MOLECULAR AND CELLULAR BIOLOGY,
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Mar. 2001, p. 1854–1865 Vol. 21, No. 5
HIRA, the Human Homologue of Yeast Hir1p and Hir2p, Is a
Novel Cyclin-cdk2 Substrate Whose Expression
Blocks S-Phase Progression
CAITLIN HALL,1DAVID M. NELSON,1XIAOFEN YE,1KAYLA BAKER,2JAMES A. DECAPRIO,2
STEVEN SEEHOLZER,1MARC LIPINSKI,3AND PETER D. ADAMS1*
Fox Chase Cancer Center, Philadelphia, Pennsylvania 191111; Dana-Farber Cancer Institute and Harvard
Medical School, Boston, Massachusetts 021152; and Institut Gustave Roussy, Villejuif, France3
Received 20 July 2000/Returned for modification 11 September 2000/Accepted 7 December 2000
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif
and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 sub-
strates, we searched the database for proteins containing both of these motifs. One such protein is human
HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces
cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2
kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent
manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at
least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA
in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in
S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where
active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase
and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.
The cyclin-dependent kinases (cdks) are key regulators of
progression through the eukaryotic cell cycle (21, 37). For
example, cyclin E- and A-cdk2 promote progression of the cell
from G1phase into and through S phase. Periodic activation of
the different cyclin-cdk complexes is largely responsible for the
characteristic sequence of cell cycle events, such as mitosis,
DNA synthesis, chromatin assembly, and other biosynthetic
processes. However, at present we do not fully understand how
periodic cyclin-cdk activation is translated into other events of
the cell cycle, e.g., S-phase-specific expression of histone genes.
This is largely due to a failure to identify many of the key
Previous studies have shown that cyclin-cdk2 kinases phos-
phorylate serine and threonine residues found within the con-
sensus S/TPXK/R, although the K or R at position ?3 relative
to the phosphoacceptor site is not absolutely required (38, 55,
57, 62). However, it has been appreciated for some time that
the cyclin subunit plays a role in further controlling and re-
stricting substrate specificity beyond this simple requirement
(43). For example, cyclin A but not cyclin E binds to the E2F1
transcription factor (13, 26, 61), allowing cyclin A-cdk2 to
phosphorylate E2F1’s heterodimeric partner, DP1. Recently,
we and others gained novel insight into the role played by the
cyclin in substrate recognition. Specifically, we identified a
short sequence motif that is present in a number of cyclin-cdk2
substrates and is both necessary for and promotes the phos-
phorylation of these substrates (1, 6, 65). This motif, which has
a conserved RXL at its core, is entirely distinct from the
S/TPXK/R phosphoacceptor site. Figure 1a shows a list of
proteins that have been shown to have functional RXL cyclin-
cdk2-binding sequences (1, 2, 6, 14, 30, 33, 46, 47, 65). This list
includes many substrates, such as E2F1, p107, pRB, cdc6, and
the human papillomavirus E1 protein.
Figure 1a indicates that a number of cyclin-cdk2-inhibitory
proteins, namely, p21cip1, p27kip1, and p57kip2, also contain
RXL motifs that are required for efficient binding to the cyclin-
cdk2 complex and inhibition of kinase activity (1, 6, 65). The
RXL motifs derived from substrates and cyclin-cdk2 inhibitors
appear to be functionally equivalent. For example, when
a 10-amino-acid sequence containing the RXL motif of ei-
ther p21cip1or E2F1 was fused to the C terminus of a poorly
phosphorylated retinoblastoma tumor suppressor protein mu-
tant (pRB) which lacks its own RXL motif, phosphorylation
was restored (2). Based on these and other biochemical studies
it was proposed that the RXL motifs of both substrates and
inhibitors serve as docking sites for the binding of the cyclin-
cdk2 kinase. This was confirmed by X-ray crystallographic
structural data for cyclin A-cdk2 complexed to p27kip1or a
p107-derived peptide (5, 45). Both crystal structures and sub-
sequent studies in which mutations were introduced into cyclin
A showed that the RXL motif and surrounding residues inter-
act with a hydrophobic patch on cyclin A (5, 45, 50). The fact
that both substrates and inhibitors have a common sequence
that is required for interaction with the cyclin-cdk2 complex
implies that the inhibitors function, at least in part, by com-
peting with substrates for access to the kinase.
We performed a database search to identify other proteins
that contain putative RXL cyclin-cdk2-targeting sequences and
S/TPXK/R phosphoacceptor sites. One gene product identified
through this screen was a protein previously named HIRA
(28). The primary sequence of HIRA contains an RXL cyclin-
cdk2-binding motif, two potential cyclin-cdk2 phosphorylation
* Corresponding author. Mailing address: Fox Chase Cancer Cen-
ter, 7701 Burholme Ave., Philadelphia, PA 19111. Phone: (215) 728-
7108. Fax: (215) 728-3616. E-mail: email@example.com.
sites that conform to the S/TPXK/R best-fit consensus, and
another 13 S/TP motifs that might also be targets of cyclin-cdk2
kinases. The HIRA gene was originally cloned due to its pres-
ence within a region of human chromosome 22q11.2 that is
deleted in most patients with DiGeorge syndrome (DGS), a
syndrome that results from haploinsufficiency of one or more
gene products (28). The name HIRA is derived from its amino-
acid sequence homology to the products of two Saccharomyces
cerevisiae genes, HIR1 and HIR2, that encode cell cycle-regu-
lated transcriptional repressors of histone gene expression (11,
Here we present data to show that human HIRA is a cyclin-
cdk2 substrate that is phosphorylated in S phase. In addition,
consistent with the notion that human HIRA is a cell cycle
regulator, we have shown that ectopic expression of HIRA
causes cell cycle arrest in S phase. Taken together, these results
indicate that HIRA is a novel cyclin-cdk2 substrate and cell
MATERIALS AND METHODS
Cell culture and transfections. U2OS cells were cultured and transfected as
described previously (1).
Plasmid construction. Wild-type (WT) pGEX2T-E2F1, pGEX2T-E2F1[?24],
pRC-CMVp21, pGEX2T-RB[792–928], pGEX2T-RB[792–829]E2F1Cy, and
pGEX2T-RB[792–829]E2F1CyMut have been described previously (1, 2). pBS-
Cyclin A was a gift of Ed Harlow.
pGEX2T-HIRA[421–729], pGEX2T-HIRA[421–729]?RXL, pGEX2T-RB[792–
829]HIRACy, pGEX2T-RB[792–829]HIRACyMut, pcDNA3-HAHIRA[1–1017],
pcDNA3-HAHIRA[421–729], pcDNA3-HAHIRA[421–729]?RXL, pcDNA3-HA-
HIRA[421–729]T555A, pcDNA3-HAHIRA[520–1017], and pcDNA3-HA-HIRA
[1–1017] were made by standard molecular-biology techniques. All plasmids were
confirmed by direct sequencing and/or restriction digestion. Construction methods
are available on request.
Peptides. Synthetic peptides were purchased from Biosynthesis, Inc. (Lewis-
Antibodies. The anti-simian virus 40 (SV40) T-antigen antibody (419) and the
anti-cyclin A antibody (C160) were gifts from Ed Harlow. The antihemagglutinin
(HA) antibody (12CA5) was purchased from Roche. The anti-cdk2 antibody
(M2) and the anti-p21cip1(sc-187) antibody were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, Calif.). The anti-phosphothreonine-proline an-
tibody (P-thr-pro-101) was purchased from Cell Signaling Technology (Beverly,
The anti-HIRA monoclonal antibodies (WC15, WC19, WC117, and WC119)
were raised against glutathione S-transferase (GST)-HIRA[421–729] in mice
according to standard protocols (19). The anti-HIRA polyclonal antisera (D32
and D34) were raised in rabbits against GST-HIRA[421–729] according to stan-
dard protocols (19).
The anti-phosphothreonine 555 antiserum (D44) was raised in a rabbit. The
phosphopeptide containing threonine 555 (CLSPSVLTpTPSK; synthesized by
the Howard Hughes Medical Institute peptide synthesis facility at Harvard Med-
ical School) was coupled to keyhole limpet hemocyanin (KLH) using a Pierce kit.
Antiserum to the KLH-conjugated phosphopeptide was generated according to
standard protocols. The rabbit serum was used without further purification.
GST fusion protein purification and binding assays. Recovery and purifica-
tion of GST fusion proteins for use in kinase assays was as described previously
Recovery and purification of GST fusion proteins for use in assays of binding
to radiolabeled in vitro translation products were as described previously (1),
except that HD buffer (20 mM HEPES [pH 7.3], 110 mM potassium acetate,
5 mM sodium acetate, 2 mM magnesium acetate, 0.5 mM EGTA, 0.05% digi-
tonin, 1 mM dithiothreitol, 5 ?g of leupeptin/ml, 10 ?g of aprotinin/ml) was sub-
stituted for NETN (120 mM NaCl, 50 mM Tris [pH 8], 1 mM EDTA, 0.5%
In vitro kinase assays. Cyclin A and cdk2 immunoprecipitates and kinase
assays were performed essentially as described previously (1).
