Chz1, a Nuclear Chaperone for Histone H2AZ
Ed Luk,1,* Ngoc-Diep Vu,1Kem Patteson,2Gaku Mizuguchi,1Wei-Hua Wu,1Anand Ranjan,1
Jonathon Backus,1Subhojit Sen,1Marc Lewis,2Yawen Bai,1and Carl Wu1,*
1Laboratory of Biochemistry and Molecular Biology, National Cancer Institute
2Division of Bioengineering and Physical Science, Office of Research Services, Office of the Director
National Institutes of Health, Bethesda, MD 20892, USA
*Correspondence: firstname.lastname@example.org (E.L.), email@example.com (C.W.)
The histone variant H2AZ marks nucleosomes
flanking the promoters of most genes of bud-
ding yeast. The incorporation of H2AZ into
chromatin is dependent on the SWR1 complex,
which catalyses the replacement of conven-
tional histone H2A with H2AZ. In cells, the
pool of unincorporated histone H2AZ has previ-
ously been found in association with Nap1,
a chaperone for conventional histone H2A-
H2B. Here, we report the discovery of Chz1,
a histone chaperone that has preference for
H2AZ and can also deliver a source of the his-
tone variant for SWR1-dependent histone re-
placement. Bacterially expressed Chz1 forms
a heterotrimer with H2AZ-H2B, stabilizing the
association of the histone dimer. We have iden-
tified a conserved motif important for histone
variant recognition within the H2AZ-interacting
domain of Chz1. The presence of this motif in
other metazoan proteins suggests that H2AZ-
specific chaperones may be widely conserved.
In eukaryotic cells, the packaging of DNA into chromatin
provides a dynamic system for controlling chromosome
processes such as transcription, DNA replication, repair,
and recombination. The fundamental repeating unit of
chromatin organization, the nucleosome, consists of al-
most two superhelical turns of DNA wrapped around an
octameric histone core made of two of each histone
eral, the bulk of chromatin is assembled from the major or
canonical class of histones, which are synthesized during
S phase. By contrast, minor nonallelic variant histones are
synthesized and incorporated into nucleosomes through-
out the cell cycle. These histone variants have important
functions in gene expression and chromosome metabo-
lism (Kamakaka and Biggins, 2005).
Outside of chromatin, both canonical and variant his-
in the soluble fraction of cell extracts. Histone chaperones
constitute an evolutionarily diverse group of acidic pro-
teins that playa keyrolein coordinating chromatin assem-
bly of the major histones and their variants (Polo and Al-
mouzni, 2006). Initially discovered in Xenopus eggs as
nucleoplasmin (Laskey et al., 1978), histone chaperones
physiological salt conditions, presumably by shielding the
strong electrostatic charges between the basic histones
and DNA phosphates, which would otherwise cause
aggregation. Despite the general property of charge neu-
tralization, there is some specificity in histone chaperone
interactions with H2A-H2B and H3-H4 histones. For ex-
ample, nucleoplasmin and Nap1 preferentially bind to
H2A-H2B, whereas specificity for H3-H4 is shown by
N1/N2, CAF1, HIRA/Hir, and Asf1 (Loyola and Almouzni,
Histone chaperones perform critical functions in a vari-
ety of physiological settings. CAF-1 is a key factor in the
assembly of nascent chromatin onto newly replicated
of the replication machinery (Moggs et al., 2000; Smith
and Stillman, 1989). CAF-1 and Asf1 function synergisti-
cally in chromatin assembly during DNA replication and
repair (Gaillard et al., 1996; Linger and Tyler, 2005; Mello
et al., 2002; Tyler et al., 1999). In addition, histone chaper-
ones also have critical functions in replication-indepen-
dent histone transactions that are associated with tran-
scription. As such, Asf1 has also been implicated in
nonreplicative histone disassembly and reassembly asso-
ciated with promoter and transcribed regions (Adkins
et al., 2004; Korber et al., 2006; Schwabish and Struhl,
2006). Another chaperone, FACT, mediates H2A-H2B
eviction and reassembly during RNA polymerase elonga-
tion through nucleosomes (Orphanides et al., 1999;
sively complexed with the histone H3.3 variant that is
implicated in histone replacement during transcription
(Ahmad and Henikoff, 2002; Tagami et al., 2004). Interest-
ingly, a number of multicomponent ATP-dependent chro-
matin remodeling complexes of the SWI/SNF superfamily
contain integral subunits that have histone chaperone-like
properties, suggesting the participation of these compo-
nents in mediating histone transactions during chromatin
remodeling (Polo and Almouzni, 2006).
Our laboratory has been investigating the deposition
pathway of the Saccharomyces cerevisiae histone H2AZ
Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc. 357
variant (Htz1), which isincorporated into one or two nucle-
osomes flanking nucleosome-free promoters throughout
the yeast genome (Guillemette et al., 2005; Li et al.,
2005; Raisner et al., 2005; Zhang et al., 2005). The site-
specific incorporation of Htz1 in vivo is dependent on
the SWR1 chromatin remodeling complex (Korber and
Horz, 2004), which catalyzes the in vitro replacement of
Htz1 on nucleosome arrays in an ATP-dependent manner
(Mizuguchi et al., 2003). It has been previously shown that
a fraction of unincorporated Htz1-H2B dimers in cell ex-
tracts is complexed with Nap1, a well-known chaperone
for H2A-H2B (Kobor et al., 2004; Mizuguchi et al., 2003),
and the Nap1-Htz1-H2B complex is capable of providing
Htz1 for SWR1-mediated histone replacement in vitro
(Mizuguchi et al., 2003). Nap1 has also been shown to in-
teract with the recombinant H2AZ-H2B dimer and facili-
tate its noncatalytic exchange in vitro (Park et al., 2005).
