Cooperative action of TIP48 and TIP49 in H2A.Z exchange catalyzed by acetylation of nucleosomal H2A

Abstract

H2A.Z is an evolutionarily conserved H2A variant that plays a key role in the regulation of chromatin transcription. To understand
the molecular mechanism of H2A.Z exchange, we purified two distinct H2A.Z-interacting complexes termed the small and big complexes
from a human cell line. The big complex contains most components of the SRCAP chromatin remodeling and TIP60 HAT complexes,
whereas the small complex possesses only a subset of SRCAP and TIP60 subunits. Our exchange analysis revealed that both small
and big complexes enhance the incorporation of H2A.Z-H2B dimer into the nucleosome. In addition, TIP60-mediated acetylation
of nucleosomal H2A specifically facilitates the action of the small complex in the H2A.Z exchange reaction. Among factors
present in the small complex, we determined that TIP48 and TIP49 play a major role in catalyzing H2A acetylation-induced H2A.Z
exchange via their ATPase activities. Overall, our work uncovers the previously-unrecognized role of TIP48 and TIP49 in H2A.Z
exchange and a novel epigenetic mechanism controlling this process.

Full text

Published online 20 August 2009 Nucleic Acids Research, 2009, Vol. 37, No. 18 5993–6007
doi:10.1093/nar/gkp660
Cooperative action of TIP48 and TIP49 in H2A.Z
exchange catalyzed by acetylation of
nucleosomal H2A
Jongkyu Choi, Kyu Heo and Woojin An*
Department of Biochemistry and Molecular Biology, University of Southern California, Norris Comprehensive
Cancer Center, Los Angeles, CA 90033, USA
Received May 28, 2009; Revised July 21, 2009; Accepted July 24, 2009
ABSTRACT
H2A.Z is an evolutionarily conserved H2A variant
that plays a key role in the regulation of chromatin
transcription. To understand the molecular
mechanism of H2A.Z exchange, we purified two
distinct H2A.Z-interacting complexes termed the
small and big complexes from a human cell line.
The big complex contains most components of the
SRCAP chromatin remodeling and TIP60 HAT
complexes, whereas the small complex possesses
only a subset of SRCAP and TIP60 subunits. Our
exchange analysis revealed that both small and big
complexes enhance the incorporation of H2A.Z-H2B
dimer into the nucleosome. In addition, TIP60-
mediated acetylation of nucleosomal H2A speci-
fically facilitates the action of the small complex in
the H2A.Z exchange reaction. Among factors
present in the small complex, we determined that
TIP48 and TIP49 play a major role in catalyzing
H2A acetylation-induced H2A.Z exchange via their
ATPase activities. Overall, our work uncovers the
previously-unrecognized role of TIP48 and TIP49 in
H2A.Z exchange and a novel epigenetic mechanism
controlling this process.
INTRODUCTION
In eukaryotes, genomic DNA is packaged into a com-
plex nucleoprotein structure called chromatin. The
fundamental unit of chromatin is the nucleosome, which
is composed of 147 bp of DNA wrapped around a histone
octamer of two H2A-H2B heterodimers and a H3-H4
tetramer (1,2). Three major remodeling processes that
regulate DNA accessibility in this repressive chro-
matin state are post-translational modifications of his-
tones, ATP-dependent remodeling of chromatin, and
incorporation of histone variants into chromatin (3–5).
Recent studies demonstrated that histone variants play
an important role in regulating gene expression and
other DNA-templated cellular processes (6). H2A.Z is
one of the evolutionarily conserved H2A variant and
comprises about 5–10% of total cellular H2A (7).
H2A.Z is expressed and integrated into chromatin
independently of DNA replication and is essential for
viability in many organisms, such as Tetrahymena,
Drosophila, and mice (8–10).
The crystal structure of an H2A.Z-containing
nucleosome indicates that H2A.Z confers a destabilization
of interaction between the H2A.Z-H2B dimer and the
H3–H4 tetramer (11). In agreement with these results,
H2A.Z-containing nucleosomes exhibited more salt and
heat-induced destabilization than canonical nucleosomes
(12–14). However, more recent biophysical studies showed
that integration of H2A.Z into nucleosomes has a positive
effect on nucleosome stability (15,16). At the functional
level, ChIP-chip analyses across the yeast genome
indicated that H2A.Z is preferentially localized to specific
sites within the promoter regions (12,17,18). This
promoter localization of H2A.Z is remarkably specific,
and appears to establish unique promoter architecture
for transcription regulation (19–21). In this regard, a
repressive role of H2A.Z in transcription is supported by
its association with heterochromatin binding protein 1-a
(HP1a) (22). However, the promoter-localized H2A.Z has
also been implicated for gene activation by facilitating
chromatin remodeling and recruitment of transcriptional
machinery at gene promoter regions (23,24). A possible
role of H2A.Z in transcription is further supported by
the discovery that H2A.Z is dissociated from nucleosomes
upon initiation of gene transcription in yeast and human
cells (12,25).
Recent studies revealed that several chromatin
remodeling activities have an ability to catalyze the
replacement of H2A.Z for H2A within canonical
nucleosomes. In yeast, the SWR1 complex containing
swi2/snf2-related ATPase Swr1 has been shown to
incorporate H2A.Z-H2B dimers into nucleosomes in an
*To whom correspondence should be addressed. Tel: +1 323 442 4398; Fax: +1 323 442 4433; Email: woojinan@usc.edu
ßThe Author 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses?
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