Recombinant cyclin A-cdk2 complexes were expressed in and purified from
insect Sf9 cells essentially as described previously (7).
Immunoprecipitation, Western blotting, and treatment with ?-phosphatase.
Immunoprecipitations were performed essentially as described previously (1, 2),
except that in immunoprecipitations of endogenous cellular HIRA the cells were
lysed in 1 ml of EBC (50 mM Tris [pH 8], 0.5% Nonidet P-40, 100 mM NaF, 0.2
mM sodium orthovanadate, 50 ?g of phenylmethylsulfonyl fluoride/ml, 10 ?g of
aprotinin/ml, 5 ?g of leupeptin/ml)–500 mM NaCl per 10 cm-diameter plate of
Western blotting was performed essentially as described previously (1).
Immunoprecipitates were treated with ?-phosphatase as described previously
MS analysis. Immunoprecipitated HIRA protein was purified by sodium do-
decyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with
Coomassie brilliant blue R250. HIRA was excised, destained, equilibrated in 0.1
M NH4HCO3, reduced with dithiothreitol, alkylated with iodoacetamide, and
hydrolyzed in 0.02 ?g of trypsin (porcine; Promega)/ml in 0.05 M NH4HCO3.
Tryptic peptides were extracted with 50% CH3CN–5% formic acid. The super-
natants were reduced in vacuo and applied to a 0.32 (inner diameter)- by 150-mm
C18column (Symmetry 300; Waters Corporation) equilibrated with 5% CH3CN–
0.1% HO-acetic acid. A linear gradient (5 to 80% CH3CN) was developed with
a Waters Alliance 3690 separation module. Output from the column was directed
to the LCQ quadrupole ion trap mass spectrometer (Finnigan) equipped with
the stock microelectrospray ion source. The mass spectrometry (MS)/MS data
were analyzed using the SEQUEST program.
FACS. Two-color fluorescence-activated cell sorting (FACS) to determine the
DNA content of cells transiently transfected with a plasmid encoding CD19 was
performed as described previously (1). Two-color FACS to assay 5?-bromode-
oxyuridine (BrdU) incorporation of cells transiently transfected with CD19 was
performed essentially as described previously (59). Briefly, for 1 h prior to
harvesting cells were pulsed with 10 ?M 5?-BrdU. Cells were harvested, stained
with phycoerythrin-conjugated CD19, and fixed in 70% ethanol essentially as
described previously. Fixed cells were washed in phosphate-buffered saline (PBS)
fixed again in PBS–1% paraformaldehyde–0.01% Tween 20, and then treated
FIG. 1. HIRA contains a putative cyclin-cdk2-binding sequence.
(a) Alignment of the RXL motifs of cell cycle control proteins. Black
boxes, identical residues; grey boxes, conserved residues. The numbers
at the N- and C-terminal ends of each sequence indicate the numbers
of the residues in the protein’s full-length primary sequence. There are
two RXL motifs in p21cip1 (N and C) and at least two in pRB (870 and
886). (b) Schematic of human HIRA primary sequence. Black boxes,
7 WD 40 repeats; grey box, RXL motif (residues 629 to 631), Ps, two
potential cyclin-cdk2 phosphorylation sites that conform to the best-
fit consensus of S/TPXK/R: threonine 555 (TPSK) and serine 687
VOL. 21, 2001 HIRA IS A NOVEL CYCLIN-cdk2 SUBSTRATE1855
with DNase I and stained with anti-5?-BrdU fluorescein isothiocyanate. Samples
were analyzed on a Becton Dickinson FACScan.
Immunofluorescence. For staining of endogenous HIRA, cells grown on cov-
erslips were washed once in EBC–120 mM NaCl and twice in PBS and then fixed
in PBS–4% paraformaldehyde. The staining of ectopically expressed HIRA after
cells were transiently transfected was performed similarly except that cells grown
on coverslips were washed twice in PBS and then fixed directly in PBS–4%
paraformaldehyde. Staining with primary and secondary antibodies and DAPI
(4?,6?-diamidino-2-phenylindole) was essentially as described previously (19).
To identify novel substrates of cyclin A- or E-cdk2 kinases,
we performed a search of the SWISSPROT database for pro-
teins that contain a cyclin-cdk2-docking motif (RXL) and con-
sensus cyclin-cdk2 phosphorylation sites (S/TPXK/R). This
search was carried out using the PatScan program (www
One protein identified through this search, HIRA, was of par-
ticular interest due to its previously described homology to two
S. cerevisiae proteins, Hir1p and Hir2p, that are known to play
a role in control of cell cycle-regulated transcription of histone
genes. Sequence comparisons indicate that HIRA is the best
candidate identified to date to be a human ortholog (functional
equivalent) of Hir1p and Hir2p. Figure 1 a shows an alignment
of the putative cyclin-cdk2-binding motif of HIRA (amino
acids 626 to 633) with the previously characterized cyclin-cdk2-
binding motifs of other human cell cycle control proteins. In
addition to the RXL motif, the HIRA primary sequence con-
tains 2 putative cyclin-cdk2 phosphorylation sites that conform
to the consensus S/TPXK/R (threonine 555 and serine 687), 13
other S/TP motifs that might also serve as cyclin-cdk2 phos-
phorylation sites, and 7 WD repeats (Fig. 1b) (28).
Several RXL-containing cyclin-cdk2 substrates stably bind to
cyclin-cdk2 complexes in a manner that requires the RXL
motif (1, 6, 14, 33, 46, 47, 65). To determine whether HIRA
similarly binds to cyclin A, GST fused to residues 421 to 729
of HIRA (GST-HIRA[421–729]) was tested for binding to in
vitro-translated35S-labeled cyclin A. Residues 421 to 729 of
HIRA contain the RXL motif and both S/TPXK/R cyclin-cdk2
phosphorylation sites (Fig. 1b). As shown in Fig. 2a, GST-
HIRA[421–729] efficiently bound to cyclin A whereas GST
alone or a HIRA mutant containing a four-alanine substitu-
tion in place of the KRKL of the RXL (GST-HIRA[421–729]
?RXL) did not. Similarly, and as described previously, WT
GST-E2F1, but not a mutant lacking the RXL motif (GST-
E2F1?24), bound to cyclin A in this assay (26). All of the WT
and mutant proteins were present in the assay mixture at com-
parable levels (data not shown). Thus, like that of E2F1, HIRA
binding to cyclin A was dependent on an intact RXL cyclin-
Efficient phosphorylation of p107 and E2F1 in vitro by cy-
clin-cdk2 kinase requires that each substrate have an intact
RXL motif (1). We next asked whether HIRA was phosphor-
ylated in vitro by cyclin-cdk2 kinases and whether this too
required an intact RXL cyclin-cdk2-binding motif. Cyclin-cdk2
kinase was immunopurified from asynchronously growing
U2OS cells and tested for its ability to phosphorylate equal
amounts of purified GST-HIRA[421–729] and GST-HIRA
[421–729]?RXL. Cyclin-cdk2 kinase efficiently phosphorylated
the former substrate but not the latter (Fig. 2b). Thus, cyclin-
cdk2 kinase phosphorylates HIRA in vitro and, as for p107 and
E2F1, this requires an intact RXL motif. However, cdk2 com-
plexes immunopurified from asynchronously growing cells con-
sist of cyclin A-cdk2 and cyclin E-cdk2. Therefore we directly
compared the abilities of purified recombinant cyclin A-cdk2
and cyclin E-cdk2 expressed in Sf9 cells to phosphorylate GST-
HIRA[421–729] and, as a control, GST-RB[792–928]. Although
under these particular conditions the ability of each kinase to
phosphorylate GST-HIRA[421–729] was less then its ability to
phosphorylate GST-RB[792–928], both cyclin A- and E-cdk2
efficiently phosphorylated GST-HIRA[421–729] in vitro (Fig.
We have previously shown that short synthetic peptides con-
taining functional RXL motifs will compete with the full-length
proteins for binding to the cyclin-cdk2 complex (1, 6). If the
RXL motif of HIRA serves to target the protein to the cyclin-
cdk2 kinase in a manner similar to that for the RXL motifs of
E2F1 and p107, then a peptide containing the RXL motif of
E2F1 should block phosphorylation of HIRA in vitro. To test
this, kinase assays were performed with cyclin-cdk2 complexes
and GST-HIRA[421–729] or GST-RB[792-928] as a substrate
in the absence or presence of a short synthetic peptide encom-
passing the RXL cyclin-cdk2-binding motif of E2F1. The pep-
tide efficiently blocked phosphorylation of both substrates (Fig.