However, a histone chaperone with preference for
H2AZ has not been identified. Here, we report the discov-
ery of a chaperone for the yeast Htz1 that we have termed
Chz1. We show that the functions of Chz1 overlap with
Nap1 and a minor group of Htz1-H2B chaperones. We
have characterized the Chz1-Htz1-H2B complex by
biophysical techniques and identified the key region of
Chz1 that specifies recognition of the Htz1 variant. The
presence of this region in the human HIRIP3 protein raises
the possibility that H2AZ-specific chaperones could be
Yer030w (Chz1) Is a Major Associating Factor
of Htz1-H2B Dimers
In the course of purifying soluble epitope-tagged Htz1-
H2B (Htz1FLAG-H2B) dimers from yeast whole-cell ex-
tracts, we consistently observed a major ?35 kDa protein
that cosedimented with Htz1FLAG-H2B in the light frac-
tions of a glycerol gradient (Figure 1A, left, lanes 2–4).
Interestingly, this 35 kDa polypeptide was not previously
detected in glycerol gradient fractions of Htz1FLAG-H2B
preparations purified in an identical manner (Mizuguchi
et al., 2003). However, consistent with previous findings,
the SWR1 chromatin remodeling complex and the Nap1
histone chaperone were observed to cosediment with
(Figure 1A, left, and Figure S1 in the Supplemental Data
available with this article online). We traced the source
of the discrepancy to different protein-staining proce-
dures—Coomassie blue staining was used in the present
study, whereas our previous work relied on a silver-
staining protocol described in Wray et al. (1981). The
underrepresentation of the 35 kDa polypeptide by the
ysis of immunopurified Htz1FLAGcomplexes before or
after additional fractionation on a glycerol gradient (Fig-
ure 1B and Figure 1A, right).
To identify the 35 kDa associating protein, we excised
the corresponding gel bandand subjected itto in-gel tryp-
sin digestion and mass spectrometry. The analysis clearly
revealed anuncharacterized yeastproteinencodedbythe
ORF YER030w (70% coverage from peptide spectra;
Figure 1C), in addition to the comigrating Yaf9 and Swc6
subunits of the SWR1 complex. To confirm the identity,
we immunopurified Htz1FLAGfrom a YER030w deletion
strain (yer030wD) and showed by SDS-PAGE and Coo-
massie blue staining that the polypeptide disappeared
from the collection of Htz1FLAG-associated polypeptides
(Figure 1D). In agreement with a previous global localiza-
tion study (Huh et al., 2003), immunofluorescence micros-
copy showed that the bulk of Chz1 resides in the cell
nucleus (Figure S2). We conclude that YER030w encodes
the 35 kDa factor, and have named it Chz1 (chaperone for
Chz1 Preferentially Associates with Htz1-H2B
in a Complex
Toprovideadditional evidencefortheinteraction between
Chz1 and Htz1-H2B, we raised polyclonal antibodies
against Chz1 for immunoprecipitation of native Chz1
from the soluble fraction of whole-cell extracts. As shown
by Western blotting, a-Chz1 antibodies were able to pull
down histones Htz1FLAGand H2B from extracts of wild-
type, but not from chz1D mutant strains (Figure 2A). To
detect the full range of proteins associated with Chz1,
we analyzed immunoprecipitates by SDS-PAGE and
Coomassie blue staining and observed only four specific
polypeptides after subtraction of proteins from mock pu-
rification—Chz1 itself and histones Htz1FLAG, H2B, and
H2A, but not H3 and H4 (Figures 2B and 2C). Interestingly,
Chz1 is associated preferentially with Htz1FLAGover H2A
(Figure 2C). To further investigate this preferential binding,
we immunoprecipitated Htz1FLAGand Hta1FLAG(H2AFLAG)
from yeast extracts in parallel experiments with a-FLAG
antibodies and compared the amount of copurifying
Chz1. The results confirmed that native Chz1 is preferen-
tially associated with histone Htz1 over Hta1 (Figure 2D).
Furthermore, with the use of an in vitro binding assay,
we found that the Htz1-H2B dimer exhibits a more robust
interaction (persisting at higher ionic strength) with a
recombinant protein A-Chz1 fusion polypeptide than
H2A-H2B (Figure 2E).
Chz1 and Nap1 Are Major, Redundant Htz1-H2B
The discovery of the interaction between Chz1 and Htz1-
H2B led us to examine its functional relationship to Nap1,
a well-defined histone chaperone that binds to both his-
tone H2A-H2B and Htz1-H2B dimers (Ishimi et al., 1983;
Ito et al., 1996; Mizuguchi et al., 2003; Park and Luger,
2006). Accordingly, we analyzed the relative distribution
of Htz1-H2B between Chz1 and Nap1 and the resulting
changes when either histone chaperone was eliminated
by deletion of the corresponding gene. As shown by glyc-
erol gradient fractionation and SDS-PAGE, immunopuri-
fied Htz1FLAG-H2B from wild-type cells is distributed in
two main fractions of roughly equal abundance (fractions
358 Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc.
Chz1 Histone Chaperone Prefers H2AZ-H2B
3and 5), cosedimenting withthe peaks of Chz1 and Nap1,
respectively (Figure 3A). When purified from chz1D cells,
the peak of Htz1FLAG-H2B was lost from fraction 3,
whereas the peak in fraction 5 became dominant, cosedi-
menting with the peak of Nap1 (Figure 3B). When purified
from nap1D cells, the peak of Htz1FLAG-H2B was lost from
fraction 5, whereas the peak in fraction 3 became domi-
nant, cosedimenting with the peak of Chz1 (Figure 3C).