ATP-dependent manner (26–28). A subsequent study
conducted in Drosophila showed that TIP60-mediated
acetylation of nucleosomes plays a role in replacement
of nucleosomal phospho-H2Av (the drosophila H2A.Z/
H2A.X homolog) with unmodified H2Av (29). So far,
two different activities were identified for H2A.Z exchange
in human cells. SRCAP complex was purified and shown
to include multiple subunits, one of which is Swr1-related
chromatin remodeling protein SRCAP (30). In vitro
exchange experiments indicated that the SRCAP complex
is able to exchange H2A with H2A.Z in the reconstituted
nucleosome (31). p400 is another human homologue of
yeast Swr1 found in human TIP60 complex, and shown
to have a catalytic activity for the exchange of H2A.Z into
the promoter regions of p53 target genes (32). The fact
that SRCAP and TIP60 complexes share several subunits
(e.g. TIP48, TIP49, Actin, YL1) supports the participation
of these components in regulating the H2A.Z exchange
process.
To further understand the molecular mechanisms that
regulate the incorporation of H2A.Z into the nucleosome,
we purified and characterized two H2A.Z-associated
complexes: the H2A.Z big complex containing most
SRCAP and TIP60 subunits and the H2A.Z small
complex containing only a subset of SRCAP and TIP60
subunits. In our exchange assays, we found that both
small and big complexes can moderately promote
the replacement of nucleosomal H2A with H2A.Z.
Significantly, TIP60-mediated acetylation of nucleosomal
H2A facilitates H2A.Z incorporation catalyzed by the
small complex, but not by the big complex. More
importantly, we show that TIP48 and TIP49 present in
the small complex are sufficient to recapitulate H2A.Z
exchange capabilities of the entire complex. These results
provide new insight into the role of TIP48/TIP49 ATPase
in the exchange of H2A.Z, which is facilitated by
TIP60-mediated H2A acetylation.
MATERIALS AND METHODS
Purification and identification of H2A.Z complexes
The establishment of HeLa cell lines that stably express
epitope-tagged H2A.Z (f/h:H2A.Z) and affinity
purification of the H2A.Z complex were conducted by
following essentially the same procedures as described in
our recent study (33). The affinity-purified H2A.Z
complex (0.3 ml) was further purified by 4.7 ml 15–40%
glycerol gradient sedimentation as described (26). The
purified H2A.Z complexes were identified by USC Mass
Spectrometry and Proteomics Core Facility. Experimental
details, including plasmid construction, cell line
establishment and protein purification/identification, can
be found in the Supplementary Data.
Preparation of recombinant histones and TIP48/TIP49
For the preparation of H2A.Z histone octamers and
dimers, H2A.Z cDNA was PCR-amplified and inserted
into the NdeI and BamHI sites of pET11a (for untagged
H2A.Z), pET15b (for His-tagged H2A.Z/h:H2A.Z) or
pET11d (for FLAG-tagged H2A.Z/f:H2A.Z). H2A.Z
proteins were expressed in bacteria (Rosetta
(DE3)pLysS, Novagen) and affinity-purified using Ni
+
-
NTA (for h:H2A.Z) and M2 agarose (for f:H2A.Z)
beads. For histone octamer preparation, untagged
H2A.Z and canonical H2B, H3 and H4 were prepared
as described (34,35), dissolved in 8 M guanidium solution
by rotating at room temperature and combined for
octameric reconstitutions by renaturation. For H2A.Z-
H2B dimer preparation, His-tagged or FLAG-tagged
H2A.Z was renatured together with H2B. The refolded
histones were passed through Sephacryl S300 column
(GE healthcare) to remove unreconstituted histones.
H2A–H2B and H2A.X-H2B dimers were prepared
essentially as described (34,36). To prepare recombinant
TIP48 and TIP49, full-length cDNA sequences encoding
human TIP48 and TIP49 were PCR amplified from 293T
cell mRNA and subcloned into the NdeI and BamHI sites
of pET11d together with a FLAG tag at their amino
termini. FLAG-tagged TIP48 (f:TIP48) and TIP49
(f:TIP49) were expressed in bacteria and purified using
M2 agarose beads. For reconstitution of TIP48/TIP49
complex, equal amounts of TIP48 and TIP49 were
mixed at BC150 binding buffer and further purified by
15–40% glycerol gradient sedimentation.
Reconstitution of mononucleosomes
Mononucleosomes were reconstituted with 50biotinylated
207 bp DNA fragments containing 601 nucleosome
positioning sequence (37,38) and either canonical or
H2A.Z-containing histone octamers by salt dialysis
method (39). The reconstituted nucleosomes were purified
by sedimentation in a 5–30% (vol/vol) glycerol gradient to
remove free DNA and core histones.
Histone acetyltransferase and ATPase assays
For histone acetyltransferase (HAT) assays, free histones
(1 mg) or reconstituted mononucleosomes (0.5 mg) were
incubated with the initial H2A.Z complex in the presence
of 2.8 mM[
3
H] acetyl-CoA or 10 mM cold acetyl-CoA and
analyzed by 15% SDS–PAGE and western blot analysis
(34). All antibodies used for western blot analysis were
from Millipore. ATPase assays were performed as
described previously (40) by using the purified H2A.Z
complexes (6 ml) or recombinant TIP48/TIP49 (150 ng) in
the presence of 0.05 mCi of [g-
32
P] ATP for 30 min at 308C.
Free phosphate and ATP were separated by 12%
polyacrylamide gel (19:1) containing 7 M urea in a 1
TBE buffer.
Histone exchange assays
Exchange assays were performed as recently described
(26,36) with minor modifications. Briefly, regular
mononucleosomes (150 ng DNA equivalents) immobilized
on streptavidin-conjugated Dynabeads M-280 (Dynal)
were incubated with H2A.Z-S or H2A.Z-B complex, in
exchange reaction buffer (25 mM HEPES, pH 7.6,
0.37 mM EDTA, 0.35 mM EGTA, 5 mM MgCl2, 1 mM
DTT, 70 mM KCl, 10% glycerol, 0.02% NP-40 and
0.1 mg/ml BSA) for 60 min at 308C in the absence or
presence of 1 mM ATP. Note that both H2A.Z-S and
5994 Nucleic Acids Research, 2009, Vol. 37, No. 18

H2A.Z-B complexes provide FLAG/HA-H2A.Z and H2B
for exchange reactions. Beads were concentrated on a
magnetic particle concentrator (Dynal) and washed three
times with washing buffer (25 mM HEPES, pH 7.6,
0.1 mM EDTA, 0.5 mM EGTA, 5 mM MgCl2, 1 mM
DTT, 70 mM KCl, 10% glycerol, 0.02% NP-40 and
0.1 mg/ml BSA). The supernatant was also collected
from the initial exchange reaction for analysis of free
unincorporated H2A.Z. Nucleosomal and free H2A.Z
proteins collected from the exchange reaction were
subjected to western blot analysis using anti-FLAG
antibody (Sigma). Histone exchange assays using the
acetylated nucleosomes were as described above, except
that nucleosomes were pre-acetylated by the initial
H2A.Z complex in HAT reaction buffer containing
cold acetyl-CoA (10 mM, Sigma) for 1 h before their
immobilization onto Dynabeads. Immobilized nucleo-
somes were washed extensively with 150 mM exchange
buffer (25 mM HEPES, pH 7.6, 0.37 mM EDTA,
0.35 mM EGTA, 5 mM MgCl2, 1 mM DTT, 150 mM
KCl, 10% glycerol, 0.02% NP-40 and 0.1 mg/ml BSA).
For exchange reactions, immobilized nucleosomes was
mixed with the TIP48/TIP49 complex (150 ng) in the
exchange reaction buffer for 20 min at 308C, and then
incubated with h:H2A.Z-H2B, h:H2A.X-H2B or f:H2A-
H2B dimers (400 ng each) for 60 min at 308C.
Nucleosomal proteins were analyzed by immunoblot
using anti-His (Novagen) and anti-FLAG (Sigma)
antibodies.
Chromatin isolation and mononucleosome
immunoprecipitation
Chromatin was purified from 293T cells as previously
described (41), but after expression of FLAG-tagged
H2A.Z (f:H2A.Z). For preparation of H2A.Z
mononucleosomes, nuclei of HeLa cells stably expressing
f/h:H2A.Z were first digested with micrococcal nuclease
(MNase) (0.6 U, Sigma), and preparation of mono-
nucleosome fractions was confirmed by 2% agarose gel
electrophoresis of nucleosomal DNA purified from the
MNase digestion reaction. To isolate mononucleosomes
containing f/h:H2A.Z, the entire mononucleosome
fraction was further subjected to immunoprecipitation
using M2-agarose beads. After overnight incubation,
beads were washed five times with BC300, and bead-
bound mononucleosomes were eluted from M2 agarose
by using FLAG peptide (200 ng/ml). The eluted mono-
nucleosomes were analyzed on 15% SDS-gel
electrophoresis.
TIP49 depletion and RT–qPCR
For TIP49 depletion, 293T cells were transfected with 25
nM of the validated TIP49 siRNA (Ambion, siRNA ID
#13702 or #13514) using siPORT NeoFX (Ambion) and
incubated for 96 h. The expression level of TIP49 in
siRNA-transfected cells was determined by western blot
analysis using anti-TIP49 antibody (Santa Cruz). The real
time quantitative PCR was performed using an iCycler iQ
Real-Time Detection System with the iQ SYBR Green
supermix (Bio-Rad). The primers used for qPCR are as
listed in the Supplementary Data (Supplementary
Table S1). The specificity of the amplification reactions
were monitored by melting curve analysis and sub-
sequently by agarose gel electrophoresis. The threshold
cycle (C
t
) value for each gene was normalized to the C
t
value for b-actin. All samples were run in triplicate. For
the analysis of histone incorporation on chromatin after
TIP49 knockdown, the cells were initially transfected
with TIP49 siRNA for 48 h and then retransfected
with plasmids expressing f:H2A.Z, f:H2A.X, or f:H2A
for another 48 h. Chromatin was isolated as described
above, and incorporation of ectopic histones was analyzed
by western blot analysis with anti-FLAG antibody.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) assays were
performed as described previously (42) with minor
modifications. See the Supplementary Data for exper-
imental details and primer sequences (Supplementary
Table S2).
RESULTS
Free H2A.Z stably associates with components of
SRCAP and TIP60 complexes
In an effort to enhance our understanding of H2A.Z
exchange machinery, we have generated a cell line
constitutively expressing the FLAG/HA-tagged version
of H2A.Z (Figure 1A). Ectopic H2A.Z was found to be
mainly localized in the nucleus as identified by immuno-
fluorescence analysis with an anti-FLAG antibody
(Supplementary Figure S1). Nuclear extracts were
prepared from the cell line and initially fractionated on
a phosphocellulose P11 column. The 1.0 M and 1.2 M
KCl fractions containing ectopic H2A.Z were combined
and further purified by M2-agarose affinity chroma-
tography under stringent conditions (300 mM KCl, 0.1%
Nonidet P-40) (Figure 1A). Coomassie blue stain analysis
of elution fractions revealed the association of ectopic
H2A.Z with 14 prominent bands that were not detectable
with control HeLa nuclear extract (Figure 1B, lane 2).
Mass spectrometric analysis of these 14 major bands
identified a number of proteins, most of which are
subunits of SRCAP chromatin remodeling and/or TIP60
HAT complexes as summarized in Figure 1B. We further
confirmed the mass spectrometry results by western blot
analysis using available antibodies (Figure 1C). Since
the H2A.Z complex contains TIP60, which has been
characterized as a HAT (43,44), we first asked whether
the H2A.Z complex has HAT activity. To exclude the
effect of any pre-existing modifications of native histones,
we prepared recombinant histone octamers after
expression of individual histones in bacteria for our
HAT assays (Supplementary Figure S2, lanes 1–5). We
found that the H2A.Z complex can acetylate H2A, H3
and H4 in H2A-containing histone octamers (Figure 1D,
lane 3) and H2A.Z, H3 and H4 in H2A.Z-containing
histone octamers (Figure 1F, lane 3). Since previous
studies with TIP60 detected the same substrate specificity
Nucleic Acids Research, 2009, Vol. 37, No. 18 5995