2d). In contrast, a peptide of identical amino acid composition
but of scrambled sequence blocked phosphorylation of neither
substrate. Thus the RXL motifs of HIRA and E2F1 compete
with one another, suggesting that they are functionally equiv-
Previously, Harlow and coworkers showed that the cyclin-
cdk2-binding domain of E2F1 potentiates phosphorylation of
E2F4 (14). Likewise, we showed that the RXL motifs of E2F1
and p21cip1potentiated the phosphorylation of a poorly phos-
phorylated variant of the retinoblastoma tumor suppressor
protein (pRB) that is mutated to remove all of its RXL motifs
(pRB[792–829]) (2). Therefore, as a final test of the function-
ality of the HIRA RXL motif we tested whether it would also
promote phosphorylation of pRB[792–829]. As shown previ-
ously, when pRB was expressed and purified as a GST fusion
protein, its C terminus (GST-RB[792–928]) was efficiently
phosphorylated by cyclin-cdk2 in vitro whereas GST-RB[792–
829] was not (2). Phosphorylation was restored when 10 amino
acids encompassing the RXL motif of either E2F1 or HIRA
were fused to the C terminus of GST-RB[792–829] (GST-RB
[798–829]E2F1Cy and GST-RB[792–829]HIRACy, respec-
tively) (Fig. 2e). In contrast, fusion of either peptide with
a mutation in the RXL motif did not restore phosphoryla-
tion (GST-RB[792–829]E2F1CyMut and GST-RB[792–829]
HIRACyMut). Thus, the RXL of HIRA can promote phos-
phorylation of a heterologous protein. Taken together these
studies clearly show that, at least in vitro, the HIRA RXL is
functionally similar to the RXL of a known cyclin-cdk2 sub-
To facilitate subsequent studies of the endogenous protein
in vivo, antibodies were raised to HIRA as described in Ma-
terials and Methods. Four monoclonal (WC15, 19, 117, and
119) and two polyclonal antibodies (D32 and D34) were ob-
tained. As determined by Western blotting, each of these ef-
ficiently detected HA-tagged HIRA[421–729] that was ecto-
pically expressed in U2OS cells (Fig. 3a and data not shown).
Furthermore, in extracts of untransfected cells, each antibody
1856HALL ET AL.MOL. CELL. BIOL.
detected a polypeptide with a molecular weight corresponding
to that predicted for HIRA. Immunoprecipitation followed by
Western blot analysis showed that each antibody reacted with
the same polypeptide (Fig. 3b and data not shown), confirming
that each antibody recognizes HIRA.
Using these antibodies we tested whether endogenous HIRA
was phosphorylated in vivo. Extracts derived from U2OS cells
were immunoprecipitated with anti-HIRA monoclonal anti-
body WC15 and the washed immunoprecipitates were treat-
ed with or without ?-phosphatase and fractionated by SDS-
PAGE. Western blotting with WC119 showed that treatment
with ?-phosphatase resulted in increased mobility of HIRA,
indicating that the protein was phosphorylated (Fig. 4a). When
HA-HIRA[421–729] was ectopically expressed in U2OS cells,
it too was phosphorylated, as determined by a ?-phosphatase-
dependent increase in mobility in SDS-PAGE (Fig. 4b).
If HIRA is an in vivo substrate of cyclin-cdk2 kinases, then
it should be phosphorylated in vivo on residues that are con-
sensus cyclin-cdk2 phosphorylation sites and that are phos-
phorylated by cyclin-cdk2 kinase in vitro. To test this, we raised
a rabbit polyclonal serum (D44) against a phosphopeptide
containing a phosphothreonine that corresponds to threonine
555 (one of the two residues that conforms to the cyclin-cdk2
consensus, S/TPXK/R). An enzyme-linked immunosorbent as-
say showed that the rabbit immune, but not preimmune, serum
specifically reacted with the phosphorylated but not the un-
phosphorylated peptide (data not shown). Subsequently, im-
munoprecipitation analysis showed that the anti-phosphothre-
onine 555 serum reacted with GST-HIRA[421–729] only after
the protein had been incubated with cyclin A-cdk2 and mag-
nesium-ATP (Fig. 5a). Thus, the anti-phosphothreonine 555
serum reacted only with GST-HIRA[421–729] phosphorylated
by cyclin A-cdk2, presumably on threonine 555. To determine
whether threonine 555 was phosphorylated on HIRA in vivo
and whether this phosphorylation was dependent on the RXL
motif, U2OS cells were transfected with a plasmid encoding
HA-HIRA[421–729] or a mutant either lacking the RXL
motif (HA-HIRA[421–729]?RXL) or containing a substitu-
tion of alanine for threonine 555 (HA-HIRA[421–729]T555A).
As shown in Fig. 5b, phosphorylation of HA-HIRA[421–729]
on threonine 555 was readily detectable. In contrast, phosphor-
ylation was undetectable on HA-HIRA[421–729]?RXL or
HIRA[421–729]T555A. Thus ectopically expressed HIRA was
phosphorylated on threonine 555 and, like phosphorylation by
cyclin A-cdk2 in vitro, this required an intact RXL cyclin-cdk2-
Next we asked whether the endogenous HIRA protein was
phosphorylated on threonine 555. Extracts from U2OS cells
were immunoprecipitated with an antibody to total HIRA, the
anti-phosphothreonine 555-specific serum, or control antibod-
ies. As shown in Fig. 5c, the phosphoamino acid-specific anti-
body immunoprecipitated the endogenous protein. However,
the phosphoamino acid-specific antibody precipitated a rela-
tively small proportion of total HIRA protein, and this is con-
FIG. 2. The RXL motif of HIRA directs binding to and phosphor-
ylation by cyclin-cdk2 kinases. (a) HIRA binds to cyclin A, and this re-
quires the RXL motif. In vitro-translated
incubated with GST (lane 1), GST-HIRA[421–729] (lane 2), GST-
HIRA[421–729]?RXL (lane 3), GST-E2F1 (lane 4), and -GST-E2F1
?24 (lane 5). The bound proteins were fractionated by SDS-PAGE
and visualized by autoradiography. Arrowhead, cyclin A. (b) HIRA is
phosphorylated by cyclin-cdk2 kinases, and this requires the RXL
motif. Extracts of U2OS cells were immunoprecipitated with antibod-
ies to cdk2 (lane 3 to 7) or SV40 large T antigen (control [con.]; lanes
1 and 2) and used in kinase assays with 0.1 or 1 ?g of GST-HIRA
[421–729] or GST-HIRA[421–729]?RXL as substrates, as indicated.
The phosphoproteins were fractionated by SDS-PAGE and visualized
by autoradiography. Arrowhead, phosphorylated GST-HIRA[421–729].
(c) Phosphorylation of HIRA by purified recombinant cyclin A- and
E-cdk2 in vitro. Cyclin A- and E-cdk2 were expressed in and purified
from Sf9 cells, and increasing amounts were used to phosphorylate 1
?g of GST-RB[792–928] (lanes 1 to 3 and 7 to 9) and GST-HIRA
[421–729] (lanes 4 to 6 and 10 to 12), as indicated. The reactions were
stopped by addition of 3? Laemmli sample buffer, and the phospho-
proteins were separated by SDS-PAGE. Arrowheads, phosphorylated
HIRA and RB; asterisk, autophosphorylated cyclin A. (d) Phosphor-
ylation of HIRA by cyclin-cdk2 is blocked by a peptide containing the
RXL motif of E2F1. Extracts of U2OS cells were immunoprecipitated
with antibodies to cdk2 (lanes 2 to 6 and 8 to 12) or SV40 large T
antigen (control; lanes 1 and 7) and used in kinase assays with 1 ?g of
GST-HIRA[421–729] (lanes 7 to 12) or GST-RB[792–928] (lanes 1 to
6) as the substrate. Kinase assays were performed in the presence of
0.1, 1, or 10 ?g of a 10-residue synthetic peptide that spans the cyclin-
cdk2-binding sequence of E2F1 (WT E2F1; PPVKRRLDLE) or in the
absence of the peptide, as indicated. As controls, assays were per-
formed in the presence of 10 ?g of a peptide of identical amino acid
composition but scrambled sequence (Mut E2F1; lanes 6 and 12). The
phosphoproteins were fractionated by SDS-PAGE and visualized by
autoradiography. Arrowhead, GST-pRB[792–928]; asterisk, GST-
HIRA[421–729]. (e) The RXL motif of HIRA potentiates the phos-
phorylation of another poorly phosphorylated substrate when fused to
the C terminus of that substrate. Extracts of U2OS cells were immu-
noprecipitated with antibodies to cdk2 (lanes 2 to 13) or SV40 large T
35S-labeled cyclin A was
antigen (control; lane 1) and used in kinase assays with 0.1 or 1 ?g of
the indicated protein fused to GST as the substrate. The phosphopro-
teins were fractionated by SDS-PAGE and visualized by autoradiog-
raphy. Arrowheads, relevant GST-pRB fusion proteins.
VOL. 21, 2001 HIRA IS A NOVEL CYCLIN-cdk2 SUBSTRATE1857
sistent with the notion that HIRA is phosphorylated on thre-
onine 555 only under certain conditions, e.g., in specific phases
of the cell cycle. Taken together, these results show that HIRA
is phosphorylated by cyclin A-cdk2 in vitro on a site that is
phosphorylated in vivo, threonine 555.