The results indicate that Chz1 and Nap1 can reciprocally
substitute for the binding to Htz1-H2B and suggest that
these two proteins have overlapping functions. In addition
to Chz1 and Nap1, SDS-PAGE also revealed other pro-
teins of lower abundance that cofractionated with
Little or no free Htz1-H2B could be detected in gradient
sediment (see below); this is especially evident in the gra-
dient profile of Htz1FLAGcomplexes purified from chz1D
Figure 1. Yer030w (Chz1) Is a Major Associating Factor of the Htz1-H2B Dimer in Yeast Extracts
(A) Glycerol gradient profiles of Htz1 complexes. Htz1FLAGcomplexes were immunopurified from soluble yeast whole-cell extracts with the use of M2
a-FLAG agarose (Sigma). Eluted proteins were sedimented through a 15%–40% glycerol gradient. Fractions analyzed by SDS-PAGE were stained
with Coomassie blue (left) or silver (right). Fractions containing Chz1, Nap1, and the SWR1 complex are indicated. Nap1 is not apparent in the silver-
stained gel (right), as the protein is faintly stained and underloaded (see also [B] and Figure S1).
(B) The 35 kDa protein is undetectable by silver staining. Soluble Htz1FLAGcomplexes were immunopurified as in (A). Eluted proteins were analyzed
directly by SDS-PAGE and stained with silver (left) or Coomassie blue (right). Htz1-assoicated proteins identified by mass spectrometry are indicated
(Mizuguchi et al., 2003). Yer030w was previously identified by mass spectrometry of the mixture of Htz1-associated proteins but was not assigned to
a specific protein band (Mizuguchi et al., 2003).
(C) Proteins around the 35 kDa region were identified by excision of the gel band and microcapillary reverse-phase HPLC nano-electrospray tandem
mass spectrometry (mLC/MS/MS). Number of MS/MS spectra and percentage of peptide coverage are indicated.
(D)The35kDaband isYer030w(Chz1).Htz1complexes fromwild-type(WT)oryer030wD(chz1D)strainswerepurifiedasin(A)and analyzedbySDS-
PAGE and Coomassie blue staining. The asterisk (*) indicates a proteolytic product of Nap1.
The following yeast strains were used: (A) 1703 [HTZ1FLAG], (B) MBY149, (D) BY4741 [HTZ1FLAG], and 162 [HTZ1FLAG].
Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc. 359
Chz1 Histone Chaperone Prefers H2AZ-H2B
cells (Figure 3B). The results indicate that most, if not all,
unincorporated Htz1-H2B dimers are associated with his-
tone chaperones in vivo, consistent with a prior study of
mammalian cytosolic H2A-H2B complexes (Chang et al.,
In the Absence of Both Chz1 and Nap1, Other
Htz1-H2B Binding Proteins Substitute
To explore the native state of Htz1FLAG-H2B dimers in the
absence of both Chz1 and Nap1, we constructed a viable
mutant strain in which both the CHZ1 and NAP1 genes
were deleted. Soluble Htz1FLAGcomplexes were immuno-
purified from the chz1Dnap1D mutant and fractionated by
glycerol gradient centrifugation. A number of distinct pro-
teins were observed to cosediment with Htz1FLAG-H2B in
gradient fractions 3–9, as revealed by SDS-PAGE and
Coomassie blue staining (Figure 4A, bottom). To deter-
minethe identity of theseHtz1binding factors, weexcised
the major protein bands from the polyacrylamide gel and
performed peptide sequencing withthe use of mass spec-
trometry (Figure 4A, top, boxes I–III; Table S1). Box I con-
tained Spt16, the large subunit of the FACT complex,
which mediates histone exchange during transcription
2004), Kap114, a major nuclear importin/karyopherin for
thehistoneH2A-H2B dimer(Mosammaparastetal., 2001),
and the Isw1 and Ioc3 subunits of the ISW1a complex
(Vary et al., 2003) (Figure 4C). Box II contained Pob3, the
other subunit of FACT. Box III contained the peptidyl prolyl
been shown to possess histone chaperone activity (Kuzu-
hara and Horikoshi, 2004). Most of these proteins were
that copurified with Htz1FLAGwithout further size fraction-
ation (Table S1). Interestingly, whereas FACT and Kap114
could be detected in samples purified from both wild-type
and nap1Dchz1D cells, Fpr3 and Fpr4 were detected only
in the absence of Nap1 and Chz1, suggesting that this
association is conditional. None of the aforementioned
polypeptides were detectable when an untagged Htz1
strain was used for a mock purification (data not shown).
The SWR1 Complex Can Utilize Multiple
Chaperone-Bound Htz1-H2B Complexes
for Histone Replacement
Previously, we reported that the SWR1 complex catalyzes
with Htz1 in vitro (Mizuguchi et al., 2003). In those
experiments, Htz1 was supplied by a Nap1-associated
fraction of Htz1-H2B or by a ‘‘free’’ Htz1-H2B fraction,
now revised to be the Chz1-Htz1-H2B complex. We con-
firmed that a glycerol gradient fraction highly enriched
for the native Chz1-Htz1FLAG-H2B complex can indeed
provide a source of Htz1 for histone replacement, as
Figure 2. Chz1 Binds to Htz1-H2B and H2A-H2B in Yeast Extracts
(A) a-Chz1 antibodies immunoprecipitate Htz1 and H2B. a-Chz1 antibodies immobilized on protein-A agarose were used to pull down Chz1-inter-
acting proteins in whole-cell extracts prepared from wild-type and chz1D strains. Immunoprecipitated proteins (right) and 4% input (left) were ana-
lyzed by western blotting with a-FLAG (top) or a-H2B (bottom) antibodies.
