The kinase haspin is required for mitotic
histone H3 Thr 3 phosphorylation
and normal metaphase chromosome
Jun Dai,1Sammy Sultan,1Stephen S. Taylor,2and Jonathan M.G. Higgins1,3
1Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital,
Harvard Medical School, Boston, Massachusetts 02115, USA;2Faculty of Life Sciences, University of Manchester,
Manchester M13 9PT, United Kingdom
Post-translational modifications of conserved N-terminal tail residues in histones regulate many aspects of
chromosome activity. Thr 3 of histone H3 is highly conserved, but the significance of its phosphorylation is
unclear, and the identity of the corresponding kinase unknown. Immunostaining with phospho-specific
antibodies in mammalian cells reveals mitotic phosphorylation of H3 Thr 3 in prophase and its
dephosphorylation during anaphase. Furthermore we find that haspin, a member of a distinctive group of
protein kinases present in diverse eukaryotes, phosphorylates H3 at Thr 3 in vitro. Importantly, depletion of
haspin by RNA interference reveals that this kinase is required for H3 Thr 3 phosphorylation in mitotic cells.
In addition to its chromosomal association, haspin is found at the centrosomes and spindle during mitosis.
Haspin RNA interference causes misalignment of metaphase chromosomes, and overexpression delays
progression through early mitosis. This work reveals a new kinase involved in composing the histone code
and adds haspin to the select group of kinases that integrate regulation of chromosome and spindle function
during mitosis and meiosis.
[Keywords: Chromatin; centromere; mitosis; serine/threonine kinase; histone modification; chromosome
Supplemental material is available at http://www.genesdev.org.
Received September 29, 2004; revised version accepted December 17, 2004.
To facilitate cell division, the processes of chromatin
condensation, chromosome alignment on a bipolar
spindle, chromosome separation, and cytokinesis must
occur in a defined sequence. To ensure this orderly pro-
gression, the regulation of chromatin structure and
spindle activity must be precisely integrated and check-
points must be satisfied before subsequent steps are al-
lowed. Errors in coordinating these events can lead to
genomic instability and aneuploidy, contributing to the
generation of cancer and birth defects. The elucidation of
these regulatory mechanisms therefore is a major goal of
current cell biology. To date, a select group of kinases
has been found to orchestrate mitosis. In particular,
members of the cyclin-dependent kinase, Aurora, Polo,
and NIMA/Nek families phosphorylate substrates in
chromatin and at the spindle apparatus to regulate
events during cell division and to signal the outcome of
the various checkpoints (Nigg 2001).
In chromatin, the core histones H3, H2B, H2A, and H4
form nucleosomal octamers around which DNA is
wound to form the basic organizing structure of the chro-
mosome. There has been great excitement recently as
the importance of phosphorylation, acetylation, methyl-
ation, and ubiquitinylation of the core histones has been
revealed. These modifications produce a high degree of
combinatorial complexity, the so-called “histone code,”
that forms the basis of a critical regulatory system in the
control of chromatin structure (Jenuwein and Allis 2001;
Turner 2002). Modulation of chromatin structure is of
particular significance for cell division, as the chromo-
somes undergo extensive compaction prior to segrega-
Not surprisingly, histones are major targets of mitotic
kinases. For example, histone H3 is extensively phos-
phorylated at Ser 10 during mitosis and meiosis (Hendzel
et al. 1997; Prigent and Dimitrov 2003). The function of
this modification is debated, but it may facilitate chro-
matin condensation or the release of cohesin and ISWI
chromatin-remodeling ATPases (Van Hooser et al. 1998;
Andrews et al. 2003; Prigent and Dimitrov 2003; Swed-
low and Hirano 2003). A Tetrahymena strain with his-
tone H3 mutated at Ser 10 showed perturbed chromatin
condensation and abnormal chromosome segregation
E-MAIL email@example.com; FAX (617) 525-1010.
Article published online ahead of print. Article and publication date are
472GENES & DEVELOPMENT 19:472–488 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org
during meiosis and mitosis (Wei et al. 1999), while a
similar mutation in Saccharomyces cerevisiae had no
such effect (Hsu et al. 2000). Therefore, histone phos-
phorylation at Ser 10 has an important role in mitosis,
but the extent to which it is required appears to be spe-
cies dependent, perhaps because of redundancy provided
by other mitotic histone modifications (Hsu et al. 2000).