As an alternative approach to identify the residues of HIRA
that are phosphorylated in vivo, mass spectrometry was uti-
lized. In particular, we were interested in determining whether
other consensus cyclin-cdk2 phosphorylation sites, e.g., serine
687, were phosphorylated. Asynchronously growing U2OS cells
were transfected with a plasmid encoding HA-HIRA[421–
729], and the ectopically expressed protein was immunopuri-
fied with an anti-HA antibody. The protein was purified by
SDS-PAGE, extracted from the gel, proteolytically digested
with trypsin, and subjected to matrix-assisted laser desorption
ionization–time of flight (MALDI-TOF) and liquid chroma-
tography (LC)-MS/MS mass spectrometry. Both approaches
showed that the protein was phosphorylated on the residue
that corresponds to serine 687 of the full-length protein (Fig.
5d and data not shown). In this experiment we failed to obtain
unambiguous data on the phosphorylation status of threonine
555. This could be due to a number of technical aspects of the
mass spectrometry approach and cannot be taken as evidence
that this site is not phosphorylated. Taken together, the mass
spectrometry- and immunology-based approaches indicate that
HIRA is phosphorylated in vivo on both of the consensus
cyclin-cdk2 phosphorylation sites, threonine 555 and serine
If HIRA is a substrate of a cyclin-cdk2 kinase in vivo, then its
phosphorylation should be dependent on cyclin-cdk2 kinase
activity. To test whether this is the case, U2OS cells were
transiently transfected with plasmids encoding HA-HIRA
[421–729] alone or together with a plasmid encoding cyclin-
cdk2 inhibitor p21cip1. When expressed in the absence of
p21cip1, HA-HIRA[421–729] migrated as a doublet (Fig. 6a).
The more slowly migrating band results from phosphorylation
(Fig. 4b and data not shown). However, when expressed in the
presence of p21cip1, HA-HIRA[421–729] migrated as a single
band of higher mobility, indicating that the cyclin-cdk2 inhib-
itor blocks the phosphorylation of HIRA (Fig. 6a).
Next we tested whether inhibition of cyclin-cdk2 kinase
activity inhibited phosphorylation of the mapped cyclin-cdk2
phosphorylation site, threonine 555, and some of the other
potential cdk2 phosphoacceptor sites (specifically threonine
residues followed by proline). U2OS cells were transfected
with a plasmid encoding residues 520 to 1017 of HIRA (HA-
HIRA[520–1017]) in the absence or presence of a plasmid
encoding p21cip1. Residues 520 to 1017 were utilized in this
experiment because, unlike full-length HIRA and residues 421
to 729, this region of the protein does not perturb the cell cycle
(see Fig. 9; data not shown). Phosphorylation of threonine 555
was dramatically inhibited by coexpression of p21cip1, as re-
vealed by immunoprecipitation with the phosphothreonine
555-specific antiserum followed by Western blotting with
anti-HA (Fig. 6b). Likewise, p21cip1inhibited phosphorylation
of HA-HIRA[520–1017], as detected by an anti-phosphothreo-
nine-proline-specific antibody (Fig. 6b). This antibody reacts
with most phosphothreonine residues that are followed by pro-
line. Including T555, there are four such threonine residues
in HA-HIRA[520–1017]. Taken together, these experiments
show that phosphorylation of HIRA in vivo requires cyclin-
FIG. 3. Characterization of antibodies to HIRA. (a) Monoclonal
antibodies to HIRA detect HIRA by Western blotting. U2OS cells
were transfected with a plasmid encoding HA-HIRA[421–729] (odd-
numbered lanes) or with empty vector (even-numbered lanes). Cell
extracts were prepared, and 15 ?g of soluble protein per lane was
fractionated by SDS-PAGE. The proteins were Western blotted with
anti-SV40 T antigen (419), anti-HA (12CA5), or anti-HIRA (WC15,
WC19, WC117, or WC119), as indicated. Arrowhead, HA-HIRA[421-
729]. (b) Monoclonal antibodies to HIRA detect endogenous HIRA by
immunoprecipitation and Western blotting. Extracts were prepared
from U2OS cells and immunoprecipitated with anti-SV40 large T an-
tigen (419) or anti-HIRA (WC15, WC19, WC117, or WC119), as in-
dicated. The proteins were fractionated by SDS-PAGE and Western
blotted with anti-HIRA monoclonal antibody WC119. Arrowhead,
position of HIRA.
FIG. 4. HIRA is phosphorylated in vivo. (a) Endogenous cellular
HIRA is a phosphoprotein. Extracts were prepared from asynchro-
nously growing U2OS cells and immunoprecipitated with anti-SV40
large T antigen (419; lane 1) or anti-HIRA (WC15; lanes 2 and 3). The
immunoprecipitates were treated with (lane 3) or without (lanes 1 and
2) ?-phosphatase, fractionated by SDS-PAGE, and Western blotted
with anti-HIRA (WC119). Upper arrowhead, position of phosphory-
lated HIRA; lower arrowhead, position of unphosphorylated HIRA.
(b) Ectopically expressed HA-HIRA[421–729] is a phosphoprotein.
U2OS cells were transiently transfected with a plasmid encoding HA-
HIRA[421–729] (lanes 2 to 4) or empty vector (lane 1), and extracts
were prepared and immunoprecipitated with anti-HA (12CA5). The
washed immunoprecipitates were treated with (lanes 3 and 4) or with-
out (lanes 1 and 2) ?-phosphatase in the absence (lanes 1 to 3) or
presence (lane 4) of phosphatase inhibitors, fractionated by SDS-
PAGE, and Western blotted with anti-HA (12CA5). Bracket, position
1858 HALL ET AL.MOL. CELL. BIOL.
FIG. 5. HIRA is phosphorylated on two consensus cyclin-cdk2 phosphorylation sites in vivo. (a) Specificity of phosphothreonine 555-specific
antibody D44. GST-HIRA[421–729] (100 ng) was incubated without (lanes 1 and 4) or with (lanes 2, 3, and 5 to 8) recombinant cyclin A-cdk2 in
the presence of MgCl2and ATP. The relative amount of kinase is indicated (titration over a 10-fold range). The reaction mixtures were diluted
and immunoprecipitated (IP) with anti-HIRA (WC15; lanes 1 to 3), anti-phosphothreonine 555 (D44; lanes 4 to 6), or control antibodies (419;
lane 7; D44 preimmune, lane 8). The immunoprecipitates were fractionated by SDS-PAGE and then Western blotted with anti-HIRA (WC119).
Arrowhead, GST-HIRA[421–729]. (b) HA-HIRA[421–729] is phosphorylated in vivo on threonine 555. U2OS cells were transfected with plasmids
encoding HA-HIRA[421–729] (lanes 2, 5, and 6), HA-HIRA[421–729]?RXL (lanes 3 and 7), or HA-HIRA[421–729]T555A (lanes 4 and 8) or with
the empty vector (lane 1). Extracts were prepared and immunoprecipitated with anti-HA (12CA5; lanes 1 to 4), anti-phosphothreonine 555 (D44;
lanes 6 to 8), or the control antibody (D44 preimmune; lane 5). Immunoprecipitates were fractionated by SDS-PAGE and then Western blotted
with anti-HA (12CA5). Arrowhead, HA-HIRA[421–729] and mutants thereof. (c) Endogenous HIRA is phosphorylated on threonine 555 in vivo.
Extracts were prepared from asynchronously growing U2OS cells and immunoprecipitated with anti-HIRA (WC15; lane 2), anti-phosphothreonine
555 (D44; lane 4), or control antibodies (419, lane 1; D44 preimmune, lane 3). Arrowhead, HIRA. (d) HA-HIRA[421–729] is phosphorylated on
serine 687 in vivo. U2OS cells were transfected with a plasmid encoding HA-HIRA[421–729], extracts were immunoprecipitated with anti-HA
(12CA5), and the protein was further purified by SDS-PAGE. The protein band was excised, digested with trypsin, and processed for LC-MS/MS mass
spectrometry. The major ions derived from collision-induced dissociation of tryptic phosphopeptide LPIPpS687PQR (where pS687represents phospho-
serine) are indicated as y- and b-series ions (for definitions of these ions, see reference 22). The difference in the mass/charge (m/z) ratios of ions b1-5
and b1-4 and y1-3 and y1-4 corresponds to phosphoserine rather than serine, showing that the peptide is phosphorylated at this site.
cdk2 kinase activity, as would be expected if HIRA is a sub-
strate of a cyclin-cdk2 kinase.