(Band C) Chz1associateswithHtz1, H2B, and H2A. Proteins immunoprecipitatedas in(A) were analyzed bySDS-PAGEand stained withCoomassie
blue. Arrows indicate Chz1 and core histones. IgGHand IgGL, IgG heavy and light chains, respectively; histones, recombinant yeast core histones
from reconstituted octamers. (C) Histone section of lane 2 in (B). Band intensities were analyzed by Image Quant (GE Healthcare) and plotted.
(D) Htz1preferentialassociates withChz1 incell extracts. a-FLAG pull-down of extracts prepared from yeast strains expressingHtz1FLAGor Hta1FLAG
(as the sole source of the histone). Eluted proteins were fractionated on a 15%–40% glycerol gradient. The fraction (#3) highly enriched for Chz1 was
analyzed by SDS-PAGE and stained with Coomassie blue. Z and A indicate pull-down of Htz1FLAGand Hta1FLAG, respectively.
(E) Chz1 exhibits a more robust interaction with dimers of Htz1-H2B in high salt. Bacterially expressed Chz1 fused to an N-terminal His6-protein A tag
was immobilized on IgG beads (at 0.3 mM of protA-Chz1 fusion) and incubated with reconstituted dimers of Htz1-H2B or H2A-H2B (0.05 mM). Bead-
bound complexes were washed with either 0.4 M or 0.5 M NaCl. Proteins were eluted with SDS and analyzed by SDS-PAGE and Coomassie blue
The following strains were used: (A and B) BY4741 [HTZ1FLAG] and 162 [HTZ1FLAG], and (D) MBY197b and MBY227.
360 Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc.
Chz1 Histone Chaperone Prefers H2AZ-H2B
shownbytheATP-dependent transferof Htz1FLAGto aca-
nonical nucleosome array attached to magnetic beads
(Figure 4B and Figure S3). In addition, a fraction highly en-
riched for FACT-Htz1FLAG-H2B (glycerol gradient fraction
7) could also provide a source of Htz1FLAGfor the SWR1-
mediated replacement of histone H2A (Figure 4B and
can function effectively in delivering Htz1 (presumably as
tin remodeling machinery. As might be anticipated by
these findings, individual chz1D and nap1D mutants and
even the nap1Dchz1D double mutant are capable of site-
specific deposition of Htz1 into chromatin, as shown by
ChIP analysis (Figure S4). Interestingly, unlike the nap1D
strain, the chz1D mutant exhibited weak sensitivity to
MMS (Begley et al., 2002) and benomyl, but not caffeine
(Figure S5), indicating that some functions of Chz1 cannot
of the Chz1-Htz1-H2B Complex
Chz1 has a predicted molecular weight of 18 kDa but mi-
grates as a 35 kDa protein when analyzed by SDS-PAGE.
The aberrant electrophoretic mobility is more likely due to
its amino acid composition (pI 4.5) than to posttrans-
lational modifications, because bacterially expressed
Chz1 displayed the same mobility when analyzed by
SDS-PAGE. To characterize the Chz1-Htz1-H2B complex
by biochemical and biophysical methods, we reconsti-
tuted a recombinant Chz1-Htz1-H2B complex from bac-
terially expressed proteins. The recombinant Chz1-Htz1-
H2B complex was found to sediment in the same glycerol
gradient fractions as the native complex (Figure 5A, right,
fractions 2 and 3); by contrast, the recombinant Htz1-H2B
dimer was observed to sediment in lighter fractions
(Figure 5A, left, fractions 1 and 2). A reverse-phase
HPLC analysis of the recombinant Chz1-Htz1-H2B com-
plex yielded three absorbance peaks at 214 nm, whose
peak areas gave a calculated molar ratio of 0.9 Chz1 to
1.0 Htz1-H2B dimer (Figure 5B). This value is consistent
with the relative molarity of the Chz1, Htz1, and H2B
bands (0.95:1.0:1.1) in the fractionated complex, as re-
vealed by SDS-PAGE and Coomassie blue staining
(Figure 5A). Taken together, the results strongly suggest
that the Chz1-Htz1-H2B complex iscomposed of an equi-
phy showed that recombinant Chz1-Htz1-H2B migrates
as a single peak, indicative of a homogeneous complex
Htz1-H2B was functional as a source of Htz1 for ATP-
dependent histone replacement by the SWR1 complex
(Figure 5C, center). Interestingly, we also detect activity
when recombinant dimer of Htz1-H2B was used in the
SWR1 in vitro assay (Figure 5C, right).
of the Chz1-Htz1-H2B Complex
The 1:1:1 stoichiometry of the Chz1-Htz1-H2B complex
does not provide insight into its oligomeric state, as it is
compatible with a complex composed of one or multiples
of one heterotrimer. To determine the oligomeric state of
the complex, we performed equilibrium sedimentation ex-
periments with the use of analytical ultracentrifugation.
Analysis of the sedimentation behavior clearly indicated
that the Chz1-Htz1-H2B complex is best described as
a heterotrimer rather than a hexamer or a larger oligomer
(Figure 5D). We also examined the secondary structures
and the thermostability of Chz1 alone, Htz1-H2B dimers,
and the Chz1-Htz1-H2Bcomplex byusing circular dichro-
ism (CD). Interestingly, Chz1 alone yielded a typical CD
spectrum characteristic of an unfolded polypeptide chain
(Figure 6A),suggesting that Chz1 isunfolded in physiolog-
ical conditions, whereas both Htz1-H2B and Chz1-Htz1-
H2B showed similar CD spectra with double minima at
208 and 222 nm (Figures 6A and 6B), indicative of the ex-
istence of a-helical structures. When the measured CD
Figure 3. Chz1 and Nap1 Are Major, Redundant Interacting
Proteins for Htz1-H2B Dimers
(A–C) Glycerol gradient profiles of Htz1FLAG-associating polypeptides
in wild-type (A), chz1D (B), and nap1D (C) strains. Htz1FLAG-associated
complexes were purified and sedimented through a glycerol gradient
as in Figure 1A, and gradient fractions were analyzed by SDS-PAGE
and Coomassie blue staining. Arrowheads and bracket indicate peak
fractions of Htz1 in association with Chz1, Nap1, and the SWR1
The following strains were used: (A) BY4741 [HTZ1FLAG], (B) 162
[HTZ1FLAG], and (C) 5119 [HTZ1FLAG].
Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc. 361
Chz1 Histone Chaperone Prefers H2AZ-H2B
spectrum of Chz1-Htz1-H2B is compared to the sum of
the CD spectra of Chz1 and Htz1-H2B in Figure 6A, the
former exhibits lower minima, suggesting that Chz1 forms
some a-helical structures when it binds to Htz1-H2B
This conclusion was further supported by the fact that
Chz1 alone showed no cooperative transition as the CD
signal at 222 nm was monitored as a function of tempera-
tive unfolding with a Tmof ?50?C (Figure 6C). Moreover,
the melting temperature of the Chz1-Htz1-H2B complex
was ?15?C higher than the expected value for a mixture
of Chz1 and Htz1-H2B, assuming there were no inter-
actions between the two (Figure 6D). Taken together, the
results indicate that the binding of Chz1 to Htz1-H2B
enhances the stability of the complex.
A Highly Conserved Motif in Chz1 Is Important
for Interaction with Htz1-H2B
We identified a number of fungal homologs of Chz1 by
a PSI-BLAST search of the nonredundant protein data-
base (Altschul et al., 1997) using the full-length Chz1
protein sequence. Alignment of the predicted protein se-
quences showed that the N- and C termini of Chz1 family
members are relatively divergent but do contain similar
acidic stretches rich in Glu/Asp residues. A highly con-
we designate as the CHZ motif, contains invariant
charged, polar, and hydrophobic residues. We further
queried the protein database by using the conserved
CHZ motif and identified a family of metazoan proteins
sequence similarity to the CHZ motif (Figure 7A and
Figure S6). The most prominent of these is human and
vertebrate HIRIP3, which contains a CHZ motif near the
C terminus (Figure S6). HIRIP3 was previously identified
in a yeast two-hybrid screen as a protein interacting with
HIRA, the H3.3 chaperone (Tagami et al., 2004), and has
translated histones H3 and H2B (Lorain et al., 1998).
To investigate the role of the CHZ motif in binding to
Htz1-H2B in vivo, we transformed a chz1D strain with
a mutant chz1 in which Asp103 and Asn106, two invariant
The binding of mutant chz1 with Htz1FLAGwas then ana-
lyzed by a pull-down assay with a-FLAG antibodies. As
shown by western blotting, alanine substitution of the
two invariant residues in the CHZ motif substantially re-
duced binding of the mutant chz1 to Htz1FLAG(Figure 7B),
indicating that these residues are important for the inter-
action between Chz1 and Htz1. To further determine
which regions of the Chz1 polypeptide were required for
the recognition of Htz1, we expressed defined segments
of the Chz1 polypeptide as protein A-tagged fusions and
examined their binding to the Htz1-H2B dimer (Figure 7C).
As shown by SDS-PAGE and Coomassie blue staining,
residues 74–128 of Chz1 showed strong interaction with
Htz1-H2B, whereas residues 1–73 and 129–160 did not
(Figure 7D). This 55 residue region closely overlaps with
the conserved CHZ motif and is evidently sufficient for
specifying the association between Chz1 and the Htz1-
Figure 4. In the Absence of Chz1 and
Nap1, Other Histone Binding Factors
(A) Glycerol gradient profiles of Htz1-associat-
ing proteins in a chz1Dnap1D strain. Htz1-as-
sociated polypeptides were fractionated in
blue as described in Figure 1A. The top panel is
a magnified region of the upper part of lanes 4–
7. Boxes I–III indicate gel regions excised for
peptide sequencing analysis. Box I contains
Spt16, Kap114, Isw1, and Ioc3. Box II contains
Pob3, as well as the Hsp70 molecular chaper-
one proteins Ssa1, Ssb2, and Ssa2. Box III
contains Fpr3 and Fpr4.
H2B complex provides a source of Htz1-H2B
for SWR1-mediated histone exchange. Canon-
ical nucleosomal arrays assembled in vitro as
described in Mizuguchi et al. (2003) were incu-
H2B ([A]; gradient fraction 7) with or without
ATP. The arrays were washed with 0.4 M KCl,
and Htz1 incorporated into chromatin was
eluted with SDS and detected by western blot-
ting using a-Htz1 antibodies. Left lanes repre-
sent 50% of chaperoned Htz1-H2B input
used in the exchange reaction.
The strain yEL002 [HTZ1FLAG] was used in (A).
362 Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc.
Chz1 Histone Chaperone Prefers H2AZ-H2B
Previous studies of histone chaperones have demon-
strated some specificity in the association between
histone chaperones and the major classes of histones.
Recent advances have shown that this specificity extends
to the minor histone variants as well. In particular, it has
been demonstrated that the transcription-related histone
H3.3 variant in mammalian cells is associated exclusively
with the HIRA complex, whereas the major H3 histone
associated with S phase is found in complex with CAF-1
(Tagami et al., 2004). Our studies of Chz1 introduce the
first example of a histone chaperone that has preference
for a variant of the lysine-rich histones and raise the pos-
sibility that histone chaperones dedicated to other vari-
ants of the H2A family remain to be discovered.