Figure 1. Purification of H2A.Z complex from human cells. (A) Schematic summary of purification of H2A.Z complex. Nuclear extracts from
H2A.Z-expressing cells were first fractionated by P11 cation exchange column with BC buffer as indicated. The P11 1.0 and 1.2 M KCl fractions
containing ectopic H2A.Z were combined and further purified with anti-FLAG affinity chromatography as described under ‘Materials and Methods’.
The purified proteins were separated in 4–20% gradient SDS–PAGE and subjected to western blot analysis with anti-FLAG and -HA antibodies.
Lane 1, mock purification with nuclear extracts prepared from regular HeLa cells; lane 2, FLAG/HA-H2A.Z immunoprecipitation with nuclear
5996 Nucleic Acids Research, 2009, Vol. 37, No. 18

(43,44), these results strongly support that TIP60 in the
complex is the responsible HAT for this modification.
To further analyze the ability of TIP60 in the H2A.Z
complex to acetylate nucleosomes, we reconstituted
nucleosomes with 207 bp DNA fragments containing
601 nucleosome positioning sequence and recombinant
histone octamers by the method of salt gradient dialysis.
The successful reconstitution of a nucleosome on the 601
DNA fragment was confirmed by appearance of a
retarded band in nucleoprotein gels (Supplementary
Figure S2, lanes 6 and 7). As shown in Figure 1D and
F, TIP60 in the H2A.Z complex acetylates nucleosomal
H2A, H2A.Z and H4 to a level comparable to that of free
H2A, H2A.Z and H4 (lane 6 versus lane 3). However, in
contrast to free H3 substrate, nucleosomal H3 was not
acetylated by H2A.Z-associated TIP60 (lane 6 versus
lane 3). Moreover, in western blotting to check acetylation
of two lysine substrates (K5 and K9) in H2A tails and four
lysine substrates (K5, K8, K12 and K16) in H4 tails, we
detected acetylation of all these substrates in the
nucleosome by H2A.Z-associated TIP60 (Figure 1E).
Collectively, our results show that TIP60 present in the
H2A.Z complex can acetylate the nucleosome with an
intrinsic preference for H2A/H2A.Z and H4 tails.
Initial H2A.Z complex can be separated into two
different subcomplexes
To determine whether the proteins co-purified with
ectopic H2A.Z form a single distinct complex or multiple
complexes, we further purified the initial H2A.Z complex
by ultracentrifugation in a 15–40% glycerol gradient.
Silver staining and western blot analysis of each gradient
fraction revealed that the initial complex is primarily
sedimented as two peaks in fractions 8–12 and 19–24
(Figures 2A and B), indicating the presence of two distinct
subcomplexes in the initial complex. Mass spectrometry
analysis of pooled 19–24 fractions revealed that most of
SRCAP and TIP60 components are co-sedimented with
H2A.Z in this fast-migrating peak (Figure 2C, lane 2).
However, similar analysis of pooled 8–12 fractions
detected only a subset of factors, including TIP48,
TIP49, BAF53, DMAP1 and Actin, in this slow-
sedimenting peak (Figure 2C, lane 1). We have termed
two subcomplexes purified from fractions 8–12 and
19–24 as H2A.Z-S and H2A.Z-B, respectively, implying
that they are small (S) and big (B) complexes. Consistent
with results from western blot analysis of gradient
fractions (Figure 2B), we detected HAT activity only in
the big complex, which contains a high concentration of
TIP60 (Figure 2D). To further confirm the presence of two
distinct H2A.Z complexes in vivo, we fractionated the
nuclear extracts prepared from the H2A.Z stable cell
lines through a gel filtration column. Our western blot
analysis of the fractions clearly showed that ectopic
H2A.Z and its associated factors are eluted as two
broad peaks corresponding to the small and big complexes
(Supplementary Figure S3).
Both small and big complexes mildly facilitate H2A.Z
exchange
Considering that SRCAP complex and p400 have recently
been shown to exchange H2A.Z/H2B dimers for
nucleosomal H2A/H2B (31,32), we set out to determine
whether the small and big complexes can catalyze the
H2A.Z exchange through their ATP-dependent
remodeling activities. The big complex contains four
catalytic ATPase subunits, SRCAP, p400, TIP48 and
TIP49, while the small complex contains only two
ATPases, TIP48 and TIP49. Thus, we first checked the
ability of the small and big complexes to hydrolyze ATP
in the presence or absence of the 601 nucleosomes. This
analysis revealed that both complexes have similar
ATPase activities, and the ATPase activity of neither
small nor big complex depends on the presence of
nucleosomes (Figure 3A).
To analyze the ability of the small and big complexes to
catalyze H2A.Z exchange, H2A-containing nucleo-
somes were reconstituted on 50biotinylated 207 bp DNA
fragments containing 601 nucleosome positioning
sequence with recombinant core histones (as in
Supplementary Figure S2, see ‘Materials and Methods’
section for details). After immobilizing nucleosomes to
paramagnetic beads, exchange assays were performed
with small or big complex in the presence or absence of
ATP as summarized in Figure 3B. Since both small
and big complexes contain FLAG/HA-H2A.Z and H2B,
we checked the incorporation of these dimers into
immobilized nucleosomes without adding free H2A.Z-
H2B dimers. As a gel loading control, we normalized
for the levels of histone H3 on the immobilized
nucleosomes (Figure 3C, Nucleosomal H3). Probing of
the membranes with anti-FLAG antibody revealed that
incubations of small and big complexes with H2A-
containing nucleosomes induced transfer of about 10%
extracts prepared from H2A.Z expressing cells. (B) Mass spectrometric analysis of H2A.Z-associated polypeptides. After a large-scale isolation of
H2A.Z complex, the purified polypeptides were resolved in 4–20% gradient SDS–PAGE. The protein bands, that were not present in the control
lane, were excised, and identified by mass spectrometric analysis. Lane 1, the proteins purified from control HeLa cells; lane 2, the proteins purified
from H2A.Z expressing cells. The positions of the molecular mass markers (in kDa) are indicated on the left. (C) Western blot analysis of the purified
H2A.Z complex. H2A.Z complex was separated by 4–20% gradient SDS-PAGE, transferred to nitrocellulose, and probed by western analysis with
antibodies as indicated on the left. Lane 1, mock-purified control; lane 2, H2A.Z complex. (D) Acetylation of H2A histone octamer and H2A
nucleosome by H2A.Z complex. Recombinant histone octamer and nucleosome were subjected to HAT assay with [
3
H]-acetyl-CoA and the H2A.Z
complex. HAT reactions were analyzed by 15% SDS–PAGE with subsequent fluorography. Lanes 1 and 4, buffer control; lanes 2 and 5, mock-
purified control; lanes 3 and 6, H2A.Z complex. (E) H2A/H4-targeted acetylation of H2A nucleosome by H2A.Z complex. HAT assays were as in
(D), but with nucleosomes and cold acetyl-CoA. HAT reactions were analyzed by western blot analysis with the indicated antibodies. Lane 1, mock-
purified control; lane 2, H2A.Z complex. (F) Acetylation of H2A.Z histone octamer and H2A.Z nucleosome by H2A.Z complex. Assays were
identical to (D), except that H2A.Z containing octamer and nucleosome were used as substrates. Lanes 1 and 4, buffer control; lanes 2 and 5, mock-
purified control; lanes 3 and 6, H2A.Z complex.
Nucleic Acids Research, 2009, Vol. 37, No. 18 5997