Another prediction, if HIRA is an in vivo substrate of a
cyclin-cdk2 kinase, is that the protein should be phosphory-
lated when these kinases are active. Both cyclin A- and E-cdk2
are active in growing cells but not in quiescent cells (12, 18, 25,
44). Therefore, we asked whether HIRA was phosphorylated
on threonine 555, a known in vitro phosphorylation site of
cyclin-cdk2 (Fig. 5a), in growing but not quiescent cells. Ex-
tracts derived from asynchronously growing NIH 3T3 cells and
cells that had been deprived of serum for 48 h to induce
quiescence were immunoprecipitated with the anti-phospho-
threonine 555-specific antiserum (D44) and then Western blot-
ted with the anti-HIRA antibody (WC119). Although the total
amounts of HIRA protein in growing and quiescent cells were
the same, the protein was phosphorylated on threonine 555 in
growing cells only (Fig. 7a). Next, we asked whether HIRA is
phosphorylated on threonine 555 at a time in the cell cycle
consistent with it being a cyclin A- or E-cdk2 substrate. Cyclin
A- and E-cdk2 are activated in late G1and S phases after
quiescent cells are stimulated to reenter the cell cycle (18, 25,
39, 44). Extracts derived from quiescent NIH 3T3 cells or cells
refed with serum for 4, 8, or 16 h were analyzed for the
abundance of HIRA phosphorylated on threonine 555. Al-
though, the total amounts of HIRA in quiescent and stimu-
lated cells did not vary (Fig. 7c, top), phosphorylated HIRA
was only detected 16 h after serum stimulation (Fig. 7c, bot-
tom). FACS analysis indicated that phosphorylation tempo-
rally coincided with entry of the cells into S phase (Fig. 7b)
and, presumably, activation of cyclin A- and E-cdk2.
The active forms of cyclin A- and E-cdk2 are localized pre-
dominantly in the cell nucleus (39, 41). Therefore, if HIRA is
an in vivo substrate of these kinases, then it too should be
localized, at least in part, to the cell nucleus. To test whether
this is the case, U2OS cells grown on coverslips were fixed and
stained with monoclonal antibodies to HIRA or a control
antibody. Three separate monoclonal antibodies to endoge-
nous HIRA (WC15, 19, and 119) revealed a specific punctate
nuclear staining pattern (Fig. 8a, b, e, and f and data not
shown). To confirm this staining pattern, U2OS cells were
transiently transfected with a plasmid encoding HA-HIRA[1–
FIG. 6. Phosphorylation of HIRA requires cyclin-cdk2 kinase ac-
tivity. (a) Phosphorylation of HIRA is inhibited by cyclin-cdk2 inhib-
itor p21cip1. U2OS cells were transiently transfected with a plasmid
encoding HA-HIRA[421–729] (lanes 2 and 3) or empty vector (lane 1)
in the absence (lanes 1 and 2) or presence (lane 3) of a plasmid en-
coding p21cip1. Extracts were immunoprecipitated with anti-HA (12CA5),
fractionated by SDS-PAGE, and Western blotted with anti-HA (12CA5).
Upper arrowhead, phosphorylated HA-HIRA[421–729]; lower arrow-
head, unphosphorylated HA-HIRA[421–729]. (b) Phosphorylation of
HIRA on threonine 555 and threonine-proline motifs is inhibited by
cyclin-cdk2 inhibitor p21cip1. U2OS cells were mock-transfected (lane
1) or transiently transfected with a plasmid encoding HA-HIRA[520–
1017] (lanes 2 and 3) in the absence (lanes 1 and 2) or presence (lane
3) of a plasmid encoding p21cip1. Extracts were prepared and immu-
noprecipitated with anti-HA (12CA5; top), anti-phosphothreonine 555
(D44; second from top) or anti-phosphothreonine-proline (P-thr-pro-
101; third from top). The immunoprecipitates were fractionated by
SDS-PAGE and then Western blotted with anti-HA (12CA5). Bottom,
Western blot of the crude cell extracts with the anti-p21cip(SC-187).
Arrowheads, HA-HIRA[520–1017] and p21cip1.
FIG. 7. HIRA is phosphorylated on threonine 555 in S phase of the
cell cycle. (a) Extracts were prepared from asynchronous NIH 3T3
cells growing in 10% fetal bovine serum (FBS) (lanes 1 to 4) or cells
grown in 0.1% FBS for 48 h (lanes 5 to 8) and immunoprecipitated
with the antibody to HIRA (WC15; lanes 2 and 6), phosphothreonine
555 (D44 immune serum; lanes 4 and 8), or the controls (419 and D44
preimmune serum; lanes 1, 3, 5, and 7) as indicated. Immunoprecipi-
tates were Western blotted with the anti-HIRA antibody (WC119).
Arrowhead, HIRA. (b) FACS analysis to determine the DNA content
of propidium iodide-stained NIH 3T3 cells grown in 0.1% FBS for 48 h
and then refed with 10% FBS for 0, 4, 8, or 16 h, as indicated. (c)
Extracts prepared from the NIH 3T3 cells used for panel b were
immunoprecipitated with D44 immune serum and then Western blot-
ted with WC119 (bottom). An aliquot of each cell lysate (150 ?g of
protein) was Western blotted directly with WC119 (top). Arrowheads,
position of HIRA.
1860 HALL ET AL.MOL. CELL. BIOL.
1017] and stained with anti-HA antibody 12CA5. Ectopically
expressed HA-HIRA[1–1017] was present predominantly in
the nucleus, although in some cells cytoplasmic staining was
also observed (Fig. 8c, d, g, and h). This observation is consis-
tent with previous studies by Lorain and coworkers, who de-
tected endogenous HIRA as a primarily nuclear protein in
various other cell lines (32). Taken together, these observa-
tions confirm that HIRA is a predominantly nuclear protein,
and this is consistent with it being an in vivo substrate of cyclin
A- or E-cdk2.
These data clearly show that HIRA is an in vivo substrate of
cyclin A- or E-cdk2. As such, it is a likely regulator of progres-
sion through the cell cycle. Accordingly, we next tested wheth-
er HIRA was able to modulate progression through the cell
cycle. Asynchronously growing U2OS cells were transiently
transfected with a plasmid encoding HA-tagged HIRA[1–
1017], together with a plasmid encoding cell surface marker
CD19, to allow identification of the transfected cells. FACS
analysis showed that ectopic expression of HA-HIRA[1–1017]
in cells caused the percentage of cells in S phase to increase
from 45 to 75% (Fig. 9a). To confirm that this S-phase accu-
mulation was due to an arrest in S phase, cells were again
transiently transfected with plasmids encoding HA-HIRA[1–
1017] and CD19 and 1 h prior to harvesting they were pulse-
labeled with the thymidine analogue 5?-BrdU. FACS analysis
was performed to determine whether the transfected cells had
incorporated 5?-BrdU. As shown in Fig. 9b, approximately
41% of the cells transfected with the empty vector and the
plasmid encoding CD19 were 5?-BrdU positive. This percent-
age of cells in S phase is comparable to the value of 45% that
was obtained by propidium iodide staining (Fig. 9a). In con-
trast, only 3% of the cells ectopically expressing HA-HIRA[1–
1017] were 5?-BrdU positive. Thus, although ectopic expres-
sion of HA-HIRA[1–1017] caused a dramatic accumulation of
cells with DNA content corresponding to that found in S phase
(Fig. 9a), these cells did not incorporate 5?-BrdU (Fig. 9b).
Thus, they were not actively synthesizing DNA and were ar-
rested in S phase.
HIRA is named after its homology to Hir1p and Hir2p of
S. cerevisiae (28). Homologues have also been reported in
mice, chickens, Drosophila melanogaster, Caenorhabditis ele-
gans, and Schizosaccharomyces pombe (24, 31, 42, 48). Based
on comparison of the entire S. cerevisiae genomic sequence
with the Homo sapiens genome that has been sequenced to
date, HIRA is the best candidate so far to be an ortholog
(functional equivalent) of Hir1p and Hir2p. In yeast, Hir1p
and Hir2p control histone gene expression through the cell
cycle. In WT yeast, histones are transcribed periodically
throughout the cell cycle, with expression peaking in S phase.
However, hir1 and hir2 null mutants show constitutive expres-
sion of the HTA1-HTB1 locus that encodes histones H2A and
H2B (40, 52). Accounting for this phenotype, Spector and
coworkers reported that Hir1p and Hir2p are cell cycle-regu-
lated transcriptional repressors of histone genes (56). In addi-
tion, during S phase Hir1p and Hir2p are required to recruit
FIG. 8. HIRA is a nuclear protein. Asynchronously growing U2OS cells grown on coverslips were stained with a control immunglobulin G1
(IgG1) monoclonal antibody (a) or two anti-HIRA monoclonal antibodies together, WC19 and WC119 (both IgG1) (e). DNA was visualized with
DAPI (b and f). U2OS cells grown on coverslips were transfected with a plasmid encoding HA-HIRA[1–1017] and then stained with anti-HA
antibody 12CA5 (d and h). DNA was visualized with DAPI (c and g).
VOL. 21, 2001 HIRA IS A NOVEL CYCLIN-cdk2 SUBSTRATE1861
components of the SWI-SNF chromatin-remodeling complex
to yeast histone promoters, and this is required to overcome
the G1-phase repression of the promoters (11). Interestingly,
relief of Hir1p- and Hir2p-mediated transcriptional repression
requires the yeast cdk, CDC28 (56). Thus, genetic evidence
from yeast suggests that Hir1p and Hir2p might couple cyclical
cdk activity to periodic histone gene expression.