In fact, a number of highly conserved serine and threo-
nine residues that might be phosphorylated are found in
the core histones of all eukaryotes. Mitotic phosphory-
lation of Thr 11 (Preuss et al. 2003) and Ser 28 (Goto et al.
1999) of H3 and Thr 119 of Drosophila H2A (Aihara et al.
2004) have been reported.
The identities of protein kinases that phosphorylate
the histones during mitosis in vivo remain somewhat
uncertain. The best studied is aurora B, a “chromosome
passenger protein” that is located on the chromosomes
during prophase and becomes concentrated at inner cen-
tromeres by metaphase before relocalizing to the spindle
midzone at anaphase (Carmena and Earnshaw 2003).
Consistent with this, aurora B has both chromatin and
spindle-associated substrates and influences mitosis at a
number of steps. Aurora B homologs play an important
role in ensuring chromosome bi-orientation at meta-
phase by correcting mono-orientated attachments to the
spindle, and are involved in normal chromatid separa-
tion and cytokinesis (Shannon and Salmon 2002; An-
drews et al. 2003). They are also required for phosphory-
lation of the centromeric histone variant CENP-A at Ser
7 and of histone H3 at Ser 10 in many organisms (Hsu et
al. 2000; Adams et al. 2001; Giet and Glover 2001; Pe-
tersen et al. 2001; Zeitlin et al. 2001; Crosio et al. 2002;
Ditchfield et al. 2003; Hauf et al. 2003). It has not been
possible, however, to unambiguously assign the role of
mitotic histone H3 Ser 10 phosphorylation solely to au-
rora B (Nigg 2001; Prigent and Dimitrov 2003). Indeed, in
Aspergillus, mitotic histone H3 Ser 10 phosphorylation
is dependent on the kinase NIMA (De Souza et al. 2000).
In addition, kinases that bring about the phosphorylation
of other histone residues during mitosis must exist. The
nature of these key enzymes remains unclear, although
there is some evidence that aurora B, Dlk/ZIP kinase,
and NHK-1 are responsible for mitotic phosphorylation
of H3 Ser 28, Thr 11, and H2A Thr 119, respectively
(Goto et al. 2002; Preuss et al. 2003; Aihara et al. 2004).
Haspin/Gsg2 (haploid germ cell-specific nuclear pro-
tein kinase/germ cell-specific gene-2) was first identified
as a testis-specific gene in mice (Tanaka et al. 1994,
1999). We identified the human haspin gene and con-
firmed that haspin mRNA levels are highest in testis. In
addition, in both humans and mice we detected lower
levels of haspin mRNA in other organs and in all prolif-
erating cell lines tested, suggesting that expression of
haspin is not truly haploid germ cell-specific (Higgins
2001a). Genes encoding haspin homologs are present in
all major eukaryotic phyla, including yeasts, microspo-
ridia, plants, nematodes, flies, fish, amphibians, and
mammals (Higgins 2003). These haspin genes encode
proteins that contain a distinctive C-terminal putative
kinase domain and together constitute a novel eukary-
otic protein kinase family (Higgins 2001b). The N-termi-
nal portion of the haspin proteins is less conserved
among species and has no clear homology with known
domains (Tanaka et al. 1999; Yoshimura et al. 2001; Hig-
In this study we show that phosphorylation of Thr 3 of
histone H3 occurs during mitosis and that the major ki-
nase responsible for this modification in cultured cells is
haspin. Overexpression and RNA interference experi-
ments show that haspin is required for normal mitotic
chromosome alignment. Consistent with this function,
haspin associates with chromatin and spindle compo-
nents and is phosphorylated during mitosis.
Subcellular localization of haspin
To determine the subcellular localization of haspin, we
transfected HeLa cells with myc-tagged human haspin
and conducted anti-myc immunofluorescence staining.