of FLAG/HA-H2A.Z-H2B dimers into nucleosomes
(Incorporated H2A.Z, lanes 5 and 6). Interestingly, similar
experiments in the absence of ATP also showed
nucleosomal incorporation of detectable amount of
FLAG/HA-H2A.Z-H2B dimers from the complexes
(Incorporated H2A.Z, lanes 8 and 9). Such H2A.Z transfer
is most likely to be made by endogenous ATP co-purified
with the complexes as revealed in recent studies (26,44).
The effects of small and big complexes were specific, since
the control reactions with free FLAG-H2A.Z-H2B dimers
failed to show any H2A.Z incorporation (Incorporated
H2A.Z, lanes 4 and 7). Anti-FLAG immunoblot of the
supernatants collected from the exchange reactions
showed no significant change in the level of unincorporated
H2A.Z (Unincorporated H2A.Z), indicating moderate
transfer of FLAG/HA-H2A.Z-H2B dimers into the
nucleosome (note the faster mobility of FLAG-H2A.Z
in the recombinant H2A.Z-H2B dimers compared to
FLAG/HA-H2A.Z in the complexes).
Pre-acetylation of nucleosomal histones significantly
facilitates the action of small complex
Because TIP60-mediated histone acetylation is known to
facilitate ATP-dependent exchange of phosphorylated
Figure 2. Isolation of H2A.Z subcomplexes. (A) Glycerol gradient fractionation of H2A.Z subcomplexes. The H2A.Z complex was separated by
glycerol gradient centrifugation as describe under ‘Materials and Methods’ section. Total 32 fractions were collected from the top to the bottom, and
analyzed by 4–20% gradient SDS–PAGE and silver staining. Each fraction was also subjected to western blot analysis with anti-FLAG antibody.
Asterisks indicate the ectopic H2A.Z. H2A.Z-S and H2A.Z-B indicate the H2A.Z-small (S) and H2A.Z-big (B) complexes, respectively. (B) Western
blot analysis of the purified H2A.Z subcomplexes. Aliquots of fractions corresponding to H2A.Z-S and H2A.Z-B complexes were separated by
4–20% SDS–PAGE and analyzed by immunoblot with the indicated antibodies. The initial H2A.Z complex shown in the Figure 1B was used as
input. (C) Mass spectrometric analysis of H2A.Z subcomplexes. H2A.Z-S and H2A.Z-B complexes were separated by 4–20% SDS–PAGE followed
by silver staining and identification of the bands by tandem mass spectrometry. Protein size markers are indicated on the left. Lane 1, H2A.Z-S
complex; lane 2, H2A.Z-B complex. (D) HAT assay of H2A.Z subcomplexes. Assays were identical to Figure 1D, except that H2A.Z-S (lane 3) and
H2A.Z-B complexes (lane 4) described in (C) were used. Lane 1, buffer control; lane 3, mock-purified control; lane 5, initial H2A.Z complex.
5998 Nucleic Acids Research, 2009, Vol. 37, No. 18

Figure 3. Stimulatory effect of H2A acetylation on H2A.Z exchange. (A) ATP hydrolysis by H2A.Z-S and H2A.Z-B complexes. ATPase activities of
the H2A.Z-S and H2A.Z-B complexes were checked with [g-
32
P] ATP in the presence or absence of nucleosomes. ATPse assays were normalized to
the level of FLAG/HA-H2A.Z in the complexes. Lanes 1 and 2, buffer control; lanes 3 and 4, H2A.Z-S complex; lanes 5 and 6, H2A.Z-B complex.
(B) Schematic summary of H2A.Z exchange assay. Nucleosomes were reconstituted with 50biotinylated 207 bp 601 DNA fragments and immobilized
on streptavidin-conjugated Dynabeads. Immobilized nucleosomes were incubated with either free FLAG-H2A.Z-H2B dimers or the H2A.Z
subcomplexes containing FLAG/HA-H2A.Z-H2B dimers. Nucleosomal incorporation of H2A.Z was determined by western blot analysis with
anti-FLAG antibody. (C) H2A.Z exchange by H2A.Z-S and H2A.Z-B complexes in vitro. Immobilized nucleosomes were incubated with either
free FLAG-H2A.Z-H2B dimers (lanes 4 and 7) or the H2A.Z complexes containing FLAG/HA-H2A.Z-H2B dimers (lanes 5, 6, 8 and 9) in the
presence or absence of ATP for 60 min. After extensive washing of the immobilized nucleosomes, H2A.Z incorporation was determined by western
blot analysis with anti-FLAG antibody (first panel). Relative intensity of bands was quantified using Phosphorimager (Bio-Rad). For analysis of free
unincorporated H2A.Z, supernatant was collected before the washing step and 25% of the supernatant was analyzed by western blot (third panel).
Nucleosomal H3 was used as an internal loading control (second panel). The amount of H2A.Z-S and H2A.Z-B complexes was normalized based on
the level of FLAG/HA-H2A.Z present in the complexes. Results shown are from a single experiment and are representative of three independent
experiments. Lanes 1–3, input (50%) of free FLAG-H2A.Z-H2B dimer, FLAG/HA-H2A.Z-H2B dimer in H2A.Z-S complex and FLAG/HA-H2A.Z-
H2B dimer in H2A.Z-B complex. (D) H2A.Z exchange into acetylated nucleosomes in vitro. Exchange assay was identical to (C), but nucleosomes
were acetylated with the initial H2A.Z complex before immobilizing on magnetic beads and ATP was included in all reactions. (E) Preferential
exchange of H2A.Z for acetylated H2A. After H2A.Z exchange reaction, equal amounts of nucleosomes were analyzed by western blot analyses with
anti-FLAG (a-FLAG), anti-H3 (a-H3), anti-acetyl-H2A (a-Ac-H2A) and anti-H2A antibodies (a-H2A). Equal loading of nucleosomes was
confirmed by western blotting for nucleosomal H3 (a-H3). (F) Requirement of ATP hydrolysis for H2A.Z exchange. In vitro exchange reactions
were identical to lanes 7–9 in (D), but in the absence (lanes 1–3 and lanes 7–9) or presence (lanes 4–6) of ATP or pre-treatment of the H2A.Z
complexes by 0.5 U apyrase (lane 10–12).
Nucleic Acids Research, 2009, Vol. 37, No. 18 5999