A number of lines of evidence indicate that HIRA is an in
vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to
and was phosphorylated by cyclin-cdk2 kinases in vitro. Impor-
tantly, both binding and phosphorylation required an intact
RXL cyclin-cdk2-binding motif. Furthermore, the RXL of
HIRA is clearly functional in directing substrate phosphoryla-
tion since an E2F1-derived RXL-containing peptide blocked
FIG. 9. Ectopic expression of HIRA causes arrest in S phase of the cell cycle. (a) U2OS cells were transfected without (?HIRA) or with
(?HIRA) a plasmid encoding WT HA-HIRA[1–1017] together with a plasmid encoding cell surface marker pCD19. Two days after transfection
the cells were processed for two-color FACS analysis to determine the DNA content (propidium iodide staining) of the transfected cells (CD19
positive). The dot plots show DNA content of transfected and untransfected cells. Transfected cells (CD19 positive) are boxed (R1). The
histograms show DNA content of only the transfected cells. FITC, fluorescein isothiocyanate. (b) U2OS cells were transfected without (?HIRA)
or with (?HIRA) a plasmid encoding WT HA-HIRA[1–1017] together with a plasmid encoding cell surface marker pCD19. Two days after
transfection the cells were pulse-labeled for 1 h with 5?-BrdU and then processed for two-color FACS analysis to determine whether the transfected
cells were actively synthesizing DNA (5?-BrdU-positive cells). The dot plots show 5?-BrdU labeling of transfected and untransfected cells.
Transfected cells (CD19 positive) are boxed (R1). The histograms show 5?-BrdU labeling of only the transfected cells.
1862 HALL ET AL.MOL. CELL. BIOL.
phosphorylation of HIRA in vitro and the RXL motif from
HIRA strongly potentiated the phosphorylation of a poorly
phosphorylated pRB mutant when it was fused to the C ter-
minus of that substrate. It is important to note that in these in
vitro assays HIRA behaves in a manner identical to that of
other previously characterized cyclin-cdk2 substrates, such as
E2F1, p107, and pRB (1, 2, 6, 14, 30, 33, 46, 47, 65). Second, we
showed that both endogenous cellular HIRA and ectopically
expressed HIRA are phosphorylated proteins in vivo. More-
over, we showed that HIRA is phosphorylated in vivo on two
consensus cyclin-cdk2 phosphorylation sites, threonine 555 and
serine 687. At least one of these sites, threonine 555, was
phosphorylated by cyclin A-cdk2 in vitro, consistent with the
notion that a cyclin-cdk2 kinase is responsible for phosphory-
lation in vivo. Like phosphorylation by cyclin-cdk2 in vitro,
phosphorylation in vivo on threonine 555 required the RXL
motif. Third, when cellular cyclin-cdk kinase activity was in-
hibited by cyclin-cdk2 inhibitor p21cip1, the phosphorylation of
HIRA was blocked. This is consistent with the notion that
HIRA is phosphorylated directly by a cyclin-cdk2 kinase.
Fourth, we showed that HIRA’s phosphorylation was altered
in a cell cycle-dependent manner and that the protein is phos-
phorylated on threonine 555 in S phase, a time in the cell cycle
when cyclin A- and E-cdk2 kinases are active. Fifth, as shown
previously by others (32), we showed that HIRA is localized to
the nucleus where active cyclin-cdk2 complexes are found. In
summary, we have satisfied all of the necessary criteria to
propose that HIRA is an in vivo substrate of a cyclin-cdk2
kinase. At this point, our data are consistent with HIRA being
a substrate of either cyclin A-cdk2, cyclin E-cdk2, or both. A
detailed analysis of the cell cycle kinetics of HIRA phosphor-
ylation and a more detailed comparison of the sites phosphor-
ylated in vivo with those phosphorylated by these kinases in
vitro will contribute to a distinction being drawn between these
Many cyclin-cdk2 substrates are regulators of cell cycle pro-
gression. Consistent with HIRA also being a cell cycle regula-
tor, we show here that, at least when ectopically expressed in
cells, HIRA is able to modulate cell cycle progression. Specif-
ically, ectopic expression of HIRA in cells caused an arrest of
those cells in S phase. The molecular mechanism underlying
this arrest and the extent to which it reflects a physiological
function of HIRA remain to be established. However, the joint
observations that ectopic expression of HIRA arrests cells in S
phase and that HIRA becomes phosphorylated at this time are
consistent with the notion that HIRA normally regulates pro-
gression through S phase and that this activity is modulated by
phosphorylation. If so, then mutation of HIRA phosphoryla-
tion sites to nonphosphorylatable residues might be expected
to modulate the ability of HIRA to cause S-phase arrest. For
example, HIRA might normally restrain progression through S
phase and phosphorylation might overcome this and facilitate
S-phase progression. If so, nonphosphorylatable HIRA mu-
tants might more potently arrest cells in S phase. To date we
have been unable to show an effect of phosphorylation on the
ability of HIRA to regulate the cell cycle. Mutation of both
T555 and S687 to nonphosphorylatable alanine residues did
not affect HIRA activity. However, in addition to T555 and
S687, HIRA contains another 13 S/TP motif that might also be
phosphorylated by cyclin-cdk2 kinase. Indeed, mutation of
T555 and S687 to nonphosphorylatable alanine did not affect
the apparent phosphorylation of the protein as detected in
one-dimensional SDS-PAGE (Fig. 4b and data not shown),
suggesting that HIRA is phosphorylated on additional and/or
alternative sites. Current efforts are directed toward identifying
other phosphorylation sites on HIRA and determining which
of these, either alone or in combination, regulate HIRA activ-
One possible mechanism underlying the S-phase arrest is
suggested by the homology of HIRA to Hir1p and Hir2p of
yeast. Like Hir1p and Hir2p, HIRA might function as a regu-
lator of histone gene expression. In proliferating mammalian
cells, expression of histones peaks in S phase of the cell cycle
as a result of both increased gene transcription and posttran-
scriptional regulation (4, 9, 20, 53, 58). Cis-acting promoter-
specific sequences (or subtype-specific consensus sequences
[SSCEs]) in the gene promoters mediate cell cycle periodicity
of transcription (3, 9, 15, 27, 36). For example, in the promoter
of at least one copy of the human histone H2B gene an Oct-1
site is required for S-phase-specific activation, although Oct-1
DNA binding activity remains unchanged from G1to S phase
(27, 51). Recently, the cyclin E-cdk2 substrate NPAT was
shown to play a role in mediating cyclin-cdk2-dependent acti-
vation of the histone H2B and H4 promoters (34, 64). NPAT
activation of the histone H4 and H2B promoters is dependent
on the SSCE in each case and requires phosphorylation of
NPAT by cyclin E-cdk2. Therefore, NPAT is at least part of
the link between cyclin E-cdk2 activity and histone gene tran-
scription. However, although chromatin immunoprecipitation
assays and immunofluorescence showed that NPAT is located
at histone promoters in vivo, it has not been shown to bind
directly to promoter DNA, and so whether it functions directly
in transcription activation or more indirectly is not clear. Re-
gardless, undoubtedly additional regulators of histone expres-
sion in mammalian cells await identification. HIRA might be
one such regulator. If, like Hir1p and Hir2p, HIRA is a re-
pressor of histone gene expression, then the HIRA-mediated
S-phase arrest might be a consequence of depletion of free
histones. This would presumably prevent the incorporation of
newly replicated DNA into chromatin, which might be ex-
pected to block S-phase progression. However, when drawing
parallels between the function of yeast Hir1p and Hir2p and
that of human HIRA, it should be noted that there are impor-
tant differences between the regulation of histones in yeast and
that in humans. For example, although Hir1p and Hir2p serve
as regulated repressors of histones through the cell cycle (40,
52, 56), most studies of human histone promoters have found
little evidence for regulation of transcription repression. In-
stead, most human histone promoters appear to be regulated
through activation of transcription (3, 9, 15, 27, 36). Therefore,
HIRA might have other functions in S phase that are respon-
sible for the arrest. Interestingly, HIRA also has homology to
the 60-kDa subunit of CAF1 (chromatin assembly factor 1), a
protein that assembles newly replicated DNA and histones into
chromatin (23, 54). Thus, HIRA might couple cyclin-cdk2 ki-
nase activity and formation of chromatin in S phase.
The gene encoding HIRA (HIRA) was originally cloned as a
candidate for a gene whose haploinsufficiency could contribute
to the human developmental disorder DGS (28). DGS is char-
acterized by conotruncal and cardiac abnormalities and hyp-
VOL. 21, 2001 HIRA IS A NOVEL CYCLIN-cdk2 SUBSTRATE1863
oplasia of the thymus and parathyroid glands (49). These de-
fects are thought to result from abnormality of the third and
fourth pharyngeal pouches, and this in turn is thought to be a
consequence of defective early migration of neural crest cells
(49). Proper spatial development of a multicellular organism
requires fine integration of cellular proliferation and cell mi-
gration. As a cell cycle control protein, HIRA could play a role
in such processes. A number of lines of evidence are consistent
with haploinsufficiency of HIRA being responsible for at least
some of the symptoms of DGS. First, the HIRA gene falls
within the region of chromosome 22q11.2 that is commonly
deleted in DGS (the DGS critical region). Second, the protein
product of the HIRA gene is expressed at high levels in the
tissues of developing mouse and chicken embryos, which are
thought to be the sites of the primary developmental defect;
that is, the neural crest and neural-crest-derived tissues (42,
60). Third, when explanted premigratory chicken neural crest
tissue was treated with antisense oligonucleotides designed to
ablate HIRA mRNA and then reimplanted into the chicken
embryo, persistent truncus arteriosus, one of the conotruncal
abnormalities characteristic of DGS patients, was observed
(17). Finally, HIRA has been reported to interact with tran-
scription factor Pax3, and mice lacking Pax3 die in utero with
a phenotype reminiscent of that observed in DGS patients
(35). However, to date, neither HIRA nor any other gene has
been definitively characterized as a DGS gene, and it appears
that haploinsufficiency of more than one gene at 22q11.2 could
be implicated (29).
In summary, we have shown that HIRA is a novel in vivo
substrate of a cyclin-cdk2 kinase and a likely regulator of the
cell cycle. As a cyclin-cdk2 substrate, HIRA keeps company
with a relatively small number of other known proteins. Future
studies of HIRA will address the function of HIRA in cell cycle
control and might shed light on the molecular basis of the
developmental abnormalities characteristic of DGS.
The early part of this work was supported by an NIH grant (1R01
CA76120) to the laboratory of William G. Kaelin, Jr., at the Dana-
Farber Cancer Institute and Harvard Medical School, Boston. This
work was supported by grants to P.D.A. from the W. W. Smith char-
itable trust and the V-foundation. Work in the laboratory of M.L. was
supported by a grant from the Human Frontiers Science and the
Association pour la Recherche sur le Cancer.
We thank Jianmin Gan for excellent technical assistance in gener-
ation of antibodies to HIRA. We thank Jon Chernoff and Randy Strich
for critical reading of the manuscript and Wade Harper and Ken Zaret
for discussion during the course of this work. P.D.A. thanks W.G.K. for
his teaching and generous support.
1. Adams, P. D., W. R. Sellers, S. K. Sharma, A. D. Wu, C. M. Nalin, and W. G.
Kaelin, Jr. 1996. Identification of a cyclin-cdk2 recognition motif present in
substrates and p21-like cyclin-dependent kinase inhibitors. Mol. Cell. Biol.
2. Adams, P. D., X. Li, W. R. Sellers, K. Baker, X. Leng, J. W. Harper, Y. Taya,
and W. G. Kaelin, Jr. 1999. The retinoblastoma protein contains a C-termi-
nal motif that targets it for phosphorylation by cyclin/cdk complexes. Mol.
Cell. Biol. 19:1068–1080.
3. Aziz, F., A. J. van Wijnen, J. L. Stein, and G. S. Stein. 1998. HiNF-D
(CDP-cut/CDC2/cyclin A/pRB-complex) influences the timing of IRF-2-de-
pendent cell cycle activation of human histone H4 gene transcription at the
G1/S phase transition. Cell Physiol. 177:453–464.
4. Baumbach, L. L., G. S. Stein, and J. L. Stein. 1987. Regulation of human
histone gene expression: transcriptional and posttranscriptional control in
the coupling of histone messenger RNA stability with DNA replication.
5. Brown, N. R., M. E. Noble, J. A. Endicott, and L. N. Johnson. 1999. The
structural basis for specificity of substrate and recruitment peptides for
cyclin-dependent kinases. Nat. Cell Biol. 1:438–443.
6. Chen, J., P. Saha, S. Kornbluth, B. D. Dynlacht, and A. Dutta. 1996. Cyclin-
binding motifs are essential for the function of p21cip1. Mol. Cell. Biol. 16:
7. Coleman, T. R., Z. Tang, and W. G. Dunphy. 1993. Negative regulation of the
wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer. Cell
8. Dallapiccola, B., A. Pizzuti, and G. Novelli. 1996. How many breaks do we
need to CATCH on 22q11? Am. J. Hum. Genet. 59:7–11.
9. Dalton, S., and J. R. Wells. 1988. A gene-specific promoter element is
required for optimal expression of the histone H1 gene in S-phase. EMBO
10. DeLisle, A. J., R. A. Graves, W. F. Marzluff, and L. F. Johnson. 1983.
Regulation of histone mRNA production and stability in serum-stimulated
mouse 3T6 fibroblasts. Mol. Cell. Biol. 3:1920–1929.
11. Dimova, D., Z. Nackerdien, S. Furgeson, S. Eguchi, and M. A. Osley. 1999.
A role for transcriptional repressors in targeting the yeast Swi/Snf complex.
Mol. Cell 4:75–83.
12. Dulic, V., W. K. Kaufmann, S. J. Wilson, T. D. Tlsty, E. Lees, J. W. Harper,
S. J. Elledge, and S. I. Reed. 1994. p53-dependent inhibition of cyclin-
dependent kinase activities in human fibroblasts during radiation-induced
G1 arrest. Cell 76:1013–1023.
13. Dynlacht, B. D., O. Flores, J. A. Lees, and E. Harlow. 1994. Differential
regulation of E2F transactivation by cyclin/cdk2 complexes. Genes Dev. 8:
14. Dynlacht, B. D., K. Moberg, J. A. Lees, E. Harlow, and L. Zhu. 1997. Specific
regulation of E2F family members by cyclin-dependent kinases. Mol. Cell.
15. Eilers, A., H. Bouterfa, S. Triebe, and D. Doenecke. 1994. Role of a distal
promoter element in the S-phase control of the human H1.2 histone gene
transcription. Eur. J. Biochem. 223:567–574.
16. Ewen, M. E., H. K. Sluss, C. J. Sherr, H. Matsushime, J. Kato, and D. M.
Livingston. 1993. Functional interactions of the retinoblastoma protein with
mammalian D-type cyclins. Cell 73:487–497.
17. Farrell, M. J., H. Stadt, K. T. Wallis, P. Scambler, R. L. Hixon, R. Wolfe, L.
Leatherbury, and M. L. Kirby. 1999. HIRA, a DiGeorge syndrome candi-
date gene, is required for cardiac outflow tract septation. Circ. Res. 84:127–
18. Girard, F., U. Strausfeld, A. Fernandez, and N. J. Lamb. 1991. Cyclin A is
required for the onset of DNA replication in mammalian fibroblasts. Cell 67:
19. Harlow, E., and D. Lane. 1988. Antibodies: a laboratory manual. Cold Spring
Harbor Press, Cold Spring Harbor, N.Y.
20. Heintz, N. 1991. The regulation of histone gene expression during the cell
cycle. Biochim. Biophys. Acta 1088:327–339.
21. Hunter, T., and J. Pines. 1994. Cyclins and cancer. II. Cyclin D and CDK
inhibitors come of age. Cell 79:573–582.
22. Jimenez, C. R., L. Huang, Y. Qiu, and L. A. Burlingame. 1999. Mass spec-
trometry, p. 16.3.1–16.7.3. In J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W.
Speicher, and P. T. Wingfield (ed.), Current protocols in protein science.
John Wiley & Sons, Inc., New York, N.Y.
23. Kaufman, P. D., J. L. Cohen, and M. A. Osley. 1998. Hir proteins are
required for position-dependent gene silencing in Saccharomyces cerevisiae
in the absence of chromatin assembly factor I. Mol. Cell. Biol. 18:4793–4806.
24. Kirov, N., A. Shtilbans, and C. Rushlow. 1998. Isolation and characterization
of a new gene encoding a member of the HIRA family of proteins from
Drosophila melanogaster. Gene 212:323–332.
25. Koff, A., M. Ohtsuki, K. Polyak, J. M. Roberts, and J. Massague. 1993.
Negative regulation of G1 in mammalian cells: inhibition of cyclin E-depen-
dent kinase by TGF-beta. Science 260:536–539.
26. Krek, W., M. E. Ewen, S. Shirodkar, Z. Arany, W. G. Kaelin, Jr., and D. M.
Livingston. 1994. Negative regulation of the growth-promoting transcription
factor E2F-1 by a stably bound cyclin A-dependent protein kinase. Cell 78:
27. LaBella, F., H. L. Sive, R. G. Roeder, and N. Heintz. 1988. Cell-cycle regu-
lation of a human histone H2b gene is mediated by the H2b subtype-specific
consensus element. Genes Dev. 2:32–39.
28. Lamour, V., Y. Lecluse, C. Desmaze, M. Spector, M. Bodescot, A. Aurias,
M. A. Osley, and M. Lipinski. 1995. A human homolog of the S. cerevisiae
HIR1 and HIR2 transcriptional repressors cloned from the DiGeorge syn-
drome critical region. Hum. Mol. Genet. 4:791–799.
29. Lindsay, E. A., A. Botta, V. Jurecic, S. Carattini-Rivera, Y. C. Cheah, H. M.
Rosenblatt, A. Bradley, and A. Baldini. 1999. Congenital heart disease in
mice deficient for the DiGeorge syndrome region. Nature 401:379–383.
30. Lisztwan, J., A. Marti, H. Sutterluty, M. Gstaiger, C. Wirbelauer, and W.
Krek. 1998. Association of human CUL-1 and ubiquitin-conjugating enzyme
CDC34 with the F-box protein p45(SKP2): evidence for evolutionary con-
servation in the subunit composition of the CDC34-SCF pathway. EMBO J.
31. Llevadot, R., G. Marques, M. Pritchard, X. Estivill, A. Ferrus, and P.
1864HALL ET AL.MOL. CELL. BIOL.
Scambler. 1998. Cloning, chromosome mapping and expression analysis of
the HIRA gene from Drosophila melanogaster. Biochem. Biophys. Res.
32. Lorain, S., J. P. Quivy, F. Monier-Gavelle, C. Scamps, Y. Lecluse, G. Al-
mouzni, and M. Lipinski. 1998. Core histones and HIRIP3, a novel histone-
binding protein, directly interact with WD repeat protein HIRA. Mol. Cell.
33. Ma, T., N. Zou, B. Y. Lin, L. T. Chow, and J. W. Harper. 1999. Interaction
between cyclin-dependent kinases and human papillomavirus replication-
initiation protein E1 is required for efficient viral replication. Proc. Natl.
Acad. Sci. USA 96:382–387.
34. Ma, T., B. A. van Tine, Y. Wei, M. D. Garrett, D. Nelson, P. D. Adams, J.
Wang, J. Qin, L. T. Chow, and J. W. Harper. 2000. Cell cycle-regulated
phosphorylation of p220(NPAT) by cyclin E/Cdk2 in Cajal bodies promotes
histone gene transcription. Genes Dev. 14:2298–2313.
35. Magnaghi, P., C. Roberts, S. Lorain, M. Lipinski, and P. J. Scambler. 1998.
HIRA, a mammalian homologue of Saccharomyces cerevisiae transcrip-
tional co-repressors, interacts with Pax3. Nat. Genet. 20:74–77.
36. Meergans, T., W. Albig, and D. Doenecke. 1998. Conserved sequence ele-
ments in human main type-H1 histone gene promoters: their role in H1 gene
expression. Eur. J. Biochem. 256:436–446.
37. Morgan, D. O. 1997. Cyclin-dependent kinases: engines, clocks, and micro-
processors. Annu. Rev. Cell Dev. Biol. 13:261–291.
38. Nigg, E. 1991. The substrates of the cdc2 kinase. Semin. Cell Biol. 2:261–270.
39. Ohtsubo, M., A. M. Theodoras, J. Schumacher, J. M. Roberts, and M.
Pagano. 1995. Human cyclin E, a nuclear protein essential for the G1-to-S
phase transition. Mol. Cell. Biol. 15:2612–2624.
40. Osley, M. A., and D. Lycan. 1987. Trans-acting regulatory mutations that
alter transcription of Saccharomyces cerevisiae histone genes. Mol. Cell. Biol.
41. Pines, J., and T. Hunter. 1991. Human cyclins A and B1 are differentially
located in the cell and undergo cell cycle-dependent nuclear transport.
J. Cell Biol. 115:1–17.
42. Roberts, C., S. C. Daw, S. Halford, and P. J. Scambler. 1997. Cloning and
developmental expression analysis of chick Hira (Chira), a candidate gene
for DiGeorge syndrome. Hum. Mol. Genet. 6:237–245.
43. Roberts, J. M. 1999. Evolving ideas about cyclins. Cell 98:129–132.
44. Rosenblatt, J., Y. Gu, and D. O. Morgan. 1992. Human cyclin-dependent
kinase 2 is activated during the S and G2 phases of the cell cycle and
associates with cyclin A. Proc. Natl. Acad. Sci. USA 89:2824–2828.
45. Russo, A. A., P. D. Jeffrey, A. K. Pattern, J. Massague, and N. P. Pavletich.
1996. Crystal structure of the p27Kip1cyclin-dependent-kinase inhibitor
bound to the cyclin A-Cdk2 complex. Nature 382:325–331.
46. Saha, P., Q. Eichbaum, E. D. Silberman, B. J. Mayer, and A. Dutta. 1997.
p21cip1and Cdc25A: competition between an inhibitor and an activator of
cyclin-dependent kinases. Mol. Cell. Biol. 17:4338–4345.
47. Saha, P., J. Chen, K. C. Thome, S. J. Lawlis, Z. H. Hou, M. Hendricks, J. D.
Parvin, and A. Dutta. 1998. Human CDC6/Cdc18 associates with Orc1 and
cyclin-cdk and is selectively eliminated from the nucleus at the onset of S
phase. Mol. Cell. Biol. 18:2758–2767.
48. Scamps, C., S. Lorain, V. Lamour, and M. Lipinski. 1996. The HIR protein
family: isolation and characterization of a complete murine cDNA. Biochim.
Biophys. Acta 1306:5–8.
49. Schinke, M., and S. Izumo. 1999. Getting to the heart of DiGeorge syn-
drome. Nat. Med. 10:1120–1121.
50. Schulman, B. A., D. L. Lindstrom, and E. Harlow. 1998. Substrate recruit-
ment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin
A. Proc. Natl. Acad. Sci. USA 95:10453–10458.
51. Segil, N., S. B. Roberts, and N. Heintz. 1991. Mitotic phosphorylation of the
Oct-1 homeodomain and regulation of Oct-1 DNA binding activity. Science
52. Sherwood, P. W., S. V. M. Tsang, and M. A. Osley. 1993. Characterization of
HIR1 and HIR2, two genes required for regulation of histone gene transcrip-
tion in Saccharomyces cerevisiae. Mol. Cell. Biol. 13:28–38.
53. Sive, H. L., N. Heintz, and R. G. Roeder. 1984. Regulation of human histone
gene expression during the HeLa cell cycle requires protein synthesis. Mol.
Cell. Biol. 12:2723–2734.
54. Smith, S., and Stillman. 1989. Purification and characterization of CAF-1, a
human cell factor required for chromatin assembly during DNA replication
in vitro. Cell 58:15–25.
55. Songyang, Z., S. Blechner, N. Hoagland, M. F. Hoekstra, H. Piwnica-Worms,
and L. C. Cantley. 1994. Use of an orientated peptide library to determine
the optimum substrates of protein kinases. Curr. Biol. 4:973–982.
56. Spector, M. S., A. Raff, H. DeSilva, K. Lee, and M. A. Osley. 1997. Hir1p and
Hir2p function as transcriptional corepressors to regulate histone gene tran-
scription in the Saccharomyces cerevisiae cell cycle. Mol. Cell. Biol. 17:545–
57. Srinivasan, J., M. Koszelak, M. Mendelow, Y.-G. Kwon, and D. S. Lawrence.
1995. The design of peptide based substrates for the cdc2 protein kinase.
Biochem. J. 309:927–931.
58. Stein, G. S., J. L. Stein, A. J. Van Wijnen, and J. B. Lian. 1996. Transcrip-
tional control of cell cycle progression: the histone gene is a paradigm for the
G1/S phase and proliferation/differentiation transitions. Cell Biol. Int. 20:41–
59. Tough, D. F., and J. Sprent. 1996. Measurement of T and B cell turnover
with 5-BrdU, supplement 18, p. 4.7.1–4.7.6. In J. E. Coligan, A. M. Kruis-
beek, D. H. Margulies, E. M. Shevach, and W. Strober (ed.), Current pro-
tocols in immunology. John Wiley & Sons, Inc., New York, N.Y.
60. Wilming, L. G., C. A. Snoeren, A. van Rijswijk, F. Grosveld, and C. Meijers.
1997. The murine homologue of HIRA, a DiGeorge syndrome candidate
gene, is expressed in embryonic structures affected in human CATCH22
patients. Hum. Mol. Genet. 6:247–258.
61. Xu, M., K. A. Sheppard, C. Y. Peng, A. S. Yee, and H. Piwnica-Worms. 1994.
Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA-binding ac-
tivity of E2F-1/DP-1 by phosphorylation. Mol. Cell. Biol. 14:8420–8431.
62. Zhang, J., R. J. Sanchez, S. Wang, C. Guarnaccia, A. Tossi, S. Zahariev, and
S. Pongor. 1994. Substrate specificity of cdc2 kinase from human HeLa cells
as determined with synthetic peptides and molecular modelling. Arch. Bio-
chem. Biophys. 315:415–424.
63. Zhao, J., B. K. Kennedy, B. D. Lawrence, D. A. Barbie, A. G. Matera, J. A.
Fletcher, and E. Harlow. 2000. NPAT links cyclin E-Cdk2 to the regulation
of replication-dependent histone gene transcription. Genes Dev. 14:2283–
64. Zhao, J., Dynlacht, T. Imai, T. Hori, and E. Harlow. 1998. Expression of
NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry. Genes
65. Zhu, L., E. Harlow, and B. D. Dynlacht. 1995. p107 uses a p21cip1-related
domain to bind cyclin/cdk2 and regulate interactions with E2F. Genes Dev.
VOL. 21, 2001HIRA IS A NOVEL CYCLIN-cdk2 SUBSTRATE 1865