MINIREVIEW
Chromatin assembly
Cooperation between histone chaperones and ATP-dependent nucleosome
remodeling machines
Jessica K. Tyler
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado Health Sciences Center, Denver,
CO, USA
Chromatin is a highly dynamic structure that plays an
essential role in regulating all nuclear processes that utilize
the DNA template including DNA repair, replication,
transcription and recombination. Thus, the mechanisms by
which chromatin structures are assembled and modified are
questions of broad interest. This minireview will focus on
two groups of proteins: (a) histone chaperones and (b) ATP-
dependent chromatin remodeling machines, that co-operate
to assemble DNA and histone proteins into chromatin. The
current understanding of how histone chaperones and ATP-
dependent remodeling machines coordinately assemble
chromatin in vitro will be discussed, together with the
growing body of genetic evidence that supports the role of
histone chaperones in the cell.
Keywords: chromatin; histone chaperone; nucleosome
remodeling; acetylation; DNA replication; DNA repair;
CAF-1; ACF; ASF1; Rad53.
INTRODUCTION TO CHROMATIN
The eukaryotic genome is packaged into a nucleoprotein
structure known as chromatin. The basic repeating unit of
chromatin, the nucleosome core particle, comprises
approximately two turns of DNA wrapped around two
molecules of each core histone protein; H2A, H2B, H3 and
H4 ([1]; and reviewed in [2]). Nucleosomes are regularly

chromatin in vivo. Other aspects of chromatin assembly,
including cell cycle regulation of histone chaperone
function and their role in epigenetic inheritance are
covered in a series of recent reviews and will not be
addressed here [6–11].
CHROMATIN ASSEMBLY IN THE CELL
Little is known about how chromatin is assembled in the
cell. We do know that the majority of chromatin assembly
occurs immediately following DNA replication, where
nucleosomes are disrupted by the passage of the replication
machinery (reviewed in [2,7]). The naked daughter strands
of newly replicated DNA appear to be rapidly assembled
into chromatin by a multistep process that involves the
initial deposition of histones H3 and H4 followed by
incorporation of two histone H2A-H2B dimers to complete
Correspondence to J. Tyler, Department of Biochemistry
and Molecular Genetics, School of Medicine B121,
University of Colorado Health Sciences Center,
4200 East Ninth Avenue, Denver CO 80262, USA.
Fax: + 1 303 3155467, Tel.: + 1 303 3158163,
E-mail:
Abbreviations: ACF, ATP-utilizing chromatin assembly
and remodeling factor; ASF1, antisilencing function 1;
CAC1, chromatin assembly complex 1; CAF-1, chromatin assembly
factor 1; CHRAC, chromatin accessibility complex; ISWI, imitation
switch; NAP-1, nucleosome assembly protein 1; NURF, nucleosome
remodeling factor; PCNA, proliferating cell nuclear antigen; RSF,
remodeling and spacing factor.
Dedication: This Minireview Series is dedicated to Dr Alan Wolffe,
deceased 26 May 2001.

histones onto the DNA, but has recently been shown to
facilitate the regular spacing of nucleosomes during
chromatin assembly in vitro [20].
CHROMATIN ASSEMBLY IS MEDIATED
BY HISTONE CHAPERONES
Key insight into the mechanism of chromatin assembly in
the cell has been provided by biochemical studies of
histone–DNA interactions. Histone proteins are rich in
positively charged basic amino acids that have an intrinsic
affinity for the negatively charged phosphate groups in
DNA; mixing histones and DNA in vitro at physiological
salt concentration leads to the rapid formation of undefined
insoluble aggregates (Fig. 1A). However, the presence of
additional anionic factors that shield the charge of the
histones from DNA, allow chromatin assembly to occur in
a regulated and ordered manner (Fig. 1B). It would appear
that additional negatively charged molecules can act as
histone chaperones to allow H3-H4 tetramers to bind to
DNA first, due to their higher affinity for DNA as
compared to H2A-H2B dimers (Fig. 1B). The subsequent
deposition of H2A-H2B dimers is likely to be driven by the
higher affinity of H2A-H2B for subnucleosomal particles
comprising DNA and H3-H4, as compared to their affinity
for either a histone chaperone or DNA [21]. As such,
chromatin assembly that is mediated by histone chaperones
in vitro mimics the stepwise process that occurs in the cell.
Using biochemical approaches to search for physiolog-
ically relevant histone chaperones has led to the identifica-
tion of numerous molecules that can bind to histone
proteins and facilitate chromatin assembly in vitro (reviewed

Almost any molecule that could shield the basic charge of
histone proteins from DNA, including pectin, RNA,
polyglutamic acid and even salt, was found to function as
a histone chaperone in vitro with little or no relevance for the
assembly of chromatin in vivo. However, the use of cell-free
chromatin assembly systems coupled to ongoing DNA
replication has been more productive in identifying histone
chaperones in vitro that also appear to assemble chromatin
in the cell, as discussed later.
ATP-REMODELING IS INTRINSIC
TO CHROMATIN ASSEMBLY
Histone chaperones are not sufficient to generate regular
arrays of nucleosomes with 180–200 bp spacing in vitro.
Instead, histone chaperones lead to the assembly of
irregularly spaced or closely spaced nucleosome arrays
in vitro (Fig. 1B). Presumably, factors in addition to histone
chaperones are required in order to generate the physiolog-
ically spaced arrays of nucleosomes that are seen in the cell.
Indeed, it has long been known that chromatin assembly in
crude extracts requires ATP hydrolysis in order to generate
regular arrays of physiologically spaced nucleosomes [24].
Accordingly, biochemical fractionation of crude Drosophila
embryo extracts (a rich source of chromatin assembly
factors due to the rapid rounds of DNA replication and
concomitant chromatin assembly that occur during early
embryogenesis) identified a second key component of
the chromatin assembly machinery: an ATP-dependent
chromatin remodeling factor (reviewed in [25]). This
ATP-dependent chromatin remodeling factor was found
independently by two groups and was termed ACF (for

appears to require close cooperation between histones,
DNA, ACF and a histone chaperone.
Similar protein complexes to Drosophila ACF have since
been identified in humans and Xenopus [31–34]. However, a
human counterpart called the remodeling and spacing
factor (RSF) exhibits some striking functional distinctions
from ACF. Purified RSF is sufficient to deposit histones
and space nucleosomes during chromatin assembly, in the
absence of a histone chaperone [20]. In addition, each RSF
molecule only assembles one DNA molecule into chroma-
tin, whereas each ACF molecule can assemble multiple
DNA molecules into chromatin [20,27]. These differences
may be due to the ability of RSF to bind to histones H3-H4
and simultaneously act as a histone chaperone and a
remodeling factor [20].
IDENTIFICATION OF HISTONE
CHAPERONES THAT MEDIATE DNA
REPLICATION-COUPLED CHROMATIN
ASSEMBLY
The majority of chromatin assembly is tightly coupled to
DNA replication in the cell. Accordingly, chromatin
assembly systems that preferentially assemble chromatin
onto replicating DNA templates have been developed using
crude protein extracts derived from Xenopus oocytes and
human cells [35,36]. This replication-dependent chromatin
assembly occurs in a stepwise manner in vitro, mirroring the
stepwise assembly of chromatin onto newly replicated DNA
observed in the cell (Fig. 2; [35,37]). Fractionation of the
human cell extract identified a heterotrimeric protein
complex termed chromatin assembly factor-1 (CAF-1)

DNA into chromatin in vitro in the absence of CAF-1
2270 J. K. Tyler (Eur. J. Biochem. 269) Ó FEBS 2002
[42,45,46]. It has recently been shown that ASF1 binds
directly to CAF-1 in vivo and in vitro [47]. Therefore, the
ability of ASF1 to facilitate chromatin assembly preferen-
tially onto newly replicated DNA in vitro is likely mediated
by CAF-1 targeting ASF1 to the DNA replication fork
(Fig. 3; [47]).
Genetic analyses in yeast support the biochemical
evidence that ASF1 is a histone chaperone that functions
during the assembly of newly synthesized DNA into
chromatin [42–44]. Although ASF1 is not an essential gene
in budding yeast, asf1 mutants grow slowly due to an
elongated G
2
/M phase of the cell cycle; this is extended to an
apparent arrest in G
2
/M phase under various conditions of
stress [42–44]. The sensitivity of asf1-mutant yeast to
hydroxyurea (a reagent that depletes the endogenous
nucleotide pools in the cell) indicates that under conditions
when DNA synthesis is compromised, ASF1 may be
required in order to generate chromosomes that are com-
petent to pass through G
2
/M phase. The delay in progres-
sion through G
2
/M phase of asf1 mutants is reminiscent of a

the physical interaction between ASF1 and CAF-1 in vivo
[47] indicates that the codependence between ASF1 and
CAF-1 is probably not an in vitro artifact. Instead, there
may be additional histone chaperones in vivo that are
functionally redundant with ASF1 and/or CAF-1 that are
either missing or inactive in the cell-free assembly system.
WHAT ARE THE ADDITIONAL HISTONE
CHAPERONES?
There are probably additional histone chaperones in the
cell, as yeast lacking both the ASF1 and CAF-1 histone
chaperones are viable [42]. No sequence motifs have yet
been identified to indicate a potential function as a histone
chaperone. Therefore, we have had to rely on biochemical
fractionation and genetic analyses in order to identify novel
histone chaperones. Biochemical approaches have identified
Fig. 2. Model for chromatin assembly at the DNA replication fork.
In vivo studies have indicated that the DNA replication machinery
(large red oval) displaces all the histones from the parental chromatin.
The two daughter DNA duplexes are rapidly assembled into chro-
matin in a stepwise manner from a mixture of old histones and de novo
synthesized histones. H3-H4 tetramers (large yellow-coloured ovals)
are deposited first, followed by two H2A-H2B dimers (small cream-
coloured ovals) to complete the nucleosome. The newly synthesized H3
and H4 proteins have a specific conserved pattern of acetylation in the
cell (indicated by ÔAcÕ) and are deacetylated soon after nucleosome
formation. Also indicated are chromatin assembly factors that have
been identified biochemically and may function to mediate chromatin
assembly in vivo: histone chaperones for newly synthesized H3 and H4
(ASF1 and CAF-1) and for H2A-H2B (NAP-1). ATP-hydrolysis is
required for chromatin assembly, and may reflect a role for the ATP-

personal communication).
It is interesting to note that budding yeast can tolerate
loss of individual histone chaperones much more than
higher eukaryotes. For example, whereas ASF1 mutants
are viable in yeast, ASF1 is an essential gene product in
Drosophila (Y. Moshkin, J. Kenisson & F. Karch,
Department of Zoology and Animal Biology, University
of Geneva, Geneva, Switzerland, personal communica-
tion). The ability of yeast to survive without ASF1 may
reflect the fact that yeast has a more open chromatin
structure than higher eukaryotes, which in turn may be
related to the subtle destabilization of the structure of the
yeast core nucleosome particle as compared to that of
metazoans [4]. In addition, it suggests that higher eukar-
yotes may be subject to extra levels of regulation of their
nuclear functions by chromatin structure.
DNA REPAIR MAY REQUIRE HISTONE
CHAPERONES
DNA repair is the second major site of DNA synthesis in
the cell, and it may also entail concomitant chromatin
assembly. Accordingly, CAF-1 has been implicated in the
repair of UV-induced DNA damage. Yeast disrupted for
CAF-1 function are sensitive to UV irradiation [58].
Furthermore, CAF-1 stimulates chromatin assembly fol-
lowing nucleotide excision repair in vitro [59] and is recruited
to chromatin following UV irradiation in vivo [60]. Pheno-
typic analyses of yeast with disrupted ASF1 have indicated
that ASF1 also plays a role in the response to UV
irradiation that is apparent in the absence of CAF-1 [42].
More strikingly, budding yeast with disrupted ASF1 are

tates the formation of nucleosomes and their regular
spacing along the DNA. However, many questions remain
unanswered. For example, what is the identity of the other
histone chaperone(s) for H3-H4, H2A-H2B and H1 in the
cell? How do histone chaperones and ATP-remodeling
machines function together? How are specific chromatin
structures re-established onto the daughter DNA duplexes
so as to maintain patterns of programmed gene expression
through multiple generations? What is the role of histone
chaperones in DNA repair? The answers to these questions
will provide important insight not only into the mechanism
of chromatin assembly, but also insight into the influence of
Fig. 4. Model for the regulation of ASF1 by the DNA damage check-
point machinery. (A)Rad53isboundtoASF1intheabsenceofan
activated DNA damage checkpoint, preventing ASF1 from binding
histones and assembling chromatin. (B) Following activation of the
DNA damage checkpoint, Rad53 becomes phosphorylated. ASF1 is
released from the phosphorylated Rad53, where it can then bind to
histones and assemble chromatin.
2272 J. K. Tyler (Eur. J. Biochem. 269) Ó FEBS 2002
chromatin assembly on gene expression, DNA replication,
repair, recombination, growth and development.
ACKNOWLEDGEMENTS
I would like to thank F. Karch and R. Kamakaka for sharing results
prior to publication. I am highly grateful to Les Krushel, Josh Ramey,
Jeff Linger and Susan Howar for critical reading of this manuscript.
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