H2Av with unmodified H2Av in Drosophila (29), it is
possible that TIP60 present in the purified H2A.Z complex
cooperates with small and/or big complexes for H2A.Z
exchange. Thus, having found that TIP60 in the initial
H2A.Z complex can efficiently acetylate nucleosomal
H2A/H4 (Figure 1D), we performed exchange assays
after acetylation of the nucleosome by the initial H2A.Z
complex. When nucleosomes were pre-acetylated by
TIP60, big complex-induced incorporation of H2A.Z-
H2B dimer was only slightly enhanced (Figure 3D,
Incorporated H2A.Z, lane 9 versus lane 6). In striking
contrast, identical assays using small complex showed
about five-fold increase in H2A.Z exchange
(Incorporated H2A.Z, lane 8 versus lane 5), indicating
that TIP60-induced pre-acetylation is required for full
action of small complex, but not of big complex. Since
the pre-acetylated nucleosomes are free of the initial
complex components (Supplementary Figure S4, lanes
1–4), the observed changes in H2A.Z incorporation is
rather specific for the small complex and not due to the
initial complex left from HAT reaction. To determine
whether acetylated H2A proteins are preferentially
replaced by H2A.Z, we also analyzed equivalent amounts
of nucleosomes by western blot analysis after exchange
reactions. As shown in Figure 3E, western blot intensities
perceived for H2A and acetyl H2A are decreased to a
comparable extent after exchange reactions, strongly
supporting the selective dissociation of acetylated H2A
upon H2A.Z incorporation.
We next tested whether ATP hydrolysis is required for
acetylation-dependent incorporation of H2A.Z. As was
the case for the acetylation-independent exchange
reactions (Figure 3C), the small complex was capable of
catalyzing acetylation-mediated H2A.Z exchange even
in the absence of ATP, although a slight stimulation
of H2A.Z exchange was observed by including free ATP
(Figure 3F, Incorporated H2A.Z, lanes 2 and 5). Such
H2A.Z exchange is most likely due to endogenous ATP
copurified with the small complex, since the exchange
reaction without exogenous ATP was significantly
inhibited when apyrase was added in the beginning of
the exchange assays (lane 8 versus lane 11). Big complex,
having a much weaker activity in catalyzing acetylation-
mediated H2A.Z exchange reaction, also showed a
detectable inhibition on its activity upon apyrase
treatment (lane 9 versus lane 12). When apyrase treatment
was tried for acetylation-independent exchange reactions,
we could also observe, albeit to a lesser extent, reduction
of H2A.Z incorporation (Supplementary Figure S5).
Acetylation of H2A-K5 facilitates small complex-mediated
H2A.Z exchange
The aforementioned results showing the stimulatory effect
of nucleosome acetylation on small complex-induced
H2A.Z exchange suggest that acetylation of specific
lysine substrates could be responsible for this stimulation.
To address this issue, we repeated the exchange assays
using nucleosomes reconstituted with mutant histones.
Since TIP60 present in the initial H2A.Z complex has an
intrinsic ability to acetylate nucleosomal H2A and H4
(Figure 1D and E), we mutagenized the major acetylation
sites of H2A and H4 (K5 and K9 of H2A and K5, K8,
K12 and K16 of H4). Western blot analyses confirmed
that these mutations completely blocked acetylations of
H2A-K5/K9 and H4-K5/K8/K12/K16 by TIP60 in the
initial complex (Figure 4A and C). When the effect of
H4 mutations on acetylation-mediated H2A.Z exchange
was examined, the mutant H4 nucleosome showed
H2A.Z incorporation comparable to that with the wild-
type nucleosome (Figure 4B, lanes 5 and 6 versus lanes 8
and 9). In contrast, the exchange assay with the H2A-
mutated nucleosome failed to show acetylation-facilitated
incorporation of H2A.Z into the nucleosome (Figure 4D,
lane 5 versus lane 8). More importantly, similar exchange
reactions with the nucleosome containing K5 mutant form
of H2A also showed significant repression in H2A.Z
exchange (lane 5 versus lane 11), indicating that H2A-
K5 acetylation is necessary for optimal incorporation of
H2A.Z. The fact that the K5 mutation did not change the
level of acetylation of K9 (Figure 4C, lane 8) strongly
suggests that K9 acetylation alone is not sufficient for
small complex-induced H2A.Z exchange. Overall, these
results establish a primary role of acetylation of H2A-
K5 in facilitating the action of small complex for H2A.Z
exchange.
TIP48 and TIP49 are catalytic subunits of the
small complex
Although the exchange assays described above emphasize
the specific role of the small complex in acetylation-
facilitated H2A.Z exchange, it is unclear which factors
within the complex are mainly responsible for the
exchange reaction. Because ATP hydrolysis is necessary
for the small complex-induced H2A.Z exchange
(Figure 3F) and because TIP48 and TIP49 are the only
ATPase activities of the small complex (Figure 2C), we
speculated that TIP48 and TIP49 in the small complex
are crucial for the acetylation-facilitated exchange
reaction. We first conducted ATPase assays with
recombinant TIP48 and TIP49 which were expressed in
bacteria and purified using their FLAG epitope tags
(Supplementary Figure S6A). In agreement with previous
studies (45), when either TIP48 or TIP49 was incubated
with ATP, they were unable to show catalytic activity
for ATP hydrolysis (Figure 6A, lanes 2 and 3).
However, when we used TIP48/TIP49 complex that was
reconstituted and fractionated by glycerol gradient
centrifugation (Supplementary Figure S6B), the complex
showed a strong ATPase activity (Figure 5A, lane 4),
confirming that both proteins are required for ATP
hydrolysis.
We next prepared free H2A.Z-H2B dimers
(Supplementary Figure S6C) and examined the H2A.Z
exchange following their incubation with the immobilized
nucleosomes in the presence of TIP48 and/or TIP49.
Significantly, we found that TIP48 and TIP49 can closely
recapitulate the action of the small complex in the
H2A.Z exchange reaction by facilitating incorporation
of H2A.Z-H2B dimers into acetylated nucleosomes
(Figure 5B, lane 8). Addition of a nonhydrolyzable ATP
6000 Nucleic Acids Research, 2009, Vol. 37, No. 18

analog ATP-g-S reduced H2A.Z exchange activity of
TIP48/TIP49 complex to the background level
(Supplementary Figure S7, lane 4). Parallel exchange
assays with TIP48 or TIP49 alone failed to show
nucleosomal incorporation of H2A.Z-H2B dimers
(Figure 5B, lanes 10 and 11), indicating that they play a
combinatorial role in H2A.Z exchange. The finding that
the stimulatory effect of pre-acetylation was lost upon
mutation of H2A-K5 confirms a requirement of the
modification, per se (Figure 5C, lane 4). To assess the
specificity of action of TIP48 and TIP49, we also extended
exchange assays to canonical H2A and another H2A
variant H2A.X (Supplementary Figure S6C). In sharp
contrast, TIP48 and TIP49 were unable to facilitate
incorporations of H2A.X-H2B and H2A–H2B dimers
into nucleosomes (Figure 5D), supporting highly selective
action of TIP48 and TIP49 for H2A.Z exchange. Hence,
we conclude that TIP48 and TIP49 in the small complex
are sufficient to catalyze acetylation-facilitated incorpora-
tion of H2A.Z into the nucleosome.
Acetylation of H2A-K5 stabilizes the nucleosome-TIP49
interaction
While our results emphasize the critical role of H2A-K5
acetylation in H2A.Z-H2B dimer integration into
Figure 4. Dominant role of H2A-K5 acetylation for H2A.Z exchange. (A) HAT assay with recombinant nucleosomes containing wild-type or mutant
H4. HAT assay was identical to Figure 1E, but using nucleosomes reconstituted with intact (lanes 1 and 2) or lysine mutated (lanes 3 and 4) H4.
Reaction products were subjected to western blot analysis with an antibody recognizing acetylated H4. (B) Minimal effect of H4 acetylation on
H2A.Z exchange. In vitro exchange assay was identical to Figure 3D, but using nucleosomes reconstituted with intact (lanes 1–6) or lysine mutated
(lanes 7–9) H4. Nucleosomes were unmodified (lanes 1–3) or acetylated (lanes 4–9) by TIP60 in the initial H2A.Z complex. Nucleosomal
incorporation of H2A.Z was analyzed by western blot analysis with anti-FLAG antibody (a-FLAG). Equal loading of nucleosomes was confirmed
by western blotting for nucleosomal H3 (a-H3). (C) HAT assay with recombinant nucleosomes containing wild-type or mutant H2A. HAT assay was
identical to Figure 4A, but using nucleosomes reconstituted with intact (lanes 1, 2, 5 and 6), K5/K9 mutated (lanes 3 and 4) or K5 mutated (lanes 7
and 8) H2A. HAT reactions were subjected to immunoblot analysis with antibodies recognizing acetylated K5 or K9 of H2A. (D) Significant effect of
H2A acetylation on H2A.Z exchange. In vitro exchange assay was identical to Figure 4B, but using nucleosomes containing intact (lanes 1–6), K5/K9
mutated (lanes 7–9) or K5 mutated (lanes 10–12) H2A. Again, histone H3 was used as loading control.
Nucleic Acids Research, 2009, Vol. 37, No. 18 6001

nucleosomes, how this acetylation facilitates H2A.Z
exchange reaction is unclear. One possibility is that K5
acetylation positively affects interaction of the nucleosome
with TIP48/TIP49, which in turn allows a stable action
of TIP48/TIP49 during H2A.Z exchange process. Thus,
we analyzed the ability of TIP48 or TIP49 to interact
with a fixed concentration of the H2A nucleosome
immobilized to magnetic beads. As shown in Figure 5E,
Figure 5. Exchange of H2A.Z-H2B dimer by TIP48 and TIP49. (A) ATPase activity of recombinant TIP48 and TIP49. ATPase assay was performed
with recombinant TIP48/TIP49 and [g-
32
P] ATP, and ATP hydrolysis was examined by 12% polyacrylamide gel (19:1) containing 7 M urea in a 1
TBE buffer. Lane 1, buffer control; lane2, recombinant TIP48; lane 3, recombinant TIP49; lane 4, recombinant TIP48/TIP49 complex. (B) H2A.Z
exchange by TIP48, TIP49 or TIP48/TIP49 complexes in vitro. H2A.Z exchange activity of recombinant TIP48 and/or TIP49 was checked by using
unmodified (lanes 1–4) or pre-acetylated (lanes 5–12) nucleosomes in the presence (lanes 3, 4, 7 and 8–12) or absence (lanes 1, 2, 5 and 6) of ATP as
described in ‘Materials and Methods’ section. Nucleosomal incorporation of His-H2A.Z was analyzed by western blot analysis with anti-His
antibody (a-His, upper panel). Free unincorporated H2A.Z was determined by western blot analysis of the supernatant (25%) with anti-His antibody
(a-His, lower panel). Nucleosomal H3 was used as an internal loading control. Results are representative of three independent experiments. Lanes 1,
3, 5, 7 and 9–12 are reactions without TIP48 and/or TIP49. (C) Effect of H2A acetylation on H2A.Z exchange by TIP48/TIP49 complexes. In vitro
exchange assay was identical to lanes 7–8 in Figure 6B, but using nucleosomes containing intact (lanes 1 and 2) or K5 mutated (lanes 3 and 4) H2A.
Nucleosomal incorporation of His-H2A.Z was analyzed by western blot analysis with anti-His antibody (a-His). Nucleosomal H3 (a-H3) was used as
an internal loading control. (D) Specific action of TIP48/TIP49 complex on H2A.Z exchange. In vitro exchange assays were performed with
acetylated nucleosomes as in Figure 6B, using h:H2A.Z/H2B dimer (lanes 2 and 3), h:H2A.X/H2B dimer (lanes 5 and 6) and f:H2A/H2B dimer
(lanes 8 and 9). Nucleosomal incorporation of dimers was analyzed by western blot analysis with the indicated antibodies (upper panel). Free
unincorporated dimers were determined by western blot analysis of the supernatant (25%) with the indicated antibodies (lower panel). Nucleosomal
H3 was used as an internal loading control (second panel). Results are representative of three independent experiments. Lanes 1, 4 and 7, input
(25%) of free dimers. (E) Positive effect of acetylation of H2A-K5 on interaction of TIP49 with nucleosome. The unmodified (lanes 3, 6, 7, 9 and 11)
or acetylated (lanes 10 and 12) nucleosomes containing wild type (lanes 6, 7, 9 and 10) or K5 mutated (lanes 11 and 12) H2A were immobilized on
magnetic beads and incubated with FLAG-TIP48 (lane 6), FLAG-TIP49 (lane 7) or FLAG-TIP48/TIP49 complex (lanes 9–12). The interaction of
TIP48/TIP49 with nucleosomes was analyzed by immunoblot with anti-FLAG antibody. The immobilized 50biotinylated 207 bp DNA fragments
containing 601 nucleosome positioning sequence (lane 2) was also included to determine relative DNA binding affinity of TIP48/TIP49. Histone
H3 was used as a loading control. Results are representative of three independent experiments. Lane 1, input of FLAG-TIP48; lane 2, input of
FLAG-TIP49; lane 5, input of FLAG-TIP48/TIP49 complex.
6002 Nucleic Acids Research, 2009, Vol. 37, No. 18

TIP48 and TIP49 both showed direct interactions with
immobilized nucleosomes, although TIP49 appears to
have much higher affinity to nucleosomes (lanes 6 and
7). To investigate cooperative binding of TIP48 and
TIP49 to nucleosomes, we conducted identical binding
assays with TIP48/TIP49 complex. As expected from
individual binding assays, TIP49 was able to bind to
nucleosomes, but somewhat surprisingly, TIP48 did not
show any detectable binding to the nucleosome (lane 3).
A possible explanation of these results is that TIP48 and
TIP49 both bind to the same surface of the nucleosome.
Thus, TIP48 interaction is less stable in the presence of its
binding partner TIP49, which shows high affinity binding
to nucleosomes. These results also hint at a transient
action of TIP48 onto the TIP49-bound nucleosome in
H2A.Z exchange reaction. Interestingly, the immobilized
601 DNA template also showed no detectable binding of
TIP48 and TIP49 (lane 2), suggesting that TIP48 and
TIP49 recognize unique platforms within the structural
context of the nucleosome. Given the demonstrated role
of pre-acetylation in H2A.Z exchange, we next assessed
the interaction of TIP48/TIP49 following TIP60-mediated
acetylation of nucleosomes. Significantly, there was an
apparent increase in TIP49-nucleosome interaction after
pre-acetylation of nucleosomes (lane 9 versus lane 10).
Moreover, similar binding assays with nucleosomes
bearing K5 mutant form of H2A failed to show stimula-
tory effects of TIP60 in TIP49-nucleosome interaction
(lane 11 versus lane 12), indicating that H2A-K5 acetyla-
tion distinctly regulates TIP49 recruitment. Again, no
stable interaction was observed for TIP48 in the reactions
(lanes 9–12). These binding experiments were repeated
three times and reproducibly showed at least 2-fold
increase in TIP49-nucleosome interaction upon H2A-K5
acetylation. Taken together, these results suggest that
acetylation of nucleosomal H2A at K5 is critical for
a stable association of TIP49 during TIP48/
TIP49-mediated H2A exchange process.
TIP48 and TIP49 are required for H2A.Z deposition
into chromatin in vivo
To assess the in vivo relevance of our in vitro results,
we asked whether H2A.Z exchange is altered when
endogenous TIP48 and TIP49 are down-regulated using
the siRNA approach. Since TIP48 or TIP49 alone has no
ATPase activity and both proteins are required for H2A.Z
exchange (Figure 5B), we knocked down only TIP49 to
check the catalytic activity of TIP48/TIP49 complex for
H2A.Z exchange. As confirmed by real-time PCR
analysis, the transfection of cells with TIP49 siRNA
suppressed TIP49 mRNA expression to levels equivalent
to <30% of that observed in cells transfected with control
siRNA (Figure 6A, left panel). Western blot analysis with
anti-TIP49 antibody also verified a significant decrease in
TIP49 expression, but no change in Actin expression, after
TIP49 siRNA treatment (Figure 6A, right panel),
confirming the specificity of siRNA-induced repression
of TIP49. After 48 h depletion of TIP49, cells were
transfected with FLAG-H2A.Z, -H2A.X or -H2A for
another 48 h, and chromatin was isolated from cell
nuclei. Our western blot analysis of chromatin prepared
from cells transfected with the control siRNA confirmed
the incorporation of ectopic H2A.Z into cellular nucleo-
somes (Figure 6B, lane 1). However, the nucleosomal
incorporation of ectopic H2A.Z was significantly
decreased in cells transfected with TIP49 siRNA (lane 2).
Similar assays with ectopic H2A.X and H2A did not
show any effect of TIP49 siRNA on their incorporation
into cellular nucleosomes (lanes 3–6), underscoring the
specific action of TIP48/TIP49 for H2A.Z exchange in
living cells.
As H2A.Z is known to be localized at the p21 promoter
(32), we next checked whether TIP48 and TIP49 are
required for H2A.Z incorporation into this promoter
region by ChIP analysis after siRNA-mediated depletion
of cellular TIP49. Consistent with recent results (32),
H2A.Z was enriched at the distal p53 response element
as well as the TATA transcription initiator region, albeit
a higher level of H2A.Z was detected in the distal response
region (Figure 6C). However, in a parallel ChIP analysis
after treatment of cells with TIP49 siRNA, a significant
reduction of H2A.Z peaks at both p53 response element
and transcription initiator regions was observed
(Figure 6C). The entire experiment was repeated with a
different siRNA and similar results were obtained
(Supplementary Figure S8). Thus, although other factors
may also contribute to cellular H2A.Z exchange, these
results confirm that TIP49 is essential for cellular
deposition of H2A.Z into distinct chromatin regions.
DISCUSSION
Recent studies in yeast revealed that H2A.Z can be
incorporated into chromatin as H2A.Z-H2B dimers by
the SWR1 complex, many subunits of which are
homologous to the subunits of INO80 complex. The
cellular requirement of SWR1 in H2A.Z exchange was
further confirmed by gene expression studies that
showed a considerable overlap of genes regulated by
H2A.Z and SWR1 (26). In human cells, SRCAP and
p400 ATP-dependent chromatin remodeling complexes
were identified as a counterpart of yeast SWR1 and
found to catalyze H2A.Z exchange reaction (31,32).
However, these complexes seem mutually exclusive and
exert their actions in distinct chromatin regions during
specific cellular processes, as exemplified by p21 gene-
specific action of p400 (32). Such a targeted action
would predict that there might be additional activities
and mechanisms to regulate the incorporation of H2A.Z
into the nucleosome.
In this study, we have purified H2A.Z-containing
complexes from a human cell line to dissect potential
molecular mechanisms employed for the deposition of
histone variant H2A.Z into the nucleosome. Taking
advantage of our established purification method, we
demonstrated that free H2A.Z can form two distinct
complexes, which we named small and big complexes.
The big complex consists of most components of
SRCAP and TIP60 complexes, whereas the small com-
plex contains only several components of SRCAP and
Nucleic Acids Research, 2009, Vol. 37, No. 18 6003

TIP60 complexes. In comparison with a recent study
indicating that free H2A.Z is associated primarily with
SRCAP complex (30,31), it is somewhat surprising to
find the TIP60 components in our purification. These
differences may reflect the use of different cell types
(HeLa versus 293/FRT cells) and the differences in
nuclear extract preparation and purification procedures.
That TIP60 components are bona fide subunits of the
H2A.Z-containing big complex is further supported by
(i) their copurification with H2A.Z via ion-exchange
chromatography and immunoaffinity purification steps
and (ii) their co-sedimentation with H2A.Z and SRCAP
components of the purified big complex on glycerol
gradients.
In accord with recent studies with SRCAP and p400,
the big complex containing these activities facilitated
incorporation of H2A.Z into the nucleosome. Since the
small complex lacks SRCAP and p400, one would
expect that the complex does not have function in
H2A.Z incorporation. However, our exchange assays
clearly indicate that the small complex can also exchange
H2A.Z into the nucleosome by replacing canonical H2A.
Figure 6. Requirement of TIP48/TIP49 for H2A.Z deposition into chromatin in vivo.(A) Knockdown of TIP49 using TIP49 siRNA. 293T cells were
transfected with TIP49-targeted siRNA (Si-Tip49) or negative control siRNA (Si-NC), and repression of TIP49 expression was confirmed by RT–
qPCR and western blotting. The mRNA levels of TIP49 were normalized to the b-actin mRNA, which was unaffected by TIP49 siRNA transfection.
Whole-cell lysates of the cells transfected with TIP49 or negative control siRNA were analyzed by immunoblot with anti-TIP49 and anti-actin
antibodies. Error bars indicate the mean SD of results from three independent experiments. (B) Repressive effect of TIP49 knockdown on H2A.Z
incorporation. 293T cells were initially depleted TIP49 for 48 h and transfected with FLAG-H2A.Z (f:H2A.Z, Lanes 1 and 2), FLAG-H2A.X
(f:H2A.X, lanes 3 and 4) or FLAG-H2A (f:H2A, lanes 5 and 6) for 48 h. Chromatin was isolated from the transfected cells, and the amount of
chromatin was normalized by western blot analysis of nucleosomal histone H3 (lower panel). Nucleosomal incorporations of f:H2A.Z, f:H2A.X and
f:H2A were analyzed by western blot analysis with anti-FLAG antibody (a-FLAG). Results are representative of three independent experiments.
(C) TIP48/TIP49-dependent incorporation of H2A.Z at the p21 promoter region. ChIP analysis of the p21 promoter was performed as described in
‘Materials and Methods’ section. Lines (A–H) under the diagram of p21 promoter region indicate segments used for qPCR. Error bars indicate the
mean SD of results from three independent experiments. The white boxes and black box indicate two known p53 response elements (p53RE1 and
p53RE2) and TATA box, respectively. (D) Summary of TIP48/TIP49-induced H2A.Z exchange. TIP60 is recruited to target gene promoters for
localized acetylation of histone H2A at K5, and this acetylation mark acts as a signal for the recruitment of TIP48 and TIP49. TIP48 and TIP49 then
facilitate promoter-targeted exchange of H2A.Z to establish platform that would favor the action of chromatin remodeling factors to regulate
transcription.
6004 Nucleic Acids Research, 2009, Vol. 37, No. 18

Importantly, a more prominent feature of the small
complex is derived from our finding that TIP60-mediated
acetylation of the nucleosomal H2A stimulates the action
of the small complex, but not that of the big complex, in
H2A.Z exchange reaction. This observation significantly
enhances our understanding of the mechanism by which
H2A.Z exchange process is regulated through epigenetic
alterations in target nucleosomes. In fact, previous studies
have also shown that the H2A.Z incorporation into
specific genes or loci in yeast was facilitated by acetylation
of H3 and H4 (12,46–48). Moreover, since other
modifications, especially H3/H4 methylation, were also
enriched in H2A.Z nucleosomes (49,50), it is not unlikely
that these modifications represent another mechanism of
regulation of H2A.Z exchange reaction. Bearing this
possibility in mind, we may be able to take advantage of
our in vitro assay system to identify other epigenetic
marks and mechanisms of their action in the H2A.Z
exchange pathway.
In search of which factors in the small complex play a
major role in H2A acetylation-facilitated incorporation of
H2A.Z, we focused on TIP48 and TIP49, since they are
the only subunits with ATPase activity in the small
complex. Consistent with published results (45), our
ATPase assays with recombinant TIP48 and TIP49 clearly
showed the requirement of both proteins for ATP
hydrolysis. Further analysis of H2A.Z exchange revealed
that TIP48 and TIP49 are the catalytic activity in the
small complex responsible for H2A.Z exchange reaction.
Although there have been reports linking TIP48/TIP49 to
chromatin remodeling processes (51,52), these results
provide the first direct connection between TIP48/TIP49
and histone variant exchange. There was no observable
effect of TIP48/TIP49 on the incorporation of canonical
histone H2A and histone variant H2A.X (Figure 5D),
indicating H2A.Z-specific action of TIP48/TIP49.
Moreover, the requirement of TIP48 and TIP49 in cellular
H2A.Z exchange was supported by apparent decrease of
the incorporation of ectopic H2A.Z into chromatin after
RNAi-mediated depletion of TIP49 (Figure 6). Parallel
ChIP experiments showed that TIP49 knockdown
impaired H2A.Z deposition at the p21 promoter,
specifically at the p53 response element and transcription
initiator regions. Thus, although we cannot strictly rule
out the possibility that SRCAP and p400 act in parallel
on H2A.Z exchange pathways, such an impairment of
H2A.Z exchange at a specific chromatin region upon
TIP49 knockdown is indicative of the critical targeted
action of TIP48 and TIP49 on H2A.Z exchange.
It should be noted that both small and big complexes
possess TIP48 and TIP49, but acetylation of nucleosomal
H2A promotes only small complex-induced incorporation
of H2A.Z. Although the reason for these results is not
clear at present, one possibility is that some proteins in
big complex inhibit the activity of one or both of TIP48
and TIP49, causing H2A.Z exchange to be dependent
solely on SRCAP and p400 ATPase activities. This
negative regulator might have crucial functions for
economizing the action of TIP48/TIP49 on the cellular
H2A.Z exchange process. In agreement with this idea,
the initial H2A.Z complex, albeit consisting of small and
big complexes, induced only moderate transfer of H2A.Z-
H2B dimers into pre-acetylated nucleosomes (data not
shown). Further analysis, such as identification of these
repressive factors and their functional interaction with
TIP48/TIP49, would be helpful for understanding the
mechanisms of regulation of H2A.Z exchange.
The most striking finding from our studies was the
identification of H2A-K5 acetylation as a key regulator
for the action of TIP48/TIP49 in the H2A.Z exchange
process. Although there have been reports linking histone
acetylation to H2A.Z exchange, our use of nucleosomes
reconstituted with wild type and mutant recombinant
histones allowed us to provide the first direct connection
between H2A.Z exchange and site-specific H2A
acetylation. An immediate question raised from these
findings was how TIP60-mediated acetylation of
nucleosomal H2A acts as a positive regulator for H2A.Z
exchange. There are two potential explanations for the
effect of H2A acetylation. One possibility implies that
H2A acetylation destabilizes nucleosome structure by
reducing the affinity of H2A-H2B dimer for the H3-H4
tetramer within the nucleosome and/or inducing a
conformational change of the nucleosome. However, our
structural analyses of unmodified and acetylated
nuclesomes did not allow us to reveal any differences in
their structural characteristics (data not shown). Another
possibility is that H2A acetylation positively influences the
association of TIP48/TIP49 with the N-terminal tail of
histone H2A, thereby increasing the affinity of TIP48/
TIP49 to the target nucleosome. An intriguing observation
we made in this regard is that TIP49 preferentially
interacts with nucleosome containing K5-acetylated H2A
(Figure 5E). This result strongly suggests that H2A-K5
acetylation contributes to the action of TIP48/TIP49
during H2A.Z exchange process by stabilizing TIP49
interaction with nucleosomes. In addition, several recent
studies have suggested that H2A.Z is specifically localized
at gene promoter regions to exert its influence on trans-
cription (53,54). A possible explanation for this promoter-
targeted incorporation of H2A.Z is that recruitment of
TIP60 (or other HATs) to gene promoters through its
interaction with DNA binding factors results in increased
level of H2A acetylation and H2A.Z exchange.
Thus, taking our in vitro and in vivo observations
together with recent studies, we propose the following
model for how TIP48/TIP49 ATPase activities and
TIP60-mediated H2A acetylation promote H2A.Z
exchange (Figure 6D). In the initial step, TIP60 is
recruited to specific gene promoter regions likely through
its interaction with sequence-specific DNA-binding factors
(such as p53). Once recruited, TIP60 acetylates nucleo-
somal H2A localized in target promoters. This
promoter-targeted H2A acetylation will in turn function
as a marker to direct TIP48/TIP49-induced deposition of
H2A.Z into the promoter regions. In this way, while
TIP48/TIP49, SRCAP and p400 all have an intrinsic
ability to exchange H2A.Z, TIP48/TIP49 can adopt a
highly specific strategy for localizing H2A.Z at specific
chromatin regions. It is also worth noting that TIP48
and TIP49 have been shown to interact with the RNA
Polymerase II as well as transcription factors/coregulators
Nucleic Acids Research, 2009, Vol. 37, No. 18 6005

(TBP, ATF2 and c-myc) (55–57). Thus, interaction of
TIP48/TIP49 with transcription-related factors might
also play a role in their initial localization to acetylated
promoter regions. Further investigations of whether
different genes employ different epigenetic signals and/or
factors for H2A.Z deposition will be an exciting challenge
for us in the future.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank K. Luger (Colorado State University) for
histone expression vectors, H. Willard (Duke University)
for H2A.Z cDNA, D. Rhodes (MRC Laboratory of
Molecular Biology) for DNA template containing 207 bp
601 nucleosome positioning sequence, and R. G. Roeder
(The Rockefeller University) for anti-DMAP1, anti-
BAF53 and anti-GAS41 antibodies. We also thank
M. R. Stallcup for critical reading and helpful comments
on the manuscript, and members of the An laboratory for
instructive discussions and valuable suggestions.
FUNDING
Margaret E. Early Medical Research Grant; the James H.
Zumberge Research Grant; the National Institutes
of Health (R01GM84209). Funding for open access
charge: National Institutes of Health (R01GM84209).
Conflict of interest statement. None declared.
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