Myc-haspin was found exclusively in the nucleus during
interphase. More intense staining was evident surround-
ing subnuclear structures (Fig. 1A). A similar haspin pat-
tern was reported previously in transfected HEK293 and
COS cells, although the identity of the compartment
was not determined (Tanaka et al. 1999, 2001). Double
staining with anti-myc and antibodies to B23/nucleo-
phosmin (Fig. 1A) demonstrated that these structures are
the nucleoli. Confocal fluorescence microscopy at 37°C
of live HeLa cells transfected with EGFP-haspin con-
firmed that haspin localized to the nucleus in a pattern
similar to that of DNA. The accumulation at perinucleo-
lar regions was not as clear as that seen in fixed cells (Fig.
1B), suggesting that fixation preferentially stabilizes
In fixed mitotic cells, myc-haspin (Supplementary Fig.
1A) and EGFP-haspin (data not shown) were found asso-
ciated with the condensed chromosomes in prophase
through anaphase. In metaphase spreads, myc-haspin
was found on chromosome arms, but the most intense
staining was in centromeric regions (Fig. 1C). When we
visualized live HeLa cells at 37°C by video microscopy,
EGFP-haspin was present on the condensing chromo-
somes in prophase, and remained associated with the
condensed chromosomes throughout mitosis (Fig. 1D;
Supplementary Movie). We then examined haspin local-
ization in more detail by confocal microscopy of live
mitotic cells (Fig. 1B). In addition to the localization of
EGFP-haspin on chromosomes, it also appeared at cen-
trosomes in prometaphase through telophase. Weaker lo-
calization of EGFP-haspin to spindle fibers emanating
from the centrosome was apparent from metaphase on,
as was localization of EGFP-haspin to the midbody of
telophase cells. The localization of EGFP-haspin and
?-tubulin in fixed cells confirmed that haspin was asso-
ciated with spindle poles (Fig. 1E). EGFP alone was found
exclusively in the cytoplasm of mitotic cells (data not
To assess the localization of untagged haspin, we pro-
Haspin is a histone H3 kinase
GENES & DEVELOPMENT473
duced an affinity-purified polyclonal antibody to a pep-
tide representing human haspin amino acids 329–344. By
immunoblotting, we found the antibody was specific for
haspin in transfected HEK293 cells (Fig. 2A) and HeLa
cells (see Fig. 5A, below), but was unable to detect en-
dogenous haspin. Upon fractionation of transfected cells
into nucleus and cytoplasm-enriched fractions by hypo-
tonic lysis, haspin was found only in the nuclear fraction
(Fig. 2A). Immunofluorescence of transfected untagged
haspin in HeLa cells with this antibody confirmed nuclear
localization in interphase, as well as chromosomal asso-
ciation during mitosis (data not shown). Although EGFP-
haspin can be detected at the centrosome in fixed cells
(Fig. 1E), we have so far been unable to establish a fixa-
tion technique that allows immunostaining of haspin or
myc-haspin at the spindle or centrosome.
(A) Interphase HeLa cells transiently transfected with a vector encoding myc-haspin were analyzed by immunofluorescent staining
with anti-myc-FITC (green) and anti-nucleophosmin/B23 followed by anti-goat IgG-Cy3 antibodies (red). DNA was visualized with
Hoechst 33342. The arrowhead indicates a nontransfected cell. (B) Live cell confocal fluorescence microscopy of HeLa cells stably
transfected with a vector encoding EGFP-haspin under a doxycycline-inducible promoter and treated with doxycycline for 1 d. DNA
was visualized with the cell-permeable dye DRAQ5 (blue). Images on the left are projections of confocal image stacks and those on the
right show individual confocal sections from a single metaphase, anaphase, and telophase cell. Arrows indicate the presence of haspin
at the centrosomes, and arrowheads indicate haspin associated with the spindle. (C) Metaphase chromosome spreads from myc-haspin
transfected HeLa cells were fixed and stained with anti-myc-FITC (green) and DNA was visualized with DRAQ5 (blue). (D) HeLa cells
treated as in B were analyzed by live cell epifluorescence video microscopy at 37°C. Selected images of EGFP fluorescence (green) are
shown superimposed on corresponding bright-field images. The entire series is available as a Supplementary Movie. (E) Confocal
immunofluorescence microscopy of a metaphase HeLa cell expressing EGFP-haspin (green), treated with 0.2% Triton X-100 in PHEM
buffer followed by 3% formaldehyde. DNA was visualized with DRAQ5 (blue) and mouse anti-?-tubulin staining with anti-mouse
IgG-Cy3 (red). Arrows indicate the presence of haspin but