Unlike the near exclusivity of the interaction between
histone H3.3 and HIRA, Chz1 also displays some interac-
tions with histone H2A-H2B dimers. Conversely, Nap1,
a well-studied chaperone for conventional H2A-H2B, is
Chz1 and Nap1, the two main chaperones for Htz1-H2B,
have overlapping functions in binding to the variant his-
tone dimer. Indeed, by biochemical analysis of histone
chaperone complexes purified from mutant strains, we
found that Nap1 and Chz1 could substitute for each other
Figure 5. Bacterially Expressed Chz1, Htz1, and H2B Recon-
stitute a Functional Heterotrimer
(A) Reconstituted Htz1-H2B (left) and Chz1-Htz1-H2B (right) com-
plexes were sedimented through a 15%–40% glycerol gradient, and
fractions 1–5 were analyzed by SDS-PAGE and Coomassie blue stain-
ing as in Figure 1A. (No protein was present beyond fraction 5.) The
calculatedmolar ratio of Chz1:Htz1:H2B basedon Coomassiestaining
(B) Elution of the Chz1-Htz1-H2B complex on an analytical reverse-
phase HPLC Protein-PR column. Absorbance at 214 nm was repre-
sented as a function of elution time (minutes). The first peak was an
artifact of sample injection. The Chz1, Htz1, and H2B peaks were
identified based on comparison with elution of the individual protein.
The calculated molar ratio of Chz1:Htz1:H2B based on peak areas is
(C) The recombinant Chz1-Htz1-H2B complex provides a source of
Htz1-H2B for SWR1-mediated histone exchange. Canonical nucleo-
somal arrays assembled in vitro were incubated with purified SWR1
complex and recombinant (Rec.) or native Htz1 in the presence or
absence of ATP. Nucleosomes were washed with 0.4 M KCl, and the
incorporated Htz1 was eluted with SDS and detected by western blot-
ting with a-Htz1 antibodies.
(D) Chz1, Htz1, and H2B form a heterotrimer. L-1 (robust) regression fit
of an analytical ultracentrifugal equilibrium experiment for the reduced
mass of a complex containing the subunits Chz1, Htz1, and H2B, with
a 1:1:1 stoichiometry at 4.0?C and 14,000 rev/min. Line I corresponds
to a trimeric (Chz1-Htz1-H2B)1complex and line II to a hexameric
(Chz1-Htz1-H2B)2complex. The compositional partial specific vol-
umes were 0.69989, 0.74175, and 0.73054 cm3/g, respectively. A
solvent density of 1.03555 g/cm3at 4.0?C gave reduced masses of
5344.7, 3574.6, and 3728.2 Da, respectively, giving a total reduced
mass for a trimeric 1:1:1 complex of 12,647 Da. The experimentally
obtained value was 12,833 Da. Bottom panel shows the residuals of
Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc. 363
Chz1 Histone Chaperone Prefers H2AZ-H2B
in binding to the Htz1-H2B dimer. Interestingly, in mutants
deficient for both Chz1 and Nap1, Htz1-H2B binding is as-
sumed by a variety of known histone chaperones or his-
tone interacting factors—Kap114, FACT, Fpr3, Fpr4, and
Isw1—that normally appear to have little involvement in
the Htz1 deposition pathway. The unimpaired deposition
of Htz1 into chromatin observed in chz1D and nap1D mu-
tants is consistent with these findings. The identified inter-
actions with FACT and Isw1 suggest that these chromatin
factors, which are likely to have interactions with Htz1
subsequent to its incorporation into chromatin, may be
utilized for a predeposition function on demand. The exis-
tence of multiple chaperones for the Htz1-H2B dimer un-
derscores the highly redundant nature of this aspect of
Htz1 biology. The association with molecular chaperones
of the Hsp70 family was also observed, although the sig-
nificance of these interactions remains to be determined.
Notwithstanding redundancies, the functional overlap
between Chz1 and the other histone chaperones is likely
incomplete. Unlike Chz1, Nap1 resides in both the cyto-
plasm and the nucleus as a nucleocytoplasmic shuttling
protein (Ito et al., 1996; Miyaji-Yamaguchi et al., 2003).
Nap1 also interacts with the H2A-H2B nuclear karyo-
pherin/importin Kap114 to regulate histone nuclear import
(Mosammaparast et al., 2005). Moreover, although viable,
the mutant chz1D strain does exhibit a weak sensitivity to
MMS and benomyl, which is not shared by the nap1D
strain, indicating that there may be some unique (perhaps
locus-specific) functions of Chz1 thatcannot be fullycom-
pensated for by the substitution of Nap1 or any other his-
tone chaperone. It would be of interest to explore this
question further by comparing the genome-wide deposi-
tion of Htz1 in chz1D and nap1D strains.
Chz1 is unusual among histone chaperones in that it
lacks a folded structure when bacterially expressed in
the absence of the Htz1-H2B dimer. In contrast, other his-
tone chaperones, such as nucleoplasmin (Dutta et al.,
2001; Namboodiri et al., 2003), Asf1 (Daganzo et al.,
2003), and Nap1 (Park and Luger, 2006), assume folded
structures in the absence of histones. The functional sig-
nificance of this distinction in folding pathways is unclear.
Nevertheless, we have found that the binding of Chz1 to
Htz1-H2B results in a substantial stabilization of the
Chz1-Htz1-H2B heterotrimer. This is relevant to previous
biophysical studies of H2AZ-H2B dimers, which appear
to form the least stable of the histone folds characterized
to date (Placek et al., 2005).
We have mapped the region necessary and sufficient
for specific Htz1-H2B binding to a 55 residue polypeptide
in the central portion of Chz1. This region contains
sequences (the CHZ motif) that are conserved in fungal
orthologs of Chz1. Alanine replacement of two invariant
residues of the CHZ motif in the native yeast Chz1 protein
reduces the binding to Htz1-H2B substantially, indicating
that this element contains the key determinants for Htz1
recognition in vivo. The conservation of the CHZ motif ex-
tends to a number of metazoan proteins, including human
HIRIP3, which was previously identified by a two-hybrid
assay (Lorain et al., 1998) as an interacting protein for
HIRA, the histone H3.3-specific chaperone (Tagami et al.
2004). HIRIP3 has been shown to bind to core histones
in vitro, but binding to H2AZ has not been investigated.
We speculate that HIRIP3 may have a special preference
for human H2AZ. In this context, the reported interactions
between HIRIP3 and HIRA present additional possibilities
for coordinated transactions involving histones H3.3 and
Figure 6. Biophysical Characterization
of Chz1, Htz1-H2B, and Chz1-Htz1-H2B
(A) CD spectra of Chz1 (10 mM) (gray) and Htz1-
H2B dimer (10 mM) (black).
(B) The sum of the CD spectra in (A) (gray) and
CD spectrum of native Chz1-Htz1-H2B trimer
(10 mM) (black).
(C and D) Normalized CD signal at 222 nm as
a function of temperature for Chz1 alone and
Htz1-H2B dimer in (C), and the sum of Chz1 +
(Htz1-H2B) and Chz1-Htz1-H2B trimer in (D).
364 Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc.
Chz1 Histone Chaperone Prefers H2AZ-H2B
H2AZ in the mammalian system beyond what we have
identified in yeast.
Which structural element(s) of Htz1 might specify its
recognition by Chz1? Previously, we have shown by do-
tant for specific binding to the SWR1 complex (Wu et al.,
2005). In another domain swap experiment, we have
found that the same C-terminal a helix of Htz1 was also
able to confer preferential binding to Chz1 by a chimeric
histone H2A in which all other sequences of H2A were re-
tained (Figure S7). Hence, Chz1 represents an additional
target for the defining element of histone Htz1, the
Figure 7. A Highly Conserved CHZ Motif Is Important for Htz1-H2B Binding
(A) Multiple sequence alignments of fungal Chz1 homologs and of CHZ motifs in higher eukaryotes (inset). Alignments were generated by ClustalW
(Thompson et al., 1994). Acidic residues are in blue, basic in purple, nonpolar in red, and polar in green. The purple bar highlights the CHZmotif that is
conserved from yeast to human. Accession number of the human HIRIP3 protein is CAA11275, mouse is NP_766334, fish is XP_702126, and Arabi-
dopsis is NP_192571.Arrows correspondto the positions of truncation constructsused in(B). Arrowheads indicate the sites mutated by site-directed
mutagenesis in(C).TheCHZ1 ORFhastwoin-frame ATGcodons nearthe50end.A bioinformatic studypredictedthedownstreamATG asaprobable
start codon (Zhang and Dietrich, 2005); however, mass spectrometry identified a peptide indicating that the upstream ATG codon is utilized.
(B) Htz1FLAGpull-down from extracts prepared from yeast expressing either wild-type Chz1 or chz1D103A,N106Amutant. Coimmunoprecipitation of
Chz1 was detected by western blotting with the use of a-Chz1 antibodies. Numbers indicate normalized band intensities.
(C) Constructs of Chz1 fused to a tandem His6-protein A tag at the N terminus. Green-red box represents the tandem His6-protein A tag, Chz1 is
yellow, and CHZ motif is purple.
(D)InvitroChz1 binding assay. Full-lengthand truncated Chz1 fusions wereused inabindingassay.Bacteriallyexpressedproteins wereimmobilized
on IgG beads, incubated with Htz1-H2B (left) or H2A-H2B (right),and washed before elution by SDS as in Figure 2E. Eluted proteins were analyzed by
SDS-PAGE and Coomassie blue staining. ‘‘*’’ and ‘‘?’’ indicate the positions of the Htz1-H2B and H2A-H2B polypeptides.
The following abbreviations were used in (A): S.c., Saccharmyces cereivisae; C.g., Candida glabrata; E.g., Eremothecium gossypii; K.l., Kluyveromy-
ces lactis; C.a., Candida albicans; N.c., Neurospora crassa; and S.p., Schizosaccharomyces pombe.
The strains yEL011 [pJB0200] and yEL011 [pEL0206] were used in (B).
Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc. 365
Chz1 Histone Chaperone Prefers H2AZ-H2B
corresponding region that was first shown to be function-
ally essential in Drosophila H2AZ (Clarkson et al., 1999).
The Chz1-Htz1-H2B heterotrimer and the previously
described Nap1-Htz1-H2B complex can be considered
to represent the beginning of the pathway for Htz1 depo-
sition in chromatin. We propose that Chz1 (and Nap1) de-
livers Htz1-H2B to the SWR1 complex, where the Swc2
subunit plays a key role in recruitment of Htz1 (Wu et al.,
2005). The observed Chz1 displacement when the Chz-
Htz1-H2B complex (previously described as Htz1-H2B
[Wu et al., 2005]) is bound to Swc2 is consistent with
this view (Figure S8). This transfer from Chz1 to Swc2
(possibly involving other components of the SWR1 com-
plex), before final delivery of Htz1-H2B to the (H3-H4)2
tetramer. Evidence showing that free Htz1-H2B dimer is
an active substrate for SWR1 (Figure 5C and Ruhl et al.
) suggests that the histone chaperone does not
directly participate in the SWR1-mediated replacement
reaction; however, it is possible that the histone chaper-
one is reutilized to capture the ejected H2A-H2B dimer.
How the H2A-H2B dimer becomes evicted remains un-
clear. We have previously suggested that dimer eviction
is likely to be mediated by the displacement of DNA by
the SWR1 ATPase and by interactions between compo-
nents of the SWR1 complex and nucleosomal histones
for continued investigation of the Htz1 replacement path-
way and offer insights into the roles of histone chaperones
in mediating the increasingly important histone transac-
tions that regulate gene expression.
Experimental procedures for yeast strains and plasmid constructions,
culture conditions, protein complex purification, and antibodies pro-
duction are given in the Supplemental Data.
Glycerol Gradient Fractionation
Immunopurified Htz1 complexes (0.4 mL) were sedimentated through
a 15%–40% glycerol gradient (4.7 mL)in 25 mM HEPES-KOH (pH 7.6),
1 mM EDTA, 0.01% NP-40, and 0.3 M KCl at 243,000 3 g (RCF, max)
for 20 hr. The fraction size of most experiments is 0.5 mL, except
Figure 4A is 0.4 mL.
Purification of Recombinant Proteins
Yeast Htz1 and H2B (Htb1) were expressed in E. coli and purified by
using methods as previously described (Luger et al., 1999) with an
additional step of reverse-phase chromatography (HPLC) on a Pro-
tein-RP column (Waters). The histones were eluted with a 0%–60%
acetonitrile gradient in the presence of 0.1% trifluoroacetic acid at
a flow rate of 10 mL/min. Chz1 was extracted from E. coli transformed
with pEL0201 (as described in the Supplemental Data) with buffer E
(10 mM Tris [pH 7.0], 100 mM NaCl, and 1 mM EDTA) in the presence
of protease inhibitors. The soluble protein fraction was precleared with
SP Sepharose HP (GE Healthcare) and applied to a Q Sepharose HP
column (GE Healthcare), which was then washed with buffer F (buffer
E + 2 mM b-mercaptoethanol). Chz1 was eluted with a 0.3–0.5 M NaCl
gradient in the same buffer conditions and further purified by HPLC on
a Protein-RP column. Htz1-H2B dimer was reconstituted as described
in Placek et al., (2005).
Reconstitution of Chz1-Htz1-H2B Complex
Two milligrams of lyophilized monomers of Htz1 and H2B was
unfolded in 6 M guanidine hydrochloride, 10 mM K2HPO4(pH 7.0),
1 mM EDTA, and 2 mM b-mercaptoethanol at room temperature.
Equal concentrations (?1 mg/mL) of Chz1 were subsequently mixed
with the histones for 30 min at room temperature before dialysis
against buffer C (25 mM K2HPO4[pH 7.0], 2 M KCl, 1 mM EDTA, and
2 mM b-mercaptoethanol). The protein was concentrated on a centri-
con YM-10 column (Millipore) and separated bya Sephadex 200 10/30
GL (GE Healthcare) column in buffer D (same as C except 0.15 M KCl
was used instead of 2 M KCl).
Biophysical Characterization of Chz1-Htz1-H2B Complex
The Chz1-Htz1-H2B complex eluted from the size exclusion column
(above) was analyzed by an analytical Protein-RP column with a flow
rate of 1 mL/min. Circular dichroism (CD) spectra of Chz1 monomer,
histone dimer, and Chz1-histone trimer were collected on a J-720
spectropolarimeter (Jasco). Heat melting of each species was per-
formed by monitoring the CD signal at 222 nm as a function of temper-
ature. The samples were analyzed in buffer D at 10 mM, using a cuvette
with a path length of 0.1 cm. For analytical ultracentrifugation, please
refer to the Supplemental Data.
cility by microcapillary reverse-phase HPLC nano-electrospray tan-
dem mass spectrometry (mLC/MS/MS) on a Finnigan LCQ DECA XP
Plus quadrupole ion trap mass spectrometer.
Nucleosomal arrays assembly and histone transfer assays were con-
ducted as described in Mizuguchi et al. (2003), except various chaper-
one-associated Htz1-H2B complexes were used.
For the Chz1 binding assay, N-terminal His6-protein A-tagged full-
length or truncated Chz1 fusion proteins were expressed from E. coli
and purified by using a Ni-NTA agarose column (Qiagen) under dena-
turing conditions. The fusion proteins were refolded by dialyzing
against HEGN-0.2N (25 mM HEPES-KOH [pH 7.6], 1 mM EDTA,
10% glycerol, 0.01% NP-40, 0.2 M NaCl, and 1% [vol/vol] protease in-
hibitor cocktail). The proteins (0.3–2.5 mM) were then incubated with
10 ml of IgG-Sepharose (GE Healthcare) in 1 ml of HEGN-0.1N buffer
(same as HEGN-0.2N except 0.1 M NaCl was used) for 1 hr at 4?C.
The washed beads were then incubated with 0.05 mM of Htz1-H2B
or H2A-H2B dimer for 2 hr in 1 ml of HEGN-0.1N at 4?C and washed
four times with 1 ml of HEGN-0.4N (same as HEGN-0.1N except
0.4 MNaCl wasused).Proteinswere eluted with30 mlof Laemmli sam-
ple buffer and analyzed by SDS-PAGE and Coomassie staining.
Supplemental Data include Supplemental Experimental Procedures,
Supplemental References, eight figures, and two tables and can be
found with this article online at http://www.molecule.org/cgi/content/
We thank M. Lichten for invaluable advice and especially for guidance
in the revision of this manuscript, X. Shen for initial studies of Htz1-
associated polypeptides, W. Lane for protein microsequencing, and
members of the Wu lab for critical review of the manuscript. This
Cancer Institute. E.L is a Leukemia and Lymphoma Society fellow.
Received: August 30, 2006
Revised: December 2, 2006
Accepted: December 21, 2006
Published: February 8, 2007
366 Molecular Cell 25, 357–368, February 9, 2007 ª2007 Elsevier Inc.
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