MOLECULAR AND CELLULAR BIOLOGY, Jan. 2008, p. 140–153
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 1
Hbo1 Links p53-Dependent Stress Signaling to DNA Replication Licensing?
Masayoshi Iizuka,1,2Olga F. Sarmento,1Takao Sekiya,3† Heidi Scrable,4
C. David Allis,2‡ and M. Mitchell Smith1*
Department of Microbiology, University of Virginia Health System, Charlottesville, Virginia 229081; Department of Biochemistry and
Molecular Genetics, University of Virginia Health System, Charlottesville, Virginia 229082; Oncogene Division,
National Cancer Center Research Institute, Tsukiji, Tokyo 104-0045, Japan3; and Department of Neuroscience,
University of Virginia Health System, Charlottesville, Virginia 229084
Received 16 April 2007/Returned for modification 16 May 2007/Accepted 4 October 2007
Hbo1 is a histone acetyltransferase (HAT) that is required for global histone H4 acetylation, steroid-
dependent transcription, and chromatin loading of MCM2-7 during DNA replication licensing. It is the
catalytic subunit of protein complexes that include ING and JADE proteins, growth regulatory factors and
candidate tumor suppressors. These complexes are thought to act via tumor suppressor p53, but the molecular
mechanisms and links between stress signaling and chromatin, are currently unknown. Here, we show that p53
physically interacts with Hbo1 and negatively regulates its HAT activity in vitro and in cells. Two physiological
stresses that stabilize p53, hyperosmotic shock and DNA replication fork arrest, also inhibit Hbo1 HAT activity
in a p53-dependent manner. Hyperosmotic stress during G1phase specifically inhibits the loading of the
MCM2-7 complex, providing an example of the chromatin output of this pathway. These results reveal a direct
regulatory connection between p53-responsive stress signaling and Hbo1-dependent chromatin pathways.
The dynamic regulation of chromatin structure and function
is essential for normal cell proliferation and differentiation.
This regulation is mediated by several overlapping pathways,
including the posttranslational enzymatic modification of his-
tones, the alteration of nucleosome structure by DNA-depen-
dent ATPase complexes, and changes in the histone variant
composition of chromatin (4, 34, 58). Alterations in histone
modification enzymes, particularly histone acetyltransferase
(HAT) enzymes, have been linked to human cancer (23, 73).
Viral oncoproteins, such as adenovirus E1A or simian virus 40
large T antigen, target a number of these enzymes, including
p300, CBP, and PCAF. Furthermore, in addition to modifying
histones, these HATs can also directly acetylate and activate
tumor suppressors and key growth control transcription factors
such as p53, Rb, and E2F (24). The MYST family of histone
acetyltransferases, named for the four founding proteins in the
family (67), can also contribute to carcinogenesis and tumor
progression. The MYST proteins are part of large multisubunit
HAT complexes conserved from yeast to humans, and they
have diverse roles in gene expression, DNA replication, and
DNA repair (72). The human MOZ gene, encoding one of the
human MYST enzymes, was first identified as a translocation
fusion with CBP in acute myeloid leukemias (9). Subsequently,
a number of other translocation fusions involving the MYST
HATs MOZ and MORF and partners including CBP, p300,
MLL, and TIF2 have been identified. It is thought that the
mislocalization or misregulation of the HAT activities of these
fusions contributes to tumor formation or progression (72).
Hbo1 is a member of the MYST family of HAT enzymes and
is conserved from flies to humans. It has essential roles in DNA
replication and transcription (1, 12, 22, 32, 55, 59) and is the
catalytic subunit of at least two protein complexes comprised
of JADE1/JADE2/JADE3 paralogs, hEaf6, and either ING4
or ING5, two members of the “inhibition-of-growth” (ING)
tumor suppressor protein family (17). Hbo1 was originally
identified through its physical interactions with the human
DNA replication proteins ORC1 and MCM2 (12, 32). A crit-
ical step in DNA replication is the formation of a prereplica-
tive complex (pre-RC) involving the sequential assembly of the
origin recognition complex, Cdc6/Cdcl8, Cdtl, and the
minichromosome maintenance (MCM2-7) complex. The as-
sembly of the pre-RC on replication origins confers a license
for subsequent replication initiation. Disassembly of the
pre-RC following initiation ensures that replication occurs only
once per cell cycle (41). There is increasing evidence that
chromatin modulation plays important roles in DNA replica-
tion (for a review, see reference 63). Recently, we discovered
that Hbo1 is required for the chromatin loading of the
MCM2-7 complex, the final step in pre-RC assembly and DNA
replication licensing (31). Depletion of Hbo1 in human cells
and in Xenopus egg extracts specifically blocked MCM2-7 as-
sembly into the pre-RC and inhibited DNA replication. Fur-
thermore, this defect could be corrected in egg extracts by the
addition of excess Cdt1, a key positive regulator of pre-RC
assembly. Thus, Hbo1 function regulates the pathway, ensuring
that DNA replication occurs once, and only once, per cell
Members of the JADE and ING protein families are
thought to function, at least in part, by interacting with other
human tumor suppressors. For example, ING4 and ING5 have
been shown to physically interact with the human tumor sup-
pressor p53 and potentiate its activity (19, 56, 57). Similarly,
* Corresponding author. Mailing address: Department of Microbiol-
ogy, University of Virginia Health System, P.O. Box 800734, Charlottes-
† Present address: Mitsubishi Kagaku Institute of Life Sciences,
Machida, Tokyo 194-8511, Japan.
‡ Present address: Laboratory of Chromatin Biology, Rockefeller
University, New York, NY 10021.
?Published ahead of print on 22 October 2007.
Jade1 has been found to stabilize pVHL, the product of the
von Hippel-Lindau tumor suppressor gene, which in turn can
stabilize and activate p53 (51, 76). The tumor suppressor p53 is
a stress-induced regulatory protein which, upon a variety of
cellular stresses, can trigger cell cycle arrest or apoptosis (69).
These functions are significantly compromised by somatic mu-
tations in cancers, and approximately half of all human tumors
carry mutated alleles of p53. The best-understood role of p53
is as a sequence-specific DNA-binding transcription factor,
which regulates a number of key cell cycle and apoptotic genes
(28, 69). However, p53 may also function through transcrip-
tion-independent pathways to maintain genome integrity (33).
The best studied of these is its cytoplasmic role in regulating
apoptosis (45), but transcription-independent functions in ho-
mologous recombination, replication, and DNA damage
checkpoint responses have also been suggested (5, 15, 25, 49,
There are strong connections between chromatin regulation
and p53 function. The stability and activity of p53 are known to
be regulated by several histone modification factors, including
the p300/CBP and PCAF HAT complexes and the Set9 and
Smyd2 methylase complexes (2, 13, 24, 26, 30, 39, 53). Re-
cently, two different members of the MYST family HATs,
Tip60 and Mof, were shown to acetylate p53 and change its
transcriptional targets from genes that favor cell cycle arrest to
those that promote apoptosis (62, 64). Considerably less is
known about downstream p53-dependent pathways that might,
in turn, regulate the activity of histone modification complexes.
Thus, at present, it is unclear how such potent cellular factors
as Hbo1, ING4/5, Jade1/Jade2/Jade3, and p53 interact to reg-
ulate cell proliferation, and there are currently no known links
between Hbo1 and p53. While we were investigating the roles
of Hbo1 in DNA replication, we discovered that p53 copurifies
with protein complexes immunoprecipitated with Hbo1 anti-
sera. Here, we report studies designed to characterize the
regulation of Hbo1, its physical and functional interactions
with p53, and the stress signals that feed into the pathway. The
results of these experiments show that p53 physically interacts
with Hbo1 and down-regulates its HAT activity both in vitro
and in cells. Furthermore, we show that physiological stresses
that activate p53 are coupled to decreased Hbo1 HAT activity
in a p53-dependent manner. In particular, these results define
a previously unrecognized pathway linking the cell stress re-
sponse to replication control at the level of pre-RC assembly
via p53 and Hbo1.
MATERIALS AND METHODS
Mouse Hbo1 cDNA. A mouse dbEST cDNA (dbEST accession number
8230062; GenBank accession number BG519441), which showed extensive ho-
mology with the human Hbo1 protein by blastX search, was purchased (Research
Plasmids. His-tagged Hbo1 truncations were cloned into plasmid pET19Spe
(a gift from Masumi Hidaka). His-Hbo1(G485A) was created by PCR using
mutated primers. Glutathione S-transferase (GST)-tagged p53 deletion mutants
were PCR amplified from a wild-type human p53 plasmid (a gift from Takashi
Kohno) or mouse p53 and cloned into plasmid pET19GST (a gift from Masumi
Hidaka). The Hbo1 and p53 truncations were verified by DNA sequencing.
Antibodies and peptides. Rabbit antibodies to human Orc2 and Mcm2 were
provided by B. Stillman, and a rabbit antibody to Cdtl was obtained from H.
Nishitani. The following reagents were obtained commercially: anti-histidine
epitope tag (H-3), polyclonal antigeminin (FL-209), monoclonal anti-Cdc6 (D-l),
and anti-Mek2 (N-20) (Santa Cruz Biotechnology); monoclonal anti-p53 (BP53-
12), polyclonal anti-H2B, and monoclonal anti-ING1 (CAb3) (Upstate Biotech-
nology); anti-PMS2, anti-Mcm6, monoclonal anti-p53 (polyclonal antibody
[PAb] 1801), and monoclonal anti-C-terminal p53 (PAb 122) (BD Pharmingen);
mouse monoclonal antibody against ?-tubulin (DM1A) and ?-tubulin (GTU-88)
(Sigma); polyclonal anti-ING4 (Abcam); polyclonal anti-ING5 (Rockland Im-
munochemicals); monoclonal anti-p21 (EA10) (Calbiochem); monoclonal anti-
p53 (PAb 122) (BD Biosciences); and monoclonal anti-Mdm2 (D-7) (Santa
Cruz). Peptide L74-1 (residues 158 to 172 of human Hbo1) (32) and peptide
MBP4-14 (human myelin basic protein, residues 4 to 14) were synthesized by
Sawady Technology (Tokyo, Japan). Affinity-purified or crude rabbit polyclonal
antiserum (CS445) against Hbo1 peptide L74-1 (32) was used for Western blots.
Fusion protein GST-p53(1-393) was purchased from Santa Cruz.
Protein purification. His-tagged Hbo1(1-611) and His-Hbo1(311-611) were
expressed in bacteria and purified on heparin-Sepharose (Amersham Pharmacia)
and Ni-nitrilotriacetic acid-agarose (Qiagen). GST-tagged p53 truncations were
expressed in bacteria and purified on glutathione-Sepharose (Amersham Phar-
GST pull-down assay. Two microliters of settled and washed glutathione beads
(Amersham Pharmacia) was incubated with 2 ?g of GST-tagged protein in 0.5 ml
buffer (20 mM Tris-Cl [pH 7.5], 0.2 M NaCl, 10% glycerol, 0.05% NP-40, 1 mM
dithiothreitol) for 2 h at 4°C. After extensive washing with buffer, the beads were
resuspended in 0.5 ml buffer and incubated with 0.5 ?g of input protein for 4 h
at 4°C. After extensive washing, bound proteins were separated by sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and blotted
with antibody to the His epitope tag.
Cell lines, transfection, immunoprecipitation, and HAT assays. HeLa, MCF7,
Saos-2, and NIH 3T3 cell lines were purchased from the ATCC. For immuno-
precipitation and HAT assays or Western blot assays, 1 ?g of DNA was trans-
fected per 10-cm plate using Lipofectamine Plus according to the manufacturer’s
instructions (Life Technologies). Forty-eight hours posttransfection, cells were
lysed with buffer (20 mM Tris-Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 10%
glycerol, 0.5% NP-40). Immunoprecipitation and HAT assays were performed as
reported previously (32). Human Hat1 holoenzyme, purified from 293 cells (68),
was a gift from Alain Verreault. Ethidium bromide (400 ?g/ml) was added to
immunoprecipitation reaction mixtures and washes to prevent protein-nucleic
acid interactions (38). For peptide competition in immunoprecipitation, 2 mg/ml
of peptides was included in the immunoprecipitation and the first washing step.
For assays of HAT inhibition, GST-p53 (Santa Cruz Biotechnology) was added
to reaction mixtures prior to the addition of HAT enzymes. For p53 induction,
MCF7 cells were treated with either 1.5 mM hydroxyurea (HU) (Sigma) or 0.26
M NaCl, and mouse embryo fibroblast (MEF) cells were treated with either 10
mM HU or 0.24 M NaCl. Acetylation reactions and Western blot assays were
quantified by digitizing developed films using an Epson Perfection 4180 photo
scanner and integration of the pixel intensities of the bands.
Adenoviruses and siRNA. Information on the construction of the adenoviruses
is available upon request. Replication-deficient recombinant viruses were cre-
ated as described previously (27). Adenoviral stocks were maintained as de-
scribed previously (47) and purified by cesium chloride density gradient centrif-
ugation. Viruses were used at a multiplicity of infection of 100. SignalSilence p53
small interfering RNA (siRNA) (human specific) (catalog number 6231) was
purchased from Cell Signaling and used for the inhibition of p53 expression. The
control oligonucleotide for the siRNA experiments was siCONTROL Non-Tar-
geting siRNA no. 1 (catalog number D-001210-01-05), which was purchased
from Dharmacon. MCF7 cells were transfected with siRNA oligonucleotides
using RNAiMAX (Invitrogen) and incubated for 48 to 72 h to achieve the
inhibition of p53 expression.
Growth of MEFs. Wild-type p53?/?and homozygous p53?/?MEFs were
prepared from C57BL/6 wild-type and p53 knockout mice (Taconic) as described
previously (42). Cells were collected for HAT assays and protein analysis at
passage 3. For HAT assays, a total of 5 ? 106cells were lysed with M-PER
(Pierce) supplemented with 400 mM NaCl and a 1:100 dilution of protease
inhibitor cocktail for mammalian tissues (Sigma). A total of 2 ? 105cells were
resuspended in 2? Laemmli buffer with ?-mercaptoethanol for whole-cell pro-
tein and Western blot analyses.
Nucleotide sequence accession number. The sequences determined here were
deposited in the GenBank database under accession number DQ076247.
Hbo1 interacts with p53 in human cells. We reasoned that
tumor suppressor p53 was a strong candidate for interactions
with Hbo1, given the role of Hbo1 in cell proliferation and its
VOL. 28, 2008p53 MODULATES Hbo1 HAT ACTIVITY 141
association with ING4 and ING5 (17, 31). To test this hypoth-
esis, anti-Hbo1 immunoprecipitates were prepared from
whole-cell extracts. The proteins were resolved by SDS-PAGE
and assayed by Western blotting using a monoclonal antibody
against p53 (BP53-12). As shown in Fig. 1A (lanes 1 to 6),
Hbo1 antibody specifically coimmunoprecipitated p53 from
extracts of MCF7 cells, a cell line with wild-type p53 expres-
sion. This coimmunoprecipitation was blocked by a peptide for
the Hbo1 antigenic epitope but not by an unrelated peptide or
the inclusion of ethidium bromide to disrupt protein-DNA
In reciprocal experiments, we were unable to detect endog-
enous Hbo1 directly in immunoprecipitates prepared with anti-
p53 antibody, presumably due to the large number of p53
binding partners, the nature of the interactions, or limitations
in the sensitivities of available antibodies. Similar limitations
were reported previously for other p53 binding factors (18, 51,
60). However, when we ectopically expressed FLAG-Hbo1,
coimmunoprecipitation with p53 was readily detected (Fig. 1A,
lanes 7 to 15). Direct analysis of cell lysates by Western blotting
with rabbit anti-Hbo1 confirmed that FLAG-Hbo1 was de-
tected only in cells transfected with the FLAG-Hbo1 construct
and not after mock transfection or transfection with the empty
vector. Furthermore, a mouse monoclonal antibody to the un-
related protein PMS2, but of the same idiotype (immunoglob-
ulin G1 [IgG1]) as that of the antibody to p53, did not immu-
noprecipitate Hbo1. Together, these results demonstrate that
Hbo1 and p53 are subunits of one or more of the same protein
complexes in MCF7 cells.
Endogenous Hbo1, p53, and ING4/ING5 coprecipitate. Our
finding that p53 physically associates with Hbo1 in cells sug-
gested that Hbo1, p53, and ING4 or ING5 might all be com-
ponents of the same protein complexes in cells (17, 57). There-
fore, we sought to test this coassociation and ensure that the
interactions could be detected among the endogenous proteins
and not simply ectopically expressed tagged proteins. As shown
in Fig. 1B, immunoprecipitation of MCF7 whole-cell extracts
with anti-Hbo1 antibody specifically coprecipitated both ING4
and ING5 (lanes 1 to 3). In contrast, we found that anti-Hbo1
antibody failed to coprecipitate ING1 (data not shown), which
is part of a protein complex known to lack Hbo1 (17, 36). In
reciprocal experiments, immunoprecipitation with either anti-
ING4 or anti-ING5 antibodies also coprecipitated Hbo1 (Fig.
1B, lanes 4 to 7). In addition, p53 was clearly present in anti-
FIG. 1. Hbo1, ING4, ING5, and p53 associate in shared protein complexes in cells. (A) Hbo1 and p53 specifically coprecipitate from MCF7
whole-cell extracts. (Left) Results of Western blotting using a monoclonal anti-p53 antibody (?-p53) to detect the presence of p53 in the labeled
samples. Lane 1, 13% of the input MCF7 whole-cell extract used for the other immunoprecipitations; lane 2, control immunoprecipitation (IP)
prepared using purified rabbit IgG; lane 3, precipitates prepared with anti-Hbo1 antibody alone; lane 4, Hbo1 antibody plus the specific antigenic
peptide L74-1; lane 5, Hbo1 antibody plus a nonspecific peptide, MBP4-14; lane 6, Hbo1 antibody plus ethidium bromide (EtBr). (Right) Results
of Western blotting using anti-Hbo1 antibody to detect the presence of Hbo1 in the labeled samples. Lanes 7, 10, and 13, MCF7 cells that were
mock transfected; lanes 8, 11, and 14, cells transfected with FLAG vector without insert; lanes 9, 12, and 15, cells transfected with vector expressing
FLAG-tagged Hbo1; lanes 7 to 9, samples of 4% of the input extracts used for the other immunoprecipitations; lanes 10 to 12, immunoprecipitates
prepared with anti-p53 antibody (PAb 1801) (?-p53); lanes 13 to 15, immunoprecipitates prepared with anti-PMS2 antibody of the same IgG1
subtype as PAb 1801 (?-PMS2). (B) Hbo1 and ING4 coprecipitate, and Hbo1, ING5, and p53 coprecipitate. (Left) Results of Western blotting
of anti-Hbo1 immunoprecipitations using anti-ING4 and anti-ING5 antibodies (lanes 1 to 3). Lane 1, whole-cell extract (WCE) which was first
immunoprecipitated with anti-ING4 antibody (top row) or anti-ING5 antibody (bottom row) and then run together with the control (lane 2) and
anti-Hbo1 immunoprecipitates (lane 3) for Western blotting; lane 2, immunoprecipitates prepared with control preimmune serum; lane 3,
immunoprecipitates prepared with anti-Hbo1 antibody. (Right) Results of Western blotting using anti-Hbo1 and anti-p53 antibodies (lanes 4 to
7). Lane 4, 1% of the whole-cell extract; lane 5, immunoprecipitates prepared with control goat IgG; lane 6, immunoprecipitates prepared with
anti-ING4 antibody; lane 7, immunoprecipitates prepared with anti-ING5 antibody.
142 IIZUKA ET AL.MOL. CELL. BIOL.
ING5 immunoprecipitates but not in anti-ING4 immunopre-
cipitates. However, we cannot rule out association of p53 with
Hbo1-ING4 complexes since the antigenic epitope used to
raise the anti-ING4 antibody may overlap with the p53 binding
domain of ING4 (74). In any case, these results demonstrate
the pairwise coassociations of normal endogenous levels of
Hbo1, ING4, and ING5 and show that p53 also physically
associates with complexes containing Hbo1 and ING5.
Hbo1 directly interacts with p53 in vitro. We next sought to
determine if p53 could interact directly with Hbo1 or whether
its association might be entirely explained by its binding to
ING5. A series of full-length and truncated forms of p53,
representing known functional domains, were expressed as
GST fusions in bacteria and purified, and equivalent amounts
of protein were tested for binding to His-Hbo1 using GST
pull-down assays (Fig. 2A). As shown in Fig. 2B, full-length
GST-p53, but not GST alone, was able to pull down full-length
His-Hbo1 (lanes 1 to 3). Further mapping demonstrated that
this interaction is complex. Deleting the extreme C-terminal 30
amino acids of p53 in derivative GST-p53(1-363) improved the
relative recovery of His-Hbo1 (Fig. 2A, lanes 4 to 6), while the
most efficient copurification was observed with the 108-residue
C-terminal GST-p53(286-393) fragment (lanes 7 and 8). A
substantial portion of the latter interaction could be mapped to
the 33-residue fragment expressed in GST-p53(361-393) (Fig.
2A, lane 16). Thus, this C-terminal region appears to partially
restrict binding to Hbo1 in the context of full-length p53 but
favors interactions with Hbo1 by itself and in the context of
other C-terminal-region fragments.
We carried out similar mapping studies on Hbo1. Full-
length and truncated versions of His-HBO1 were expressed in
bacteria, purified, and assayed using equivalent amounts of
protein in each reaction mixture (Fig. 2C and D). As expected,
constructs containing the entire MYST domain retain HAT
activity, whereas those that lack the MYST domain do not.
Interestingly, Hbo1(311-611) is more active than Hbo1(1-611)
in vitro, suggesting that the N-terminal domain of HBO1 may
negatively regulate the HAT activity of the C-terminal MYST
domain. Surprisingly, GST pull-down experiments revealed
that p53 interacts with the catalytic MYST domain of Hbo1. As
shown in Fig. 2E, full-length GST-p53 was able to interact with
the Hbo1(311-611) MYST domain fragment (lanes 1 to 3).
Indeed, just the C-terminal domain represented by GST-
p53(328-393) also specifically bound to the MYST domain of
Hbo1 (Fig. 2E, lanes 4 to 6). Previous work showed that p53
also interacts with CBP, another histone acetyltransferase, but
in that case, p53 binds to the noncatalytic region of CBP
FIG. 2. p53 binds to Hbo1 directly in vitro. (A) Schematic showing
the locations of known functional domains of human p53 (shaded
boxes). Shown below are the extents of the GST-p53 fusion fragments
tested for their interactions with Hbo1, labeled “A1” to “A8.”
(B) Mapping of the Hbo1 interaction domains of human p53 by GST
pull-down. Full-length His6-Hbo1 protein was loaded at 5% (lane 1)
and 10% (lanes 4, 7, and 10) of the amounts used in the accompanying
binding reaction mixtures. Glutathione beads carrying GST only (lanes
2, 5, 8, and 11) or various GST-p53 deletion mutants (lanes 3, 6, 9, and
12 to 16) were incubated with purified His-Hbo1(1-611) protein. After
extensive washing, the bound proteins were resolved by SDS-PAGE
(10% gel) and assayed by Western blotting using rabbit antiserum to
the His6epitope tag. The individual GST-p53 fusions are labeled “A1”
to “A8” and correspond to the constructs depicted above (A). (C) The
Hbo1 constructs illustrated were expressed as His6-tagged proteins in
bacterial cells and affinity purified. Hbo1(G485A) carries a point mu-
tation predicted to be catalytically dead based on the structure of the
MYST domain (71). The constructs are labeled “C1” to “C5.” ZF, zinc
finger. (D) Results of HAT assays using the subclones shown above
(C). The affinity-purified proteins (12 pmol each) were assayed using
chicken core histones as a substrate in a filter binding assay. The
plotted values represent means and standard errors of the incorpora-
tion of [3H]acetyl-CoA into histone (n ? 3). A control without Hbo1
enzyme is labeled “N,” and the different Hbo1 enzyme fragments used
in the reaction mixtures are labeled “C1” to “C5,” corresponding to
the fragments shown above (C). (E) The C-terminal domain of human
p53 binds the MYST domain of Hbo1. Glutathione beads carrying
GST only (lanes 2 and 5), full-length GST-p53(1-393) protein A1 (lane
3), or C-terminal-domain GST-p53(328-393) protein A3 (lane 6) were
incubated with the MYST domain His-Hbo1(311-611) and processed
as described above. Five percent of the input His6-Hbo1(311-611) is
shown in lane 1, and 10% of the input is shown in lane 4.
VOL. 28, 2008 p53 MODULATES Hbo1 HAT ACTIVITY143
FIG. 3. p53 inhibits Hbo1 HAT activity in vitro and in vivo. (A) Addition of p53 inhibits Hbo1 HAT activity in vitro. The results of HAT assays
using [3H]acetyl-CoA and chicken-free core histones are shown. Control incubations with histones only (lanes 1 and 9) or with histones plus
GST-p53 only (lane 7, 12.5 pmol GST-p53; lane 8, 50 pmol GST-p53) did not give histone acetylation. However, the addition of His-Hbo1 (14
pmol) to the reaction mixtures gave strong histone acetylation (lanes 2 and 10). This activity was inhibited by the addition of GST-p53 (lane 3, 12.5
pmol; lane 4, 50 pmol) but not GST alone (lane 5, 12.5 pmol; lane 6, 50 pmol). Inhibition activity was retained in only the C-terminal regulatory
domain represented on GST-p53(328-393) (lane 11, 12.5 pmol; lane 12, 50 pmol). (B) The inhibition of Hbo1 by p53 in vitro is specific. Hbo1 HAT
activity is not inhibited by GST-p53(286-330), a fragment that does not bind well to the enzyme (Fig. 2B). HAT assays with and without the enzyme
served as positive and negative controls (lanes 1 and 2). His-Hbo1 (14 pmol) was incubated with the GST-p53(286-330) protein (lanes 3 and 4)
or GST alone (lanes 5 and 6) at 12.5 pmol (lanes 3 and 5) and 50 pmol (lanes 4 and 6). A different histone H4 acetyltransferase, Hat1, is not
inhibited by p53 in vitro (lanes 7 to 12). Purified human Hat1 (0.7 pmol) was incubated with free chicken core histones and [3H]acetyl-CoA in the
absence (lane 8) or presence (lanes 9 and 10) of GST-p53 or GST alone (lanes 11 and 12) at 12.5 pmol (lanes 9 and 11) or 50 pmol (lanes 10 and
11). Reactions were separated by SDS-PAGE and assayed by measuring the incorporation of [3H]acetate into histones (“Fluorogram”). The
144 IIZUKA ET AL.MOL. CELL. BIOL.
p53 inhibits Hbo1 HAT activity in vitro. The finding that p53
binds the catalytic domain of Hbo1 suggested that it might
regulate the HAT activity of Hbo1. To test this prediction, we
first compared the HAT activities of purified His-Hbo1 in the
presence and absence of GST-p53 in vitro. As shown in Fig.
3A, GST-p53 specifically inhibits the HAT activity of Hbo1
(lanes 2 to 6). To determine whether this inhibition correlated
with Hbo1 binding, we assayed two fragment derivatives of
p53: GST-p53(328-393), which binds strongly to Hbo1 in pull-
down assays, and GST-p53(286-330), which binds poorly to
Hbo1 (Fig. 2B, lanes 10, 11 and 14). Assays with GST-p53(328-
393) demonstrated that this derivative is sufficient to inhibit the
HAT activity of Hbo1 (Fig. 3A, lanes 9 to 12). In contrast, an
identical concentration of GST-p53(286-330) is poor at inhib-
iting Hbo1 HAT activity (Fig. 3B, lanes 1 to 6). These results
suggest that the inhibition of Hbo1 catalytic activity by p53 may
be a direct consequence of its binding to the enzyme.
Because p53 polypeptides were potential substrates for acet-
ylation by Hbo1, we considered the possibility that p53 inhib-
ited Hbo1 HAT activity by competing as substrate for limiting
amounts of Hbo1 and [3H]acetyl coenzyme A (acetyl-CoA). To
test this alternative, we assayed the ability of Hbo1 to acetylate
these substrates individually. Hbo1 strongly prefers histone
substrates, and p53 acetylation is detected only in reaction
mixtures that do not contain histones, arguing against the com-
petition model (data not shown). We also considered the pos-
sibility that p53 might appear to inhibit Hbo1 activity through
indirect mechanisms such as an associated histone deacetylase
activity or by blocking access to the N-terminal tails. In that
case, p53 would appear to inhibit the acetylation activity of any
HAT enzyme and not just Hbo1. However, we found that the
HAT activity of Hat1, a different histone H4 acetyltransferase,
was not altered in the presence of p53 (Fig. 3B, lanes 7 to 12),
arguing against these alternatives. These results show that the
inhibition of Hbo1 HAT activity by p53 in vitro is specific and
is likely to be a direct consequence of the binding interactions.
p53 inhibits Hbo1 HAT activity in cells. We next sought to
determine whether p53 can inhibit Hbo1 HAT activity in hu-
man cells by expressing the exogenous p53 alleles in p53?/?
Saos-2 osteosarcoma cells (16). Anti-Hbo1 immunoprecipi-
tates were isolated from equivalent amounts of whole-cell
extract, and both the p53 contents and HAT activities of these
immunoprecipitates were compared. As shown in Fig. 3C, p53
protein was observed only in the cells transfected with the p53
expression construct. As expected, this also resulted in the
increased expression of the Mdm2 and p21Waf1/Cip1genes, two
genes transcriptionally activated by p53. However, even though
similar amounts of Hbo1 were present in the immunoprecipi-
tates, the HAT activity of Hbo1 was inhibited more than 2.5-
fold in cells expressing p53. We also transfected Saos-2 cells
with an adenovirus expressing only the C-terminal 66 amino
acids of p53 (p53C). This fragment lacks transcriptional acti-
vation potential, and, as expected, there were no changes in
expression of the p53-activated Mdm2 or p21 gene in cells
transfected with p53C. Nevertheless, the specific HAT activity
of Hbo1 was almost entirely inhibited in cells expressing p53C
(Fig. 3D). Together with our in vitro results, these experiments
demonstrate that overexpressed p53 is capable of negatively
regulating Hbo1 activity in cells through a transcription-inde-
Hbo1 HAT activity in p53?/?and p53?/?mouse cells. Since
the HAT-specific activity of Hbo1 decreases when ectopic p53
or p53C is expressed in Saos-2 cells, we predicted that the
opposite should also be true: Hbo1 HAT activity should in-
crease when p53 is deleted from wild-type cells. MEFs isolated
from p53?/?and p53?/?mice provide a system to test this
prediction. As a first step, we characterized key aspects of
Hbo1 structure and function in the mouse. We subcloned and
sequenced an expressed-sequence-tag cDNA (dbEST acces-
sion number 8230062) for Myst2, the mouse gene encoding
Hbo1 (GenBank accession number DQ076247). The deduced
amino acid sequence is identical to that of its human counter-
part, except for the 63rd amino acid (Gln in human Hbo1 and
Pro in mouse Myst2) and is identical to the coding sequences
of the reference genomic Myst2 locus (GenBank accession
number NT_039521). Western blots of NIH 3T3 cell extracts
revealed an 83-kDa protein comigrating with human Hbo1
(Fig. 4A). Mouse Myst2 immunoprecipitates displayed HAT
activity (data not shown), and a GST fusion with the C-termi-
nal region of mouse p53 (amino acids 325 to 390) interacted
with full-length His-Hbo1 (Fig. 4B).
To assess the role of p53 in regulating Hbo1 HAT activity in
MEF cells, we examined early-passage primary embryonic fi-
broblast cells derived from p53?/?and p53?/?animals. Anal-
ysis of these cultures by flow cytometry demonstrated that they
had similar cell division cycle distributions (Fig. 4C), although
the p53?/?cultures contained a subpopulation of tetraploid
cells, as expected (8, 21). Whole-cell extracts were prepared
from third-passage cell cultures, and Hbo1 complexes were
immunoprecipitated. These Hbo1 immunocomplexes were
then assayed for HAT activity (Fig. 4D). Western blot analysis
showed that the total expression of Hbo1 was the same in both
the p53?/?and p53?/?cells. Nevertheless, the Hbo1 HAT
activity of p53?/?cells was elevated by an average of 3.7-fold ?
1.2-fold (n ? 3) compared to that of wild-type mouse cells.
This increase is consistent with p53 negatively regulating Hbo1
amounts of histone substrate were measured by Coomassie staining of the gels (“Stain”). Histone acetylation activity in these reaction mixtures
required the addition of the Hat1 enzyme as expected (lane 7). (C) Expression of p53 in cells inhibits Hbo1 acetylation activity. Saos-2 cells
(p53?/?) were transiently transfected with either empty pcDNA3.1Zeo(?) plasmid (lane 1) or a pcDNA3.1Zeo(?) clone expressing p53 (lane 2).
Whole-cell extracts were separated by PAGE and assayed by Western blotting with antibodies against p53, Hbo1, Mdm2, p21, and ?-tubulin.
Immunoprecipitates pulled down from the extracts with anti-Hbo1 antibody were assayed for HAT activity using [3H]acetyl-CoA incorporation
(“Fluorogram”) with chicken core histones as a substrate (“Stain”). (D) Expression of the C-terminal domain of p53 is sufficient to inhibit the
HAT activity of Hbo1 in cells. Saos-2 cells were transfected with either adenovirus expressing GFP (lane 1) or adenovirus expressing only
the C-terminal regulatory domain of p53, residues 328 to 393 (lane 2). Whole-cell extracts were probed by Western blotting as described
above (B), with the exception that p53C was detected by slot blot Western analysis of the chromatin-enriched fractions using an antibody
(PAb 122) specific for the C-terminal region of p53. Immunoprecipitation (IP) and HAT assays were carried out as described above (B).
VOL. 28, 2008p53 MODULATES Hbo1 HAT ACTIVITY 145
and, moreover, shows that this role is conserved between
mouse and human cells.
p53C inhibits pre-RC assembly. The direct inhibition of
Hbo1 expression by antisense knockdown or immunodepletion
specifically blocks MCM2-7 association with chromatin, a late
step in pre-RC assembly (31). We reasoned that this step might
also be inhibited by the down-regulation of Hbo1 by p53. To
test this prediction, we transfected HeLa cells with either p53C
FIG. 4. Hbo1 is negatively regulated by p53 in MEFs. (A) Whole-cell extracts of human 293T cells (lane 1) and mouse NIH 3T3 cells (lane 2)
were resolved by 12% SDS-PAGE, transferred onto a membrane, and immunoblotted with anti-Hbo1 antibody. (B) Mouse GST-tagged p53 or
GST alone was incubated with His-Hbo1 and purified on glutathione beads. The bound proteins were separated by SDS-PAGE and immuno-
blotted for His-Hbo1. His-Hbo1 was used in these assays, rather than creating a His-Myst2 construct, since Hbo1 differs from Myst2 only at residue
63, which is outside the region of interaction with p53. (C) Flow cytometry of MEF cell cultures. Primary MEF cells cultured from wild-type mouse
embryos (“p53?/?”) and p53 null embryos (“p53?/?”) were stained with propidium iodide and assayed for DNA content. The histograms of cell
number versus relative DNA fluorescence are shown. The experimentally determined cell counts are plotted as solid dots. The histograms were
fit to G1-phase (green), S-phase (gray), and G2/M-phase (blue) populations, which are plotted at half-scale below the experimental data. The
overall fit is shown by the red line. Note that the p53?/?MEF cell cultures contained both diploid cells, cycling between 1C and 2C DNA content,
and tetraploid cells, cycling between 2C and 4C DNA content (8, 21). Therefore, these cultures were treated as a mixture of cycling diploid and
tetraploid cells with similar division cycle distributions. (D) Whole-cell extracts of the p53?/?and p53?/?cells were probed with anti-Hbo1
antibody (top). Immunoprecipitates prepared with Hbo1 antibody were subjected to HAT assays with [3H]acetyl-CoA, and incorporation was
monitored by fluorography (middle). The histone substrate levels were monitored by Coomassie blue staining (bottom).
146IIZUKA ET AL.MOL. CELL. BIOL.
adenovirus or green fluorescent protein (GFP) control adeno-
virus. After 48 h of infection, the cells were fractionated to give
total cell extract and soluble cytoplasmic (S2), soluble nuclear
(S3), and chromatin-enriched (P3) fractions as described pre-
viously (43). Good fractionation was achieved, as indicated by
the predominant recovery of Mek2 in the cytoplasmic fraction
and Hbo1 in the chromatin fraction (Fig. 5). These fractions
were then subjected to SDS-PAGE, and the amounts of
pre-RC components were determined for each fraction by
Western blotting (Fig. 5). The overall levels of expression of
pre-RC components remained unchanged, with or without
p53C (Fig. 5, lanes 1 and 2). Nevertheless, the overexpression
of p53C caused a sixfold decrease in the chromatin binding of
Mcm2 and Mcm6 (lanes 5 and 6) without affecting the binding
of Orc2, Cdc6, or Cdtl. This outcome matches the effect of
depleting Hbo1 expression in human cells (31). Thus, these
results suggest that p53 can be coupled to the control of
pre-RC assembly in cells through its regulation of Hbo1 HAT
Activation of p53 by hyperosmotic stress inhibits Hbo1. A
strong prediction of this model is that physiological stress con-
ditions that activate p53 should, in turn, inhibit Hbo1 and its
downstream functions such as pre-RC formation. To test this
prediction, we first examined the consequences of high salt, a
stress known to stabilize and activate p53 (35), which can be
imposed during G1phase at the time of pre-RC assembly.
MCF7 cells were arrested at metaphase with nocodazole for
16 h and then released into fresh medium. Cells were allowed
to recover for 2 h, and the cultures were adjusted to 0.26 M
NaCl to activate p53 and incubated for an additional 6.5 h.
Flow cytometry confirmed that cells were arrested at meta-
phase by nocodazole and that both salt-treated cells and their
untreated controls exited metaphase and were in G1phase at
the time of harvest (Fig. 6A). Analysis of whole-cell extracts
demonstrated that salt treatment increased p53 levels approx-
imately 1.7-fold, as expected (Fig. 6B). We also observed a
reproducible decrease of roughly 2.5-fold in Mdm2 and p21
after 6.5 h of salt treatment. The decrease in Mdm2 is in
agreement with data from previous experiments that demon-
strated a transient decrease in Mdm2 in response to osmotic
shock, with a minimum at approximately 6 h posttreatment
(35). This decrease in Mdm2 contributes to the stabilization of
p53. The relative expression of p21 following salt stress in
synchronized MCF7 cells has not been examined previously,
but our results presumably reflect the kinetics of activation of
p53 by phosphorylation of Ser33 by p38MAPK(35). Hbo1 im-
munoprecipitates from these extracts were then assayed for
p53 association and HAT activity. As seen in Fig. 6C, although
similar amounts of Hbo1 were recovered in the precipitates
from control and salt-treated cells, salt stress increased the
amount of p53 recovered in Hbo1 complexes by approximately
3.3-fold. Furthermore, the HAT activity of Hbo1 in the treated
cells was inhibited proportionally by 3.4-fold, consistent with
the activation of p53.
We next assayed the state of pre-RC assembly in control and
salt-treated cells by monitoring the chromatin loading of
pre-RC component proteins. Total cell extracts were fraction-
ated as described above and assayed for p53 levels and pre-RC
subunits by Western blotting (Fig. 6D). We observed either no
change or a slight increase in the overall relative level of
expression of Hbo1 and pre-RC components in salt-treated
cells (Fig. 6D, lanes 1 and 2). As expected, salt treatment
increased p53 levels in the total cell extract and increased its
recovery in the chromatin fraction by 2.4-fold (Fig. 6D, lanes 1
and 2 and 7 and 8). The chromatin-bound levels of Orc2 and
Cdt1 were unaltered by salt treatment. However, the chro-
matin-bound amounts of Mcm2 and Mcm6 were strongly in-
hibited by at least 2.6-fold in extracts from the treated cells,
consistent with down-regulation of Hbo1 by activated p53 (Fig.
6D, lanes 7 and 8). These results show that physiological
stresses that stabilize p53 during the time of DNA replication
licensing result in the inhibition of Hbo1 HAT activity and the
association of MCM2-7 with chromatin.
Activation of p53 by HU arrest inhibits Hbo1. After pre-RC
assembly, DNA replication is initiated by a series of regulated
steps that result in the disassembly of the pre-RC and the
release of MCM2-7 to travel with the DNA replication fork
(3). We were curious whether p53 activation following repli-
cation initiation would also inhibit Hbo1 activity and whether
it would affect the chromatin association of MCM2-7. HU,
which inhibits ribonucleotide reductase, blocks DNA synthesis
within S phase by limiting deoxynucleotide triphosphates (66).
This stress, in turn, stabilizes p53, leading to increased protein
levels (25, 65). To test the effect of this pathway on Hbo1
activity in S phase, we treated MCF7 cells with HU and exam-
ined p53 and Hbo1 functions at time intervals after the block.
Flow cytometry of these cells showed that they were predom-
inantly in G1and S phases with underreplicated DNA (Fig.
7A). The expression of p53 was significantly increased after 6 h
of HU treatment, and p21 expression was increased as well
(Fig. 7B), consistent with previous observations (29). Hbo1
immunoprecipitates were then assayed for HAT activity and
their Hbo1 and p53 protein levels (Fig. 7C). Equivalent
amounts of Hbo1 were recovered in the immunoprecipitates
FIG. 5. A C-terminal fragment of p53 is sufficient to inhibit pre-RC
assembly of the MCM2-7 complex. HeLa cells were transfected with
adenovirus expressing either control GFP or p53C, the C-terminal
regulatory domain of p53 (amino acids 328 to 393). Cells were har-
vested and fractionated as described previously (43). Fractions were
separated by SDS-PAGE and assayed by Western blotting with anti-
bodies against the proteins indicated. The results are shown for the
total cell extracts (TCE), the soluble cytoplasmic fractions (S2), and
the chromatin-enriched fractions (P3).
VOL. 28, 2008p53 MODULATES Hbo1 HAT ACTIVITY147
from control and HU-treated cells; however, the precipitates
from HU-arrested cells were enriched approximately twofold
in the p53 protein. Along with this increased p53 association,
we observed a 2.6-fold decrease in the relative HAT activity of
immunocomplexes recovered from cells arrested with HU.
Thus, as with hyperosmotic shock, the activation of p53 by HU
stress correlated with an inhibition of Hbo1 HAT activity.
Whole-cell extracts were then fractionated as described above
to assess MCM2-7 loading under HU stress conditions (Fig.
7D). As expected, HU treatment increased p53 binding in the
chromatin faction. However, in this case, the chromatin levels
of Mcm2 and Mcm6 did not decrease, despite the down-reg-
ulation of Hbo1 activity (Fig. 7D, lanes 7 and 8). The only
pre-RC factor apparently affected by HU stress was geminin,
which displayed increased expression and association with
chromatin (Fig. 7D, lanes 1, 2, 7, and 8). This is likely due to
the normal increase in its expression during S phase and en-
richment of those cells by HU treatment (48). The continued
association of MCM2-7 with chromatin in the face of de-
creased Hbo1 activity is consistent with the disassembly of the
pre-RC and relocation of MCM2-7 to the replication fork
following replication initiation (7, 11, 37, 40).
The coupling of stress signals to Hbo1 is p53 dependent. If
p53 is mediating the Hbo1 response to salt and HU stress
signals, then the deletion of p53 should abolish this response.
To test this prediction, we first examined the effect of these
stresses on p53?/?and p53?/?MEF cells. Primary early-pas-
sage cell cultures were treated with either NaCl for 3 h or HU
for 6 h. The DNA content histograms of treated and control
cells are shown in Fig. 8A. Whole-cell extracts were prepared
from these cultures and assayed for protein levels by Western
blotting and HAT activity in Hbo1 immunoprecipitates. As
shown in Fig. 8B, both NaCl and HU stresses induced the
activation of p53 in wild-type p53?/?cells greater than sixfold
and caused an inhibition of Hbo1 HAT activity of over 2.5-
fold. In contrast, the HAT activity of Hbo1 was not inhibited by
either high salt or HU arrest in p53?/?cells. These results
demonstrate that the inhibition of Hbo1 HAT activity caused
by hyperosmotic stress and DNA replication arrest is depen-
dent on p53 function in MEFs.
FIG. 6. Salt stress in G1phase activates p53 expression, inhibits Hbo1 HAT activity, and blocks chromatin loading of the MCM2-7 complex.
(A) MCF7 cells were arrested at metaphase with nocodazole for 16 h (“Noc Arrest”). The synchronized cell cultures were then split into fresh
medium without nocodazole and allowed to release from arrest. After 2 h of recovery, one set of cells was left untreated (“Released Untreated”),
while the other was treated with 0.26 M NaCl (“Released ?NaCl”). Cells were harvested from the cultures after 6.5 h, stained for DNA, and
assayed for their cell cycle distributions by flow cytometry. (B) Effect of salt stress on the amounts of key proteins. Whole-cell extracts were
prepared from untreated (lane 1) (“?”) and NaCl-shocked (lane 2) (“?”) cultures described above (A). The extracts were separated by PAGE
and assayed by Western blotting with antibodies against p53, Hbo1, Mdm2, p21, and ?-tubulin. (C) Immunoprecipitates were prepared using
anti-Hbo1 antibody and whole-cell extracts of untreated cells (lane 1) and NaCl-shocked cells (lane 2) as described above (A). Portions of the Hbo1
immunoprecipitates were then assayed by Western blotting for the presence of Hbo1 and p53 (“p53/Hbo1 in the complex”) and for HAT activity
by [3H]acetate incorporation (“Fluorography”) into chicken core histones (“Stain”). (D) MCF7 cells, either untreated or NaCl shocked as
described above (A), were fractionated into total cell extract (TCE), a soluble cytoplasmic fraction (S2), a soluble nuclear fraction (S3), and a
chromatin-enriched fraction (P3) as described previously (43). Fractions were separated by PAGE and assayed by Western blotting with antibodies
against p53, Mcm2, Mcm6, Hbo1, Cdt1, Orc2, and Mek2.
148 IIZUKA ET AL.MOL. CELL. BIOL.
To test the p53 dependence of the pathway in human cells,
we used siRNA to block p53 expression and assayed the inhi-
bition of pre-RC assembly by osmotic shock. MCF7 cells were
transfected with an siRNA oligonucleotide directed against
p53 expression, or a nontargeted control siRNA oligonucleo-
tide, and were incubated for 48 to 72 h. The cells were then
arrested at metaphase by treatment with 50 ng/ml nocodazole
for 16 h, and the rounded mitotic cells were collected by de-
taching them from the culture surface by shaking. Flow cytom-
etry confirmed that the recovered cell population was synchro-
nized in mitosis with a G2DNA content (Fig. 9A). The cells
were then incubated in medium without extra salt for 2.5 h to
allow recovery from mitotic arrest. Cells were then treated with
0.26 M NaCl for 6.5 h and then collected for biochemical
fractionation. As shown in Fig. 9A, both the control siRNA
and p53 siRNA cell populations exited from mitosis and dis-
played similar flow cytometry profiles, with predominantly G1
DNA contents at harvest. In cells transfected with control
siRNA oligonucleotides, osmotic shock increased the amount
of p53 in the whole-cell extracts, and the levels of MDM2 and
p21 decreased (Fig. 9B, lanes 1 and 2). The chromatin associ-
ation of p53 in the P3 fraction also increased about twofold,
and in two independent experiments, the chromatin associa-
tion of Mcm2 and Mcm6 specifically decreased by approxi-
mately twofold (Fig. 9B, lanes 10 and 11). These results are in
agreement with the above-described osmotic shock experi-
ments (Fig. 6) demonstrating that pretreatment with siRNA
did not affect the p53-Hbo1 regulatory pathway. The transfec-
tion of cells with p53 siRNA successfully blocked p53 expres-
sion in total cell extracts and also reduced the association of
p53 in the P3 chromatin fractions (Fig. 9B, lanes 2, 3, 11, and
12). However, osmotic shock failed to inhibit the chromatin
loading of Mcm2 and Mcm6 when p53 expression was knocked
down by siRNA (Fig. 9A, lanes 10 to 12). These results show
that p53 is required to couple the Hbo1-dependent regulation
of pre-RC assembly to osmotic stress.
Our results identify a previously unrecognized pathway that
links stress response signaling through p53 to the output of a
histone acetyltransferase complex. In the case of salt stress
FIG. 7. DNA replication arrest in S phase with HU activates p53 expression and inhibits Hbo1 HAT activity. (A) An asynchronously dividing
culture was split and either left untreated or arrested with 1.5 mM HU treatment. Cells were harvested from the cultures after 6 h of HU treatment
and assayed by flow cytometry. The DNA content histograms are plotted as shown. (B) MCF7 cells were either left untreated (lane 1) (“?”) or
treated for 6 h with 1.5 mM HU (lane 2) (“?”) as described above (A). Whole-cell extracts were separated by PAGE and assayed by Western
blotting with antibodies against p53, Hbo1, p21, and ?-tubulin as a loading control. The pairs of Western blot bands presented in columns 1 and
2 are from nonadjacent lanes of the same gel images. (C) MCF7 whole-cell extracts as described above (A) were immunoprecipitated with
anti-Hbo1 antibody. Portions of the Hbo1 immunoprecipitates were then assayed by Western blotting for the presence of Hbo1 and p53
(“p53/Hbo1 in the complex”) and for HAT activity by [3H]acetate incorporation (“Fluorography”) into chicken core histones (“Stain”). (D) MCF7
cells, either untreated or HU arrested as described above (A), were fractionated into total cell extract (TCE) (lanes 1 and 2), a soluble cytoplasmic
fraction (S2) (lanes 3 and 4), a soluble nuclear fraction (S3) (lanes 5 and 6), and a chromatin-enriched fraction (P3) (lanes 7 and 8) (43). Fractions
were separated by PAGE and assayed by Western blotting with antibodies against p53, Mcm2, Mcm6, Hbo1, geminin, Cdt1, Orc2, and Mek2.
VOL. 28, 2008 p53 MODULATES Hbo1 HAT ACTIVITY149
during G1phase, this pathway results in the inhibition of
pre-RC assembly. These results are consistent with the previ-
ous observation that p53 can inhibit nuclear DNA replication
in vitro in Xenopus egg extracts in a transcription-independent
manner (15). In addition to p53, the tumor suppressors RB and
p16 also regulate the initiation of DNA replication (10, 50).
The Rb protein, which binds to Mcm7, inhibits DNA unwind-
ing in vitro in Xenopus extracts. The tumor suppressor p16
inhibits the chromatin association of minichromosome main-
tenance proteins at G1phase, presumably through the inhibi-
tion of G1cyclin-dependent kinase activity. Thus, tumor sup-
pressor proteins employ diverse molecular mechanisms to
modulate the initiation of DNA replication.
Using antibodies against native proteins, we found that en-
dogenous Hbo1 coprecipitates with endogenous ING4 and
ING5, in agreement with previous work using epitope-tagged
constructs (17). In addition, the Hbo1/ING5 complex also as-
sociates with p53 in MCF7 cells. We considered the possibility
that p53 and ING4/ING5 compete for binding to Hbo1. How-
ever, this is unlikely since the interaction between ING5 and
Hbo1 was not disrupted by the stabilization of p53 following
HU treatment (data not shown). At present, the functions of
ING4 and ING5 in the complexes are unclear. On one hand,
their properties strongly suggest that they are tumor suppres-
sor proteins, formally analogous to p53 in their actions (52, 57).
On the other hand, the inhibition of ING4 and ING5 expres-
sion by RNA interference in HeLa cells causes phenotypes
similar to those produced by the loss of Hbo1 (17, 31); that is,
FIG. 8. Stress regulation of Hbo1 is p53 dependent in MEFs. (A) Flow cytometry of MEF cells. MEF cell cultures from wild-type mouse
embryos (top row) and p53 null embryos (bottom row) were split into three portions and either left untreated, treated with 10 mM HU for 6 h,
or salt shocked with 0.24 M NaCl for 3 h. Cells were harvested, stained for DNA, and assayed by flow cytometry. The DNA content histograms
are plotted as described for Fig. 7A. (B) Primary MEF cell cultures from p53?/?and p53?/?animals were left untreated (lanes 1 and 4), stressed
for 3 h with 0.24 M NaCl (lanes 2 and 5), or treated for 6 h with 10 mM HU (lanes 3 and 6). Whole-cell extracts were probed for levels of Hbo1,
p53, and ?-tubulin, as a loading control, by Western blot assays. Immunoprecipitates were prepared from the extracts with anti-Hbo1 antibody and
assayed for HAT activity by [3H]acetate incorporation (“Fluorogram”) using chicken core histones as a substrate (“Stain”).
150IIZUKA ET AL.MOL. CELL. BIOL.
ING4 and ING5 apparently act as positive factors in their
respective Hbo1/ING/Jade complexes and yet also act in par-
allel with p53, which negatively regulates the Hbo1 HAT ac-
tivities of the complexes. Resolving this apparent paradox
through further molecular and genetic experiments promises
to provide novel insights into the control of cell growth and
Based on our data, and those reported previously by others,
we speculate that Hbo1 and p53 may serve to integrate and
balance conflicting mitogenic and stress signals that impinge
upon cell proliferation. Several observations support the idea
that Hbo1 responds positively to mitogenic signaling pathways
to promote replication licensing and proliferation. For exam-
ple, the cyclin-dependent kinase CDK11p58has recently been
found to bind Hbo1 and simulate its HAT activity (77). This
fits well with our observation that removing the N-terminal
serine-rich domain increases Hbo1 HAT activity (Fig. 2D).
Counterbalancing these proliferative responses, growth-inhib-
itory stress signals would result in p53 activation and thus the
inhibition of Hbo1 activity. Pre-RC assembly provides a model
example of this regulation. The activation of Hbo1 during G1
phase serves to facilitate MCM2-7 loading onto chromatin and
ensure the efficient completion of pre-RC assembly (31). How-
ever, if cells experience hyperosmotic shock during G1phase,
this activates p53 expression, which in turn inhibits Hbo1 ac-
tivity, blocking MCM2-7 loading and delaying pre-RC assem-
bly. Another example of this regulatory pathway may be the
cellular response to human cytomegalovirus infection, which
both increases the expression of p53 and prevents MCM2-7
loading and replication licensing (6, 46, 70).
FIG. 9. Osmotic stress regulation of MCM2-7 loading is p53 dependent. (A) Flow cytometry of synchronized cultures. MCF7 cells were
transfected with either control siRNA or p53-specific siRNA (“p53 siRNA”) and cultured asynchronously for 72 h (“Asynchronous”). Cells were
then arrested in mitosis with nocodazole for 16 h (“Noc Arrest”). The synchronized cell cultures were then split into fresh medium without
nocodazole and allowed to release from arrest. After 2 h of recovery, cells were then treated with 0.26 M NaCl (“Released ?NaCl”). Cells were
harvested from the cultures after 6.5 h, stained for DNA, and assayed for their cell cycle distributions by flow cytometry. (B) MCM2-7 chromatin
loading under osmotic stress is p53 dependent. MCF7 cells were fractionated into total cell extract (TCE) and soluble cytoplasmic, soluble nuclear,
and chromatin-enriched fractions as described previously (43). Fractions were separated by PAGE and assayed by Western blotting with antibodies
against p53, Mcm2, Mcm6, Hbo1, Cdt1, Orc2, and Mek2. Whole-cell extracts were also assayed for MDM2 and p21 by Western blotting. The
standard cell population was transfected with control siRNA but not shocked with NaCl (lanes 1, 4, 7, and 10). The stress control cell population
was transfected with a control siRNA and treated with 0.26 M NaCl (lanes 2, 5, 8, and 11). The stress- and p53-depleted cell population was
transfected with p53 siRNA and treated with 0.26 M NaCl (lanes 3, 6, 9, and 12).
VOL. 28, 2008 p53 MODULATES Hbo1 HAT ACTIVITY151
The extent to which this regulatory pathway influences cell
proliferation remains to be determined, but it is likely to affect
more than just replication licensing. Blocking DNA replication
fork progression with HU causes a p53-dependent down-reg-
ulation of Hbo1 activity (Fig. 8). However, in this case, the
MCM2-7 complex remains bound to chromatin. While this
result makes sense given the importance of MCM2-7 for
stalled replication forks and subsequent replication elongation
following restart (7, 11, 37, 40), it leaves open the question of
why Hbo1 is inhibited under these conditions. One possibility
is that Hbo1 has additional unknown roles during S phase, and
the results of experiments using RNA interference to inhibit
ING4 and ING5 expression support this interpretation (17). In
addition, it is also likely that other functions of Hbo1, such as
its role as a transcriptional coactivator (22, 44, 55), must be
kept in check by p53 during cellular stress responses.
The negative regulation of Hbo1 by p53 suggests that this
HAT may be a candidate oncogene, and several observations
are consistent with this idea. Hbo1 maps to 17q21, a locus that
shows frequent allelic gain in a variety of human tumors.
Moreover, the Hbo1 gene has been found to be amplified or
overexpressed in several breast cancer cell lines (14). As p53 is
deficient in many breast cancers, this would exacerbate the
oncogenic potential of Hbo1 if amplified or overexpressed.
Interestingly, retroviral tagging screens have identified two in-
dependent tumor lines in which the site of retroviral insertion
mapped to locations consistent with a dominant activation of
Hbo1 expression (61). Finally, the acetylation pattern of his-
tone H4 in many cancers is consistent with the substrate spec-
ificity of Hbo1 in vivo (17, 20).
We thank Jacques Co ˆte ´, Herbert Cohen, and Daniel Engel for
discussions; Jacques Co ˆte ´ for communicating research results prior to
publication; and Alain Verreault, Bruce Stillman, and Hideo Nishitani
We thank Rong Li for financial support to M.I. M.I. was supported
in part by UVA P30 CA44579 and the Kincaid Charitable Trust. This
work was supported by a grant-in-aid for the 2nd Term Comprehensive
10-Year Strategy for Cancer Control and a research grant for human
genome and gene therapy from the Ministry of Health and Welfare of
Japan to T.S. and grants from the National Institutes of Health to
C.D.A. (GM53512) and M.M.S. (GM60444).
1. Aggarwal, B., and B. Calvi. 2004. Chromatin regulates origin activity in
Drosophila follicle cells. Nature 430:372–376.
2. Barlev, N., L. Liu, N. Chehab, K. Mansfield, K. Harris, T. Halazonetis, and
S. Berger. 2001. Acetylation of p53 activates transcription through recruit-
ment of coactivators/histone acetyltransferases. Mol. Cell 8:1243–1254.
3. Bell, S., and A. Dutta. 2002. DNA replication in eukaryotic cells. Annu. Rev.
4. Berger, S. 2002. Histone modifications in transcriptional regulation. Curr.
Opin. Genet. Dev. 12:142–148.
5. Bertrand, P., Y. Saintigny, and B. S. Lopez. 2004. p53’s double life: trans-
activation-independent repression of homologous recombination. Trends
6. Biswas, N., V. Sanchez, and D. H. Spector. 2003. Human cytomegalovirus
infection leads to accumulation of geminin and inhibition of the licensing of
cellular DNA replication. J. Virol. 77:2369–2376.
7. Borel, F., F. Lacroix, and R. Margolis. 2002. Prolonged arrest of mammalian
cells at the G1/S boundary results in permanent S phase stasis. J. Cell Sci.
8. Borel, F., O. Lohez, F. Lacroix, and R. Margolis. 2002. Multiple centrosomes
arise from tetraploidy checkpoint failure and mitotic centrosome clusters in
p53 and RB pocket protein-compromised cells. Proc. Natl. Acad. Sci. USA
9. Borrow, J., V. Stanton, Jr., J. Andresen, R. Becher, F. Behm, R. Chaganti, C.
Civin, C. Disteche, I. Dube, A. Frischauf, D. Horsman, F. Mitelman, S.
Volinia, A. Watmore, and D. Housman. 1996. The translocation t(8;16)(p11;
p13) of acute myeloid leukaemia fuses a putative acetyltransferase to the
CREB-binding protein. Nat. Genet. 14:33–41.
10. Braden, W. A., J. M. Lenihan, Z. Lan, K. S. Luce, W. Zagorski, E. Bosco,
M. F. Reed, J. G. Cook, and E. S. Knudsen. 2006. Distinct action of the
retinoblastoma pathway on the DNA replication machinery defines specific
roles for cyclin-dependent kinase complexes in prereplication complex as-
sembly and S-phase progression. Mol. Cell. Biol. 26:7667–7681.
11. Branzei, D., and M. Foiani. 2005. The DNA damage response during DNA
replication. Curr. Opin. Cell Biol. 17:568–575.
12. Burke, T., J. Cook, M. Asano, and J. Nevins. 2001. Replication factors
MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J. Biol.
13. Chuikov, S., J. K. Kurash, J. R. Wilson, B. Xiao, N. Justin, G. S. Ivanov, K.
McKinney, P. Tempst, C. Prives, S. J. Gamblin, N. A. Barlev, and D. Rein-
berg. 2004. Regulation of p53 activity through lysine methylation. Nature
14. Clark, J., S. Edwards, M. John, P. Flohr, T. Gordon, K. Maillard, I. Gid-
dings, C. Brown, A. Bagherzadeh, C. Campbell, J. Shipley, R. Wooster, and
C. Cooper. 2002. Identification of amplified and expressed genes in breast
cancer by comparative hybridization onto microarrays of randomly selected
cDNA clones. Genes Chromosomes Cancer 34:104–114.
15. Cox, L., T. Hupp, C. Midgley, and D. Lane. 1995. A direct effect of activated
human p53 on nuclear DNA replication. EMBO J. 14:2099–2105.
16. Diller, L., J. Kassel, C. Nelson, M. Gryka, G. Litwak, M. Gebhardt, B.
Bressac, M. Ozturk, S. Baker, B. Vogelstein, and S. Friend. 1990. p53
functions as a cell cycle control protein in osteosarcomas. Mol. Cell. Biol.
17. Doyon, Y., C. Cayrou, M. Ullah, A. Landry, V. Co ˆte ´, W. Selleck, W. Lane, S.
Tan, X. Yang, and J. Co ˆte ´. 2006. ING tumor suppressor proteins are critical
regulators of chromatin acetylation required for genome expression and
perpetuation. Mol. Cell 21:51–64.
18. Enari, M., K. Ohmori, I. Kitabayashi, and Y. Taya. 2006. Requirement of
clathrin heavy chain for p53-mediated transcription. Genes Dev. 20:1087–
19. Feng, X., Y. Hara, and K. Riabowol. 2002. Different HATS of the ING1 gene
family. Trends Cell Biol. 12:532–538.
20. Fraga, M., E. Ballestar, A. Villar-Garea, M. Boix-Chornet, J. Espada, G.
Schotta, T. Bonaldi, C. Haydon, S. Ropero, K. Petrie, N. Iyer, A. Perez-
Rosado, E. Calvo, J. Lopez, A. Cano, M. Calasanz, D. Colomer, M. Piris, N.
Ahn, A. Imhof, C. Caldas, T. Jenuwein, and M. Esteller. 2005. Loss of
acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common
hallmark of human cancer. Nat. Genet. 37:391–400.
21. Fukasawa, K., T. Choi, R. Kuriyama, S. Rulong, and G. Vande Woude. 1996.
Abnormal centrosome amplification in the absence of p53. Science 271:
22. Georgiakaki, M., N. Chabbert-Buffet, B. Dasen, G. Meduri, S. Wenk, L.
Rajhi, L. Amazit, A. Chauchereau, C. Burger, L. Blok, E. Milgrom, M.
Lombe ´s, A. Guiochon-Mantel, and H. Loosfelt. 2006. Ligand-controlled in-
teraction of HBO1 with the N-terminal transactivating domain of progester-
one receptor induces SRC-1-dependent co-activation of transcription. Mol.
23. Gibbons, R. 2005. Histone modifying and chromatin remodelling enzymes in
cancer and dysplastic syndromes. Hum. Mol. Genet. 14(Spec. No. 1):R85–
24. Goodman, R., and S. Smolik. 2000. CBP/p300 in cell growth, transformation,
and development. Genes Dev. 14:1553–1577.
25. Gottifredi, V., S. Shieh, Y. Taya, and C. Prives. 2001. p53 accumulates but is
functionally impaired when DNA synthesis is blocked. Proc. Natl. Acad. Sci.
26. Gu, W., X. Shi, and R. Roeder. 1997. Synergistic activation of transcription
by CBP and p53. Nature 387:819–823.
27. Hardy, S., M. Kitamura, T. Harris-Stansil, Y. Dai, and M. Phipps. 1997.
Construction of adenovirus vectors through Cre-lox recombination. J. Virol.
28. Harris, S. L., and A. J. Levine. 2005. The p53 pathway: positive and negative
feedback loops. Oncogene 24:2899–2908.
29. Ho, C. C., W. Y. Siu, A. Lau, W. M. Chan, T. Arooz, and R. Y. Poon. 2006.
Stalled replication induces p53 accumulation through distinct mechanisms
from DNA damage checkpoint pathways. Cancer Res. 66:2233–2241.
30. Huang, J., L. Perez-Burgos, B. J. Placek, R. Sengupta, M. Richter, J. A.
Dorsey, S. Kubicek, S. Opravil, T. Jenuwein, and S. L. Berger. 2006. Re-
pression of p53 activity by Smyd2-mediated methylation. Nature 444:629–
31. Iizuka, M., T. Matsui, H. Takisawa, and M. Smith. 2006. Regulation of
replication licensing by acetyltransferase Hbo1. Mol. Cell. Biol. 26:1098–
32. Iizuka, M., and B. Stillman. 1999. Histone acetyltransferase HBO1 interacts
with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 274:
33. Johnson, T. M., E. M. Hammond, A. Giaccia, and L. D. Attardi. 2005. The
152 IIZUKA ET AL.MOL. CELL. BIOL.
p53QS transactivation-deficient mutant shows stress-specific apoptotic activ- Download full-text
ity and induces embryonic lethality. Nat. Genet. 37:145–152.
34. Kamakaka, R., and S. Biggins. 2005. Histone variants: deviants? Genes Dev.
35. Kishi, H., K. Nakagawa, M. Matsumoto, M. Suga, M. Ando, Y. Taya, and M.
Yamaizumi. 2001. Osmotic shock induces G1 arrest through p53 phosphor-
ylation at Ser33 by activated p38MAPK without phosphorylation at Ser15
and Ser20. J. Biol. Chem. 276:39115–39122.
36. Kuzmichev, A., Y. Zhang, H. Erdjument-Bromage, P. Tempst, and D. Rein-
berg. 2002. Role of the Sin3-histone deacetylase complex in growth regula-
tion by the candidate tumor suppressor p33ING1. Mol. Cell. Biol. 22:835–848.
37. Labib, K., J. A. Tercero, and J. F. Diffley. 2000. Uninterrupted MCM2-7
function required for DNA replication fork progression. Science 288:1643–
38. Lai, J., and W. Herr. 1992. Ethidium bromide provides a simple tool for
identifying genuine DNA-independent protein associations. Proc. Natl.
Acad. Sci. USA 89:6958–6962.
39. Liu, L., D. M. Scolnick, R. C. Trievel, H. B. Zhang, R. Marmorstein, T. D.
Halazonetis, and S. L. Berger. 1999. p53 sites acetylated in vitro by PCAF
and p300 are acetylated in vivo in response to DNA damage. Mol. Cell. Biol.
40. Lupardus, P., T. Byun, M. Yee, M. Hekmat-Nejad, and K. Cimprich. 2002.
A requirement for replication in activation of the ATR-dependent DNA
damage checkpoint. Genes Dev. 16:2327–2332.
41. Machida, Y., J. Hamlin, and A. Dutta. 2005. Right place, right time, and only
once: replication initiation in metazoans. Cell 123:13–24.
42. Maier, B., W. Gluba, B. Bernier, T. Turner, K. Mohammad, T. Guise, A.
Sutherland, M. Thorner, and H. Scrable. 2004. Modulation of mammalian
life span by the short isoform of p53. Genes Dev. 18:306–319.
43. Me ´ndez, J., and B. Stillman. 2000. Chromatin association of human origin
recognition complex, Cdc6, and minichromosome maintenance proteins dur-
ing the cell cycle: assembly of prereplication complexes in late mitosis. Mol.
Cell. Biol. 20:8602–8612.
44. Miotto, B., and K. Struhl. 2006. Differential gene regulation by selective
association of transcriptional coactivators and bZIP DNA-binding domains.
Mol. Cell. Biol. 26:5969–5982.
45. Moll, U. M., S. Wolff, D. Speidel, and W. Deppert. 2005. Transcription-
independent pro-apoptotic functions of p53. Curr. Opin. Cell Biol. 17:631–
46. Muganda, P., O. Mendoza, J. Hernandez, and Q. Qian. 1994. Human cyto-
megalovirus elevates levels of the cellular protein p53 in infected fibroblasts.
J. Virol. 68:8028–8034.
47. Nevins, J., J. DeGregori, L. Jakoi, and G. Leone. 1997. Functional analysis of
E2F transcription factor. Methods Enzymol. 283:205–219.
48. Nishitani, H., S. Taraviras, Z. Lygerou, and T. Nishimoto. 2001. The human
licensing factor for DNA replication Cdt1 accumulates in G1 and is desta-
bilized after initiation of S-phase. J. Biol. Chem. 276:44905–44911.
49. Notterman, D., S. Young, B. Wainger, and A. Levine. 1998. Prevention of
mammalian DNA reduplication, following the release from the mitotic spin-
dle checkpoint, requires p53 protein, but not p53-mediated transcriptional
activity. Oncogene 17:2743–2751.
50. Pacek, M., and J. C. Walter. 2004. A requirement for MCM7 and Cdc45 in
chromosome unwinding during eukaryotic DNA replication. EMBO J. 23:
51. Roe, J. S., H. Kim, S. M. Lee, S. T. Kim, E. J. Cho, and H. D. Youn. 2006.
p53 stabilization and transactivation by a von Hippel-Lindau protein. Mol.
52. Russell, M., P. Berardi, W. Gong, and K. Riabowol. 2006. Grow-ING, age-
ING and die-ING: ING proteins link cancer, senescence and apoptosis. Exp.
Cell Res. 312:951–961.
53. Scolnick, D., N. Chehab, E. Stavridi, M. Lien, L. Caruso, E. Moran, S.
Berger, and T. Halazonetis. 1997. CREB-binding protein and p300/CBP-
associated factor are transcriptional coactivators of the p53 tumor suppres-
sor protein. Cancer Res. 57:3693–3696.
54. Sengupta, S., and C. Harris. 2005. p53: traffic cop at the crossroads of DNA
repair and recombination. Nat. Rev. Mol. Cell Biol. 6:44–55.
55. Sharma, M., M. Zarnegar, X. Li, B. Lim, and Z. Sun. 2000. Androgen
receptor interacts with a novel MYST protein, HBO1. J. Biol. Chem. 275:
56. Shi, X., and O. Gozani. 2005. The fellowships of the INGs. J. Cell. Biochem.
57. Shiseki, M., M. Nagashima, R. Pedeux, M. Kitahama-Shiseki, K. Miura, S.
Okamura, H. Onogi, Y. Higashimoto, E. Appella, J. Yokota, and C. Harris.
2003. p29ING4 and p28ING5 bind to p53 and p300, and enhance p53
activity. Cancer Res. 63:2373–2378.
58. Smith, C., and C. Peterson. 2005. ATP-dependent chromatin remodeling.
Curr. Top. Dev. Biol. 65:115–148.
59. Stedman, W., Z. Deng, F. Lu, and P. Lieberman. 2004. ORC, MCM, and
histone hyperacetylation at the Kaposi’s sarcoma-associated herpesvirus la-
tent replication origin. J. Virol. 78:12566–12575.
60. Sui, G., E. B. Affar, Y. Shi, C. Brignone, N. R. Wall, P. Yin, M. Donohoe,
M. P. Luke, D. Calvo, S. R. Grossman, and Y. Shi. 2004. Yin Yang 1 is a
negative regulator of p53. Cell 117:859–872.
61. Suzuki, T., H. Shen, K. Akagi, H. Morse, J. Malley, D. Naiman, N. Jenkins,
and N. Copeland. 2002. New genes involved in cancer identified by retroviral
tagging. Nat. Genet. 32:166–174.
62. Sykes, S. M., H. S. Mellert, M. A. Holbert, K. Li, R. Marmorstein, W. S.
Lane, and S. B. McMahon. 2006. Acetylation of the p53 DNA-binding
domain regulates apoptosis induction. Mol. Cell. 24:841–851.
63. Tabancay, A. P., and S. L. Forsburg, Jr. 2006. Eukaryotic DNA replication
in a chromatin context. Curr. Top. Dev. Biol. 76:129–814.
64. Tang, Y., J. Luo, W. Zhang, and W. Gu. 2006. Tip60-dependent acetylation
of p53 modulates the decision between cell-cycle arrest and apoptosis. Mol.
65. Taylor, W., M. Agarwal, A. Agarwal, D. Stacey, and G. Stark. 1999. p53
inhibits entry into mitosis when DNA synthesis is blocked. Oncogene 18:
66. Timson, J. 1975 Hydroxyurea. Mutat. Res. 32:115–132.
67. Utley, R., and J. Co ˆte ´. 2003. The MYST family of histone acetyltransferases.
Curr. Top. Microbiol. Immunol. 274:203–236.
68. Verreault, A., P. Kaufman, R. Kobayashi, and B. Stillman. 1998. Nucleoso-
mal DNA regulates the core-histone-binding subunit of the human Hat1
acetyltransferase. Curr. Biol. 8:96–108.
69. Vogelstein, B., D. Lane, and A. Levine. 2000. Surfing the p53 network. Nature
70. Wiebusch, L., R. Uecker, and C. Hagemeier. 2003. Human cytomegalovirus
prevents replication licensing by inhibiting MCM loading onto chromatin.
EMBO Rep. 4:42–46.
71. Yan, Y., N. Barlev, R. Haley, S. Berger, and R. Marmorstein. 2000. Crystal
structure of yeast Esa1 suggests a unified mechanism for catalysis and sub-
strate binding by histone acetyltransferases. Mol. Cell 6:1195–1205.
72. Yang, X. 2004. The diverse superfamily of lysine acetyltransferases and their
roles in leukemia and other diseases. Nucleic Acids Res. 32:959–976.
73. Zhang, K., and S. Dent. 2005. Histone modifying enzymes and cancer: going
beyond histones. J. Cell. Biochem. 96:1137–1148.
74. Zhang, X., K. S. Wang, Z. Q. Wang, L. S. Xu, Q. W. Wang, F. Chen, D. Z.
Wei, and Z. G. Han. 2005. Nuclear localization signal of ING4 plays a key
role in its binding to p53. Biochem. Biophys. Res. Commun. 331:1032–1038.
75. Zhou, J., and C. Prives. 2003. Replication of damaged DNA in vitro is
blocked by p53. Nucleic Acids Res. 31:3881–3892.
76. Zhou, M. I., R. L. Foy, V. C. Chitalia, J. Zhao, M. V. Panchenko, H. Wang,
and H. T. Cohen. 2005. Jade-1, a candidate renal tumor suppressor that
promotes apoptosis. Proc. Natl. Acad. Sci. USA 102:11035–11040.
77. Zong, H., Z. Li, L. Liu, Y. Hong, X. Yun, J. Jiang, Y. Chi, H. Wang, X. Shen,
Y. Hu, Z. Niu, and J. Gu. 2005. Cyclin-dependent kinase 11(p58) interacts
with HBO1 and enhances its histone acetyltransferase activity. FEBS Lett.
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