto Newly Replicated DNA
After the establishment of the (H3–H4)2 tetramer on DNA, two H2A–H2B dimers
are deposited to complete the nucleosome. The histone chaperone involved in H2A–
H2B dimer assembly onto newly replicated DNA is not yet clear. FACT is a potential candidate for this function given its localization to replication forks and many
functional links to replication (Sect. 2.3.1). Furthermore, in vitro, FACT can deposit
H2A–H2B onto DNA (Belotserkovskaya et al. 2003). Nap1 is also a potential candidate, because it can assemble chromatin together with the chromatin remodeler
ACF in vitro (Ito et al. 1997). However, in vitro chromatin assembly and disassembly assays are quite permissive. Many negatively charged, but physiologically
irrelevant, molecules can mediate chromatin assembly and disassembly in vitro
(Tyler 2002). In contrast to the in vitro evidence for Nap1 in chromatin assembly,
the in vivo evidence indicates that Nap1 functions to remove H2A–H2B dimers
from DNA that are present in nonnucleosomal DNA interactions: loss of Nap1 leads
to an increase in H2A–H2B, but not H3 associated with chromatin in yeast (Andrews
et al. 2010). This study revealed for the first time the unexpected function of a histone
chaperone in the dissolution of improper histone–DNA interactions (Fig. 2.3).
Finally, histone H1 can be incorporated onto the linker DNA to promote higher-order
folding of the chromatin structure. This is likely mediated by the histone chaperone
NASP (Fig. 2.3). NASP forms cytosolic complexes with linker histone H1 (Alekseev
et al. 2003, 2005) and in vitro, NASP can deposit H1 onto chromatin fibers that contain
nucleosomes but have been depleted of H1, resulting in a more compact chromatin
structure (Finn et al. 2008).
Whatever the current understanding of chromatin reassembly, it is important to note
that histone chaperones do not function alone. A current model, based on reconstituted
chromatin assembly systems in vitro, suggests that ATP-dependent chromatin
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
43
remodelers play a critical role in not only the regular spacing of nucleosomes during
chromatin assembly but also in the wrapping of the DNA around the histones that are
deposited by the histone chaperones (Torigoe et al. 2011).
2.3.2.3
Reassembly of Heterochromatin
The details of how heterochromatin is reestablished during replication have not
been fully elucidated. The largest subunit of mammalian CAF-1 binds to both HP1α
(heterochromatic protein 1) (Murzina et al. 1999) and the CpG-me binding protein
MBD1 (Reese et al. 2003). MBD1 recruits the H3K9 methylase SETDB1 to CAF-1
in S phase, thus promoting H3K9 dimethylation on the newly replicated chromatin
(Sarraf and Stancheva 2004) and allowing HP1 to recognize and bind to dimethylated H3K9. That these proteins are in complex with each other at the replication
fork during S-phase suggests that CAF-1 is important in the reestablishment of
silenced chromatin domains during replication. Indeed, the association of HP1 with
CAF-1 is required for the replication of heterochromatin surrounding the centromere in mouse cells, via a process independent of histone deposition, but likely
related to methylation (Quivy et al. 2008).
In Drosophila, Nap1 may also be involved in the reestablishment or maintenance
of heterochromatin. Phenotypically, loss of Nap1 function leads to a dominant loss
of silencing in heterozygotes (Stephens et al. 2005), and is embryonic lethal in
homozygotes (Lankenau et al. 2003). Nap1 binds to HP2 (heterochromatin protein 2),
which itself binds to HP1 (Stephens et al. 2005), as well as NURF (nucleosome
remodeling factor) (Stephens et al. 2006), an ATP-dependent chromatin remodeling
complex (Tsukiyama and Wu 1995). Together, they may function to promote
heterochromatin formation.
2.4
Reassembly of Centromeric Chromatin
With few exceptions, each chromosome contains one centromere, which serves as
the site for kinetochore assembly in order to achieve equal sister chromatid segregation during mitosis. That each chromosome contains only one centromere is critical, as
multiple centromeres would lead to chromosome breakage and unequal chromosome
segregation. Centromeric nucleosomes contain a centromere-specific histone H3
variant (generically termed CenH3) encoded by CSE4 in S. cerevisiae, cnp1+ in
S. pombe, CID in D. melanogaster, HCP-3 in C. elegans, and CENP-A in H. sapiens.
One feature of CenH3 that differs from canonical H3 is the CENP-A targeting
domain (CATD) contained in loop1 and the α-2 helix of CenH3 (Vermaak et al.
2002), contributing to a more rigid structure (Black et al. 2004) relative to that
region in canonical H3. By substituting the amino acids of the CATD into the
analogous region of H3, canonical H3 can be made to function as CenH3 in vivo in
human cells (Black et al. 2007). This is because the CATD interacts with
centromere-specific histone chaperones (discussed in Sect. 2.4.1).
44
B.K. Dennehey and J. Tyler
The DNA composition of the centromere varies from organism to organism, with
most regional centromeres containing an array of CenH3-containing nucleosomes
surrounded by pericentric heterochromatin. Two exceptions are the S. cerevisiae
and C. elegans centromeres. The budding yeast centromere is a small, roughly
125 bp segment, with a centrally positioned single nucleosome (Furuyama and
Biggins 2007; Cole et al. 2011; Henikoff and Henikoff 2012). In contrast, C. elegans
chromosomes are holocentric, forming centromeres along the length of the chromosome, but with mechanisms preventing the formation of multiple microtubule
attachment sites (Maddox et al. 2004). In budding yeast, the presence of the 125 bp
centromere DNA element dictates the position of the centromere (Bloom and
Carbon 1982; Fitzgerald-Hayes et al. 1982). In most other organisms, the presence
of CenH3 serves as the epigenetic mark that defines the centromere.
Interestingly, newly synthesized CenH3 is not deposited onto the DNA during
DNA replication. Instead, the half-complement of parental CenH3 inherited onto
each daughter DNA strand during replication is sufficient for centromeric function
during mitosis, with the new CenH3 usually being incorporated during late stages
of or after mitosis, although in some species incorporation may occur during the G2
phase of the cell cycle (Fig. 2.4).
Fluorescent pulse-labeling experiments in HeLa cells have indicated that parental
CenH3 proteins are distributed to both daughter centromeres during DNA replication (Jansen et al. 2007). But unlike the replication-specific histone H3.1, which is
made prior to the onset of S phase and deposited during S phase, and H3.3 which
is synthesized throughout the cell cycle, newly synthesized CenH3 variants are typically made in G2 and deposited onto DNA outside of replication in late mitosis or
G1 (Shelby et al. 2000; Jansen et al. 2007; Schuh et al. 2007). In the yeasts, newly
synthesized CenH3 molecules are deposited in anaphase in S. cerevisiae (Pearson
et al. 2004; Shivaraju et al. 2012) and in G2 in S. pombe (Takayama et al. 2008).
In tissue culture cells, H3.3 is deposited during replication at those sites destined to
be occupied by newly synthesized CenH3 (Dunleavy et al. 2011) in G1 phase,
thereby necessitating histone exchange (i.e., removal of H3.3 from the DNA and
replacement with CenH3).
2.4.1
CenH3 Chaperones and Their Function
Currently, the known and putative CenH3 chaperones and the organisms that they
have been suggested to have roles in are: HJURP (Holiday junction recognition protein), nucleophosmin, and RbAp46/48 in humans (Dunleavy et al. 2009; Shuaib et al.
2010); FACT in chicken cells; CAL1 (chromosome alignment defect) (Schittenhelm
et al. 2010; Mellone et al. 2011) and RbAb48 in flies (Furuyama et al. 2006); Scm3
in budding yeast (Stoler et al. 2007; Dechassa et al. 2011); and spScm3 (Williams
et al. 2009), and the NASP-related protein Sim3 (Dunleavy et al. 2007) in fission
yeast. However, the strongest evidence for a function in the assembly of newly synthesized CenH3 onto centromeres in vivo exists for mammalian HJURP and yeast
Scm3, as discussed below.
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
CenH3-H4
Parental Cen4
distributed between
sister centromeres
Exchange of H3.3
for new CenH3
G1
45
S
Assembly of
place holder
H3.3-H4
H3.3-H4
H3.3-H4
M
G2
Sister chromatids
separate
Synthesis of
new CenH3
Fig. 2.4 The unusual timing of the incorporation of new CenH3 into the centromere. The parental
CenH3 (shown in yellow) is distributed between the two sister chromatids following DNA replication, such that the newly replicated centromere has only half the amount of CenH3 compared to the
parental centromere prior to DNA replication. Following DNA replication, histone H3.3 (green) is
inserted as a place-holder for the subsequent insertion of more CenH3 after mitosis
HJURP is a mammalian CenH3-specific histone chaperone that shares sequence
similarity to Scm3 at its N-terminus. HJURP and CenH3 coimmunoprecipitate from
HeLa extracts and knockdown of HJURP in tissue culture leads to loss of CenH3 at
centromeres (Shuaib et al. 2010). In vitro, HJURP binds (CenH3–H4)2 tetramers via
an N-terminal “TLTY box” within HJURP and promotes the formation of CenH3–
H4 tetrasomes (Shuaib et al. 2010). It is likely that the TLTY box of HJURP interacts with the CATD of CenH3 that specifies its centromere-specific incorporation,
because placement of the CATD on H3 allows its association with HJURP (Shuaib
et al. 2010) and the N terminus of HJURP is sufficient for CenH3–H4 but not H3.1–
H4 nucleosome assembly in vitro (Barnhart et al. 2011).
The yeast CenH3-specific histone chaperone Scm3 also binds to the CATD of
CenH3 and canonical H3 can be made to bind to Scm3 in vitro with only four residues in the α-2 helix replaced with a minimal CATD (Black et al. 2007). The Scm3
residues that interact with these CenH3 residues are conserved in human HJURP
(Zhou et al. 2011) consistent with the requirement for the CATD for the recognition
46
B.K. Dennehey and J. Tyler
of CenH3 by HJURP (Bassett et al. 2012). In vitro, budding yeast Scm3 functions
as a Cse4-specific nucleosome assembly factor (Dechassa et al. 2011) and Scm3 is
required for CenH3 placement and function at the centromere in budding (Camahort
et al. 2007; Stoler et al. 2007) and fission (Pidoux et al. 2009; Williams et al. 2009)
yeast. In S. pombe, Scm3 binds centromeres in mid-to-late anaphase and dissociates
from the spindle in early mitosis (Pidoux et al. 2009). In S. cerevisiae, the exact timing of the centromeric localization of Scm3 is debatable (Luconi et al. 2011; Mishra
et al. 2011; Xiao et al. 2011; Shivaraju et al. 2012) but is clearly tightly controlled
because overexpression of Scm3 in budding yeast leads to its constitutive localization at centromeres and subsequent chromosome loss (Mishra et al. 2011).
The precise details of how newly synthesized CenH3 is incorporated into the
centromere remain to be determined, but seem to depend upon previously localized
kinetochore proteins, as well as the selective degradation of misincorporated CenH3
from noncentromeric sites. In S. cerevisiae, Scm3 binds to Ndc10, a component of the
CBF3 (CEN–DNA-binding factor) inner kinetochore binding complex, which is
required for CenH3 localization (Hajra et al. 2006; Camahort et al. 2007). It has been
suggested that in S. pombe, Scm3 targets CenH3 to centromeres via binding to the
histone chaperone Mis16/RbAp46/48 and the inner kinetochore protein Mis18 (Pidoux
et al. 2009; Williams et al. 2009). Moreover, in Drosophila, CenH3–H4 has been copurified with RbAp48 (Furuyama et al. 2006). In accordance with a role for these proteins
in CenH3 localization, mutations in, or knockdown of, the centromere/kinetochore
proteins hMis18α/β, and Mis18BP1/KLN2 also lead to loss of CenH3 localization in
vertebrates and nematodes (Fujita et al. 2007; Maddox et al. 2007). Importantly, the
requirement for Mis18 in CenH3 localization is bypassed when LacI–HJURP fusions
are recruited to DNA via integrated LacO arrays (Barnhart et al. 2011). Like loss of
Mis18, RbAp46/48 knockdown leads to mislocalization of CenH3 in human cells
(Hayashi et al. 2004). It should be noted that RbAp46 is a component of multiple HAT
complexes, and that the loss of CenH3 at centromeres in the absence of hMis18α is
corrected by treating cells with the histone deacetylase (HDAC) inhibitor trichostatin
A, leading to the suggestion that histone acetylation primes the centromere for incorporation of new CenH3 (Fujita et al. 2007). The exact structure of the centromeric
nucleosome, i.e., whether it contains only four histone proteins (CenH3–H4–H2A–H2B)
or the usual complement of eight histone molecules, is still highly controversial, but
recent evidence suggests that the structure of the centromeric nucleosome changes
through the cell cycle (Bui et al. 2012; Shivaraju et al. 2012).
2.5
Histone Chaperones in Replication-Independent
Chromatin Disassembly and Assembly
Aside from replication-dependent assembly of histones H3.1–H4, H3.2–H4, and
H2A–H2B, there exist many histone variants that are incorporated into the chromatin outside of DNA replication. These include CenH3 (as discussed in Sect. 2.4),
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
47
Rtt109
Asf1
H2AZ/H2B
Spt6
Chz1
Ac
New H3-H4
(H3.3-H4 in metazoans)
Rtt109
Ac
Asf1
Rtt106
Hir1
New H3-H4
FACT
Transcription
machinery
H36Kme2
Transcription
machinery
FACT
Spt6
FACT
old H2A-H2B
old H3-H4
FACT
Promoter
Gene body
Fig. 2.5 Replication-independent chromatin disassembly and assembly. Some histone chaperones promote chromatin disassembly from promoter regions to enable access of the transcription
machinery to the DNA, whereas others promote chromatin reassembly onto the promoter region
to block access of the transcription machinery to the DNA. Still other histone chaperones promote the dynamic disassembly and reassembly of the chromatin to enable RNA pol II passage.
FACT reorganizes the nucleosome to either promote chromatin disassembly or chromatin reassembly, and it also holds the old histones H2A–H2B that have been removed from the DNA,
retaining them in the vicinity of the gene. New histones H3–H4 can be incorporated by the Asf1,
Rtt106, and Hir1 histone chaperones working together, but this is downregulated by methylation
of H3 on K36, in order to promote the reincorporation of the old histones. The majority of the
information shown here is derived from studies in budding yeast. Ac refers to the acetylation of
H3K56
H3.3, and the mammalian H2A variant proteins H2A.Z, H2A.X, H2A.Bbd (Barr
body deficient), and Macro H2A. The process of swapping histones incorporated
into chromatin for free histones is called histone exchange. In yeast, H3–H4
exchange occurs readily outside of DNA replication, perhaps because yeast H3 is
most similar to mammalian H3.3, which is known to destabilize nucleosomes (Jin
and Felsenfeld 2007). Due to their peripheral location on the nucleosome, H2A–
H2B exchange predominates over H3–H4 exchange. It is important to realize that
histone exchange does not necessitate splitting of the (H3–H4)2 tetramer into two
H3–H4 dimers, as the entire (H3–H4)2 tetramer can be exchanged. Also, given the
physical disruption of nucleosomes that must accompany RNA polymerase
passage, it follows that histone exchange occurs frequently in highly transcribed
regions in yeast and Drosophila. In addition to highly transcribed genes, low levels of histone exchange have also been seen at inactive yeast and Drosophila
promoters (Dion et al. 2007; Mito et al. 2007; Nakayama et al. 2007; Rufiange
et al. 2007). Finally, dynamic chromatin disassembly and reassembly occurs
during the binding and removal, respectively, of the transcription machinery from
promoters and enhancer regions, as well as during RNA polymerase passage
along the DNA (Fig. 2.5).
48
2.5.1
B.K. Dennehey and J. Tyler
(H3–H4)2 Tetramer Splitting
Despite the fact that parental (H3–H4)2 tetramers remain intact during replication
(Senshu et al. 1978; Jackson and Chalkley 1981; Jackson 1987, 1988), there is some
evidence for the splitting of the H3–H4 tetramer into H3–H4 dimers in special
circumstances (Xu et al. 2010; Katan-Khaykovich and Struhl 2011). One study,
using isotope labeling and mass spectrometry to distinguish between new and old
histones in bulk chromatin from mammalian cells, suggested that replicationdependent splitting of (H3.3–H4)2 tetramers, but not (H3.1–H4)2 tetramers occurs
(Xu et al. 2010). However, this study left open the possibility that the analyses of
H3.1 and H3.3 may not have been directly comparable, that is, the recovery of H3.3
might have been biased to particular loci. This is relevant because the incorporation
of H3.3 does not depend on replication. A subsequent study, using differentially
tagged and expressed H3 in budding yeast, followed by sequential ChIP, found no
strong evidence for replication-coupled tetramer splitting, but rather found evidence
for transcription-related tetramer splitting at highly transcribed genes (KatanKhaykovich and Struhl 2011). To explain these findings, it was suggested that the
passage of RNA Pol II, and its associated factors along chromatin might allow disruption of nucleosomes such that one H3–H4 dimer is preserved and one H3–H4
dimer is replaced (Katan-Khaykovich and Struhl 2011).
2.5.2
Exchange of H2A.Z
Histone variant H2A.Z is synthesized throughout the cell cycle (Hatch and Bonner
1988) and is incorporated into chromatin outside of S-phase. H2A.Z has been implicated in transcription, the delineation of boundary regions, heterochromatic silencing,
proper chromosome segregation, replication, and DNA repair, where it is recruited
to break sites (see Sect. 2.6). H2A.Z is enriched near the promoters of genes, often
found in the highly positioned +1 nucleosome that is immediately upstream of the
transcription start site of genes (Guillemette and Gaudreau 2006; Marques et al.
2010), some of which are highly regulated, for example, the repressed PHO5, GAL1
(Santisteban et al. 2000), and GAL1/GAL10 (Floer et al. 2010) yeast promoters.
H2A.Z can influence transcription. For example, in yeast, when the 9.4 kb VPS13
gene (large by yeast standards) is placed under the control of the GAL10 promoter,
the absence of H2A.Z leads to a decreased rate of transcriptional elongation and an
increase in nucleosome occupancy along the gene (Santisteban et al. 2011). This
result suggests that nucleosomes containing H2A.Z are more readily disassembled
during transcription than are nucleosomes containing H2A. In Drosophila, H2Av
takes on the functions of both H2A.Z and H2A.X, another histone variant that is
phosphorylated and recruited to sites of DNA damage (see Sect. 2.6). In Drosophila
S2 cells, H2Av–H2Av containing nucleosomes are enriched 3′ to both transcriptional start sites and intron–exon junctions (Weber et al. 2010).
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
49
Several different histone chaperones have been copurified with H2A.Z from
budding yeast including Nap1, Chz1, FACT, Fpr3, and Fpr4, as well as components
of the ATP-dependent chromatin remodeling complexes SWR1 (e.g., Yaf9 and Swc6)
and Isw1 (e.g., Isw1 and Ioc3) (Mizuguchi et al. 2003; Kobor et al. 2004; Luk et al.
2007). Of these, Fpr3 and Fpr4, two nonessential peptidyl-prolyl cis–trans isomerases, related to proteins with proven histone chaperone activity (Kuzuhara and
Horikoshi 2004) and the Isw1 subunits, were only detected with H2A.Z in the absence
of Nap1 and Chz1, suggesting that they do not normally interact with H2A.Z (Luk
et al. 2007). Nap1 can bind to both H2A–H2B and H2A.Z–H2B in vitro and facilitate
their exchange onto DNA (Park et al. 2005), as can Chz1, in the presence of SWR1
and ATP (Luk et al. 2007). However, Nap1, but not Chz1 is associated with H2A.Z
isolated from soluble cytoplasmic extracts and is likely to be involved in the transport
of Nap1–H2A.Z–H2B to Kap114 for import into the nucleus (Straube et al. 2010).
In contrast, Chz1 is primarily a nuclear protein (Luk et al. 2007) making Chz1 the
most likely histone chaperone involved in H2A.Z–H2B exchange in cells (Fig. 2.5).
2.5.3
Exchange of H3.3
In mammals, H3.3 serves as a place holder for CenH3 from S phase until it is
exchanged for CenH3 during G1 phase (Dunleavy et al. 2011) (see Sect. 2.4;
Fig. 2.4). H3.3 is also exchanged into the chromatin, independent of DNA replication, near the body of transcribed genes (Mito et al. 2005; Luciani et al. 2006;
Jin et al. 2009; Goldberg et al. 2010) including CpG-rich promoters, gene regulatory
sequences (Goldberg et al. 2010), rDNA repeats (Ahmad and Henikoff 2002),
telomeres, and pericentric heterochromatin (Goldberg et al. 2010). H3.3, like H2A.Z,
is synthesized and deposited onto DNA throughout the cell cycle. H3.3 nucleosomes
are inherently unstable (Jin and Felsenfeld 2007), and more so when combined with
H2A.Z (Jin et al. 2009). This instability may facilitate nucleosome clearance at CpGrich promoters and transcription factor binding sites (Jin et al. 2009).
The metazoan HIRA complex promotes assembly of H3.3–H4 during replicationindependent chromatin assembly (Tagami et al. 2004). Likewise, the yeast counterpart of HIRA, the Hir1 complex, promotes H3–H4 replication-independent
chromatin assembly (Fig. 2.5). Given that much of H3.3 exchange occurs at highly
transcribed regions, it was suggested that H3.3 incorporation would require chromatin assembly via factors also linked to transcription (Ahmad and Henikoff 2002;
Schwartz and Ahmad 2005). Indeed, mammalian HIRA and RNA Pol II reciprocally
coimmunoprecipitate, and the levels of RNA Pol II Ser5 (initiating RNA Pol II),
at certain transcription factor binding sites, correlate with the levels of HIRA
(Ray-Gallet et al. 2011). In Drosophila, GAGA factor, a zinc-finger transcription
factor that binds to GAGA-rich DNA sequences, reciprocally coimmunoprecipitates
with HIRA (Nakayama et al. 2007) and binds to FACT (Shimojima et al. 2003).
FACT, itself, enhances the deposition of histone H3.3 on nucleosomes adjacent to
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B.K. Dennehey and J. Tyler
FACT-binding sites, and together with GAGA factor, appears to help direct histone
H3.3 replacement to prevent heterochromatin spreading (Nakayama et al. 2007).
In addition to HIRA, the histone chaperone DAXX (death-domain associated
protein) and the ATPase/helicase ATRX (α thalassemia/mental retardation syndrome
X-linked) (Xue et al. 2003; Tang et al. 2004) can incorporate H3.3, particularly at
telomeres (Drane et al. 2010; Goldberg et al. 2010; Lewis et al. 2010; Wong et al.
2010). DAXX binds specifically to histone H3.3–H4 dimers via residues unique to
the H3.3 variant (Lewis et al. 2010; Elsasser et al. 2012), and the DAXX domain,
which has some similarity to Rtt106, is required for the specific binding of DAXX
to H3.3 (Drane et al. 2010). In vitro, DAXX facilitates the formation of (H3.3–H4)2
tetrasomes (Drane et al. 2010), and tetrasome formation is further enhanced by the
addition of ATRX leading to the formation of extended, nonregular, nucleosomal
arrays (Lewis et al. 2010). In mouse embryonic stem cells, HIRA, but not DAXX,
is dispensable for the localization of H3.3 at telomeres and at many transcription
factor binding sites, whereas HIRA is required for H3.3 enrichment at high CpG
content promoters, with the highest levels of enrichment correlating with highly
transcribed genes (Xue et al. 2003; Goldberg et al. 2010).
2.5.4
Transcription-Dependent Chromatin Disassembly
and Reassembly
During the transcription of coding genes, the nucleosome impedes RNA Polymerase
II (RNA Pol II) from traversing the genomic template. In vitro, DNA templates
decorated with nucleosomes interfere with the initiation (Knezetic and Luse 1986;
Lorch et al. 1987) and elongation (Izban and Luse 1991) activities of RNA Pol II.
Transcription is correlated with histone exchange as exemplified by the incorporation
of histone variants at highly transcribed regions (see Sects. 2.5.3 and 2.5.2; and
Chaps. 9 and 12) and it has been suggested that transient nucleosome gaps following
RNA Pol II passage are continually refilled via replication-independent histone H3.3
replacement (Mito et al. 2005). Here, we briefly discuss possible roles of the histone
chaperones, Asf1, FACT, and Spt6 in transcription-dependent chromatin disassembly
and reassembly, bearing in mind that these chaperones must cooperate with transcriptional activators, chromatin modifiers (e.g., acetylation complexes), and ATPdependent chromatin remodeling complexes such as INO80, CHD, SWI/SNF, and
ISWI to effect change (see Chap. 3). Additionally, although most of the studies of
histone chaperone function in chromatin assembly and disassembly during transcription have been performed in yeast, these findings are likely relevant to mammalian
cells given the conservation of these proteins and processes across species.
2.5.4.1
Asf1
There is ample evidence that the H3–H4 chaperone Asf1 is involved directly and/or
indirectly in chromatin disassembly during transcription. Yeast Asf1 is found in
transcribed regions with elongating RNA Pol II (Schwabish and Struhl 2006) and
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
51
facilitates chromatin disassembly at inducible promoters during transcriptional
activation (e.g., PHO5, PHO8, GAL1-10, and HO) (Adkins et al. 2004; Korber et al.
2006; Schwabish and Struhl 2006; Adkins et al. 2007; Gkikopoulos et al. 2009;
Takahata et al. 2009). Loss of Asf1 reduces nucleosome remodeling at the HO
promoter (Gkikopoulos et al. 2009), in a specific region (Takahata et al. 2009),
resulting in decreased cell cycle-dependent transcription of HO (Gkikopoulos et al.
2009). Deletion of ASF1 also reduces histone eviction and inhibits the incorporation
of new H3 at the highly transcribed yeast gene PMA1 (Rufiange et al. 2007). Further,
a genome-wide analysis of α-factor arrested yeast cells indicated that loss of Asf1
function had the greatest effect on transcription-dependent histone H3 exchange at
promoters, particularly those that are activated in response to α-factor, but had
little effect on the levels of basal H3 exchange at promoters (Rufiange et al. 2007).
These data suggest that Asf1 may contribute to chromatin disassembly at inducible
promoters.
The influence of Asf1 on chromatin disassembly during transcription is likely to
be indirect, as opposed to Asf1 physically removing histones from DNA, as Asf1
cannot remove (H3–H4)2 from DNA in vitro (see Sect. 2.3.1). The indirect role of
Asf1 in chromatin disassembly in yeast appears to be via Asf1–Rtt109-mediated
H3K56 acetylation, given that H3K56ac breaks two histone–DNA interactions
within the nucleosome, leading to a looser nucleosome structure (Neumann et al.
2009; Shimko et al. 2011). Indeed, the defect in promoter chromatin disassembly
from the yeast PHO5 gene during transcriptional induction in an asf1 mutant is
mimicked by mutation of the Rtt109 H3K56 HAT and is largely corrected by a
H3K56Q mutation designed to mimic H3K56 acetylation (Williams et al. 2008).
The role of Asf1 in chromatin disassembly during polymerase passage may also be
indirectly mediated by its role in H3K56 acetylation, especially given that H3K56ac
is associated with elongating RNA Pol II (Schneider et al. 2006). In this model, as
polymerase passes, old histones are replaced with new, H3K56ac-marked histones,
leading to a looser chromatin structure that facilitates additional rounds of transcription. However, in yeast, the H3K56Q mutation designed to mimic acetylation at
H3K56 cannot fully substitute for Asf1 in chromatin disassembly (Williams et al.
2008). This is consistent with the finding that eviction of unacetylated histone H3
(i.e., preexisting H3) is reduced in the absence of Asf1 (Rufiange et al. 2007).
Whether this is due to a direct role of Asf1 in chromatin disassembly or whether
Asf1 provides a sink for histones evicted during transcription initiation and/or
elongation, effectively preventing nonspecific histone–DNA association, remains to
be determined. Clearly, any role of Asf1 in chromatin disassembly would not preclude a role of Asf1 in chromatin reassembly following RNA polymerase passage.
Indeed, Asf1 promotes both H3–H4 histone eviction and deposition during RNA
Pol II elongation (Schwabish and Struhl 2006).
In addition to Asf1 being important for chromatin disassembly at inducible yeast
promoters, it also appears to influence the transcriptional repression of some genes
via chromatin assembly. At the ARG1 gene, Asf1 and Rtt109 are important for
robust transcription in inducing conditions, as well as repression in noninducing
conditions. In noninducing conditions, the association of Asf1 with histones H3–H4
and the acetyltransferase activity of Rtt109 are required to prevent high levels of
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B.K. Dennehey and J. Tyler
transcription, but neither H3K56 nor H3K9 acetylation are required for this
repression and hence, the mechanism of repression is currently unknown (Lin and
Schultz 2011). In another form of transcriptional repression, Asf1 inhibits transcription initiation from cryptic promoters within coding regions (Schwabish and Struhl
2006), presumably via a role in chromatin assembly following polymerase passage.
Similarly, deletion of ASF1 inhibits the incorporation of new H3 at PMA1 (Rufiange
et al. 2007). Likewise, in both asf1Δ and hir1Δ deletion mutants, chromatin reassembly at the PHO5 promoter is delayed and the defect in chromatin assembly in
asf1Δ, hir1Δ double mutants was no worse than the defect in either single mutant
suggesting ASF1 and HIR1 are in the same genetic pathway (Schermer et al. 2005;
Kim et al. 2007). With regard to Asf1, these findings are consistent with in vitro
chromatin assembly assays showing that yeast extracts lacking Asf1 are defective in
replication-independent nucleosome assembly (Robinson and Schultz 2003). The
most likely scenario is that Asf1 transfers histones to Hir1 and Rtt106 for their
subsequent replication-independent assembly onto the DNA. It has been shown that
mutations in Rtt106 that interfere with histone binding elevate cryptic transcription
levels, and that rtt106Δ hir1Δ mutants are no worse than either single mutant suggesting that Rtt106, Hir1, and Asf1 work together to assemble chromatin over open
reading frames in order to prevent cryptic transcription initiation (Silva et al. 2012).
Therefore, Asf1–Hir1–Rtt106-mediated replication-independent chromatin assembly
is important for restoration of chromatin following RNA Pol II passage, at least at
some genes, in yeast.
2.5.4.2
FACT
FACT appears to have roles in both the disassembly of H2A–H2B from promoters
during transcriptional activation and in the reassembly of H2A–H2B following
RNA Pol II passage (Fig. 2.5). FACT is required for the rerecruitment of transcriptional coactivators at the HO promoter (Takahata et al. 2009), and FACT is also
important for the transcription of GAL1-10 (Biswas et al. 2006; Xin et al. 2009) and
H2A–H2B disassembly from the PHO5 promoter (Ransom et al. 2009). FACT travels with elongating RNA Pol II (Mason and Struhl 2003; Saunders et al. 2003), and
the loss of FACT correlates with the loss of TBP (TATA-binding protein) at the
GAL1 promoter (Biswas et al. 2005), and with loss of TBP, TFIIB, and RNA Pol II
at several other promoters (Mason and Struhl 2003). This suggests that FACT is
important for proper transcription initiation, perhaps linking initiation and elongation (Mason and Struhl 2003). Functionally, FACT promotes the removal of one
H2A–H2B dimer from the nucleosome during RNA Pol II passage (Orphanides
et al. 1999; Kireeva et al. 2002; Belotserkovskaya et al. 2003). Nucleosome removal
during transcription could be an indirect effect of Nhp6–FACT binding, leading to
loosening of the DNA wrapped around the core octamer and “nucleosome reorganizing” (Rhoades et al. 2004; Xin et al. 2009; McCullough et al. 2011). However,
Nhp6 can also stabilize, rather than destabilize, promoter nucleosomes and coregulate
transcription in vivo through its DNA-binding activity (Dowell et al. 2010).
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
53
Taken together, the available information indicates that FACT can interconvert
nucleosomes between the canonical form and a reorganized form. In the forward
direction, reorganization destabilizes nucleosomes promoting disassembly, whereas
the reverse reaction promotes chromatin assembly (McCullough et al. 2011).
Indeed, FACT is involved in chromatin reassembly behind RNA Pol II. Loss of
Spt16 function leads to an increase in transcription from cryptic promoters (Kaplan
et al. 2003; Mason and Struhl 2003) suggesting that nucleosomes do not properly
reform after polymerase passage. In Drosophila, FACT enhances the deposition of
histone H3.3 on nucleosomes adjacent to FACT-binding sites (Nakayama et al.
2007), and in yeast, FACT appears to recycle the displaced H3 histones, thereby
preventing deposition of new histone H3 without affecting deposition of new histone
H2B (Jamai et al. 2009). This is probably linked to the ability of FACT to act as a
buffer for H3–H4 and H2A–H2B evicted from transcribed chromatin (MorilloHuesca et al. 2010). Set2-mediated dimethylation on H3 K36 also plays an important role in promoting the recycling of the old histones during RNA pol II passage
in order to reduce any cryptic initiation that stems from the presence of newly incorporated histones that carry deposition-specific histone acetylation marks (Fig. 2.5)
(Venkatesh et al. 2012).
2.5.4.3
Spt6
Yeast Spt6 is an essential histone chaperone for H3–H4 that plays a key role in
chromatin reassembly at both promoters and in gene bodies. Spt6 binds to both
(H3–H4)2 tetramers and H2A–H2B dimers with a preference for (H3–H4)2 over
H2A–H2B and assembles chromatin in vitro (Bortvin and Winston 1996). Loss of
Spt6 function leads to a more open chromatin structure in the SUC2 promoter,
suggesting that Spt6 is important for proper chromatin assembly. Indeed, Spt6 is
required for chromatin assembly over the yeast PHO5 promoter to achieve transcriptional repression (Adkins and Tyler 2006). Spt6 also assembles chromatin
within open reading frames in order to prevent transcription from cryptic promoters
(Kaplan et al. 2003; Adkins and Tyler 2006; Cheung et al. 2008), suggesting a role
in nucleosome reassembly following RNA polymerase passage (Fig. 2.5).
2.6
Histone Chaperones in Chromatin Assembly
and Disassembly During DNA Repair
DNA is continually damaged by exogenous and endogenous sources such as
ultraviolet light, free radicals, gamma rays, and mutagens. This damage must be
rapidly recognized, accessed, and repaired. Just as there are many types of DNA
lesions, there are numerous distinct DNA repair pathways. The four main pathways
are nucleotide excision repair (NER), base excision repair (BER), homologous
recombination (HR), and nonhomologous end joining (NHEJ). In both BER and
54
B.K. Dennehey and J. Tyler
NER, the DNA lesion exists only on one strand of DNA. The lesion and neighboring
nucleotides are excised and the resulting gap is filled in using the intact DNA strand
as a template. HR and NHEJ both repair breaks in the phosphodiester backbone that
occur on both strands of DNA. The more error-prone NHEJ pathway is used to
“stick” the DNA ends back together, whereas the more accurate HR pathway
“patches” the DNA break using a homologous sequence as the template.
The mechanism for repair of DNA damage in the context of chromatin has been
termed the “Access, Repair, Restore” model (Smerdon 1991). In this model, the
DNA lesion is made accessible to the DNA repair machinery by a combination of
local histone acetylation, sliding of histones along the DNA, and/or removal of histones from the DNA. After DNA repair, it is necessary to “restore” the chromatin
structure by either reassembling the chromatin or sliding the histones back over the
repaired DNA. Below, we discuss the current understanding of how histone chaperones contribute to the “Access, Repair, Restore” model.
Akin to the limited knowledge of the identity of histone chaperones that disassemble chromatin during DNA replication (Sect. 2.3.1), we do not know yet which
histone chaperones disassemble chromatin during DNA repair. In all studies
performed to date, the rate of histone removal from the DNA during double-strand
break repair is identical to the rate of DNA end processing. Indeed, it remains possible that DNA end resection itself drives the process of chromatin disassembly
during DNA repair, which in turn occurs with the aid of the ATP-dependent
chromatin remodeler INO80 in yeast (Morrison et al. 2004; van Attikum et al. 2004)
and mammalian cells (Gospodinov et al. 2011). In addition to the removal of the
histones from the DNA region that is being processed for repair, there is also
dynamic exchange of histone variants prior to and after DNA repair. For example,
dynamic H2A.Z incorporation occurs around a double-strand break in human cells
and appears to promote the repair process itself (Xu et al. 2012). The DNA damage
sensitivity, genomic instability, and impaired HR and NHEJ pathways in human
cells depleted for H2A.Z indicate the importance of H2A.Z incorporation for DNA
repair. These defects in the absence of H2A.Z incorporation around sites of doublestrand breaks may be due to that fact that nucleosomes containing H2A.Z are more
readily disassembled to promote the “access” part of the “Access, Repair, Restore”
model. Consistent with this idea, H2A.Z depletion prevented the increase in histone
solubility that occurs in response to DNA damage, that is presumably due to chromatin disassembly flanking the DNA lesion in order to enable DNA repair (Xu et al.
2012). As such, incorporation of H2A.Z into the chromatin after double-strand
DNA damage, at least in mammalian cells, facilitates the subsequent opening up of
the chromatin to enable DNA repair.
Histone H2A variant H2A.X also plays a unique role during DNA repair. H2A.X
represents about 10% of the total H2A in mammalian cells and is randomly distributed throughout the chromatin. Upon DNA damage, the H2A.X preexisting within
the chromatin flanking the DNA lesion becomes phosphorylated on serine 139
(serine 129 in yeast) by the activated DNA damage checkpoint. The phosphorylated
H2A.X facilitates signaling of the presence of the DNA damage. Accordingly, it is
important to remove the phosphorylated H2A.X from the DNA after repair is
2
Histone Chaperones in the Assembly and Disassembly of Chromatin
55
complete, in order to terminate the damage signaling. The histone chaperone FACT
performs the role of removing the phosphorylated H2A.X–H2B dimers from the
chromatin, replacing them with canonical H2A–H2B dimers (Heo et al. 2008).
The histone chaperones involved in chromatin assembly after DNA repair are
largely the same as those that assemble chromatin after DNA replication, primarily
because DNA synthesis during DNA repair uses very similar machinery to that used
during replication. Accordingly, it has been shown that chromatin assembly after
NER (Gaillard et al. 1996) and HR (Linger and Tyler 2005; Chen et al. 2008) is
mediated by the histone chaperone CAF-1 together with Asf1. The current evidence
indicates that this process is very similar to that which occurs during DNA replication (Fig. 2.3). PCNA recruits CAF-1 to site of DNA repair (Moggs et al. 2000;
Linger and Tyler 2005) and CAF-1 incorporates H3.1–H4 onto the DNA at sites of
DNA repair in mammalian cells after NER (Polo et al. 2006) and after HR in yeast
(our unpublished data). The chromatin assembly over the repaired DNA is also
facilitated by Asf1-dependent acetylation of H3K56ac, because mutation of H3K56
to mimic its permanent acetylation bypasses the requirement for Asf1 in chromatin
assembly after double-strand break repair in yeast (Chen et al. 2008). Therefore, it
seems likely that H3K56ac, via its enhanced affinity for CAF-1 (Li et al. 2008; Nair
et al. 2011; Winkler et al. 2012), helps deliver the histones to the population of
CAF-1 that is tethered to sites of DNA repair in order to promote chromatin assembly after repair. Which histone chaperones mediate the reassembly of H2A–H2B
and H1 following DNA repair is not clear, but this may also occur in a manner
similar to the chromatin reassembly following DNA replication. Arabidopsis lacking
Nap1 have defects in homologous recombination, consistent with a role for Nap1
family histone chaperones in nucleosome disassembly/reassembly during this repair
pathway (Gao et al. 2012).
2.7
Concluding Remarks
In eukaryotic cells, the assembly and disassembly of nucleosomes accompanying
genomic processes are complex operations involving many steps and proteins of
various functions. Here, we have emphasized the contribution of the histone
chaperones to these chromatin dynamics. However, it is important to remember that
histone chaperone function in chromatin assembly and disassembly is intimately
dependent on the making and breaking of histone–DNA interactions by the ATPdependent chromatin remodeling complexes (Chap. 3). Eukaryotes have evolved
specific histone chaperones for one or more histone variants and likely even specific
chaperones for old histones versus newly synthesized histones. There are also multiple histone chaperones for the same histones, but that function during distinct
genomic processes, for example replication-dependent and replication-independent
processes. These multiple chaperones also contribute to the transfer of the histones
in a precise order among many separate chaperones in order to achieve the proper
pattern of histone posttranslational modifications prior to being delivered to the
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B.K. Dennehey and J. Tyler
ultimate histone chaperone that will deposit the histones onto DNA. In addition,
those histone chaperones that promote nucleosomal histone–DNA interactions also
work together with other histone chaperones that dissolve nonnucleosomal histone–
DNA interactions in order to promote the formation and maintenance of nucleosome
structure. In budding yeast, the precise role of many chaperones is partially obscured
by the ability of another chaperone to take its place (functional redundancy),
whereas in other organisms many of the chaperones are essential, highlighting their
critical role in biology. Despite this wealth of knowledge of the growing histone
chaperone family, there are still many gaps in our understanding of the mechanisms
of histone chaperone function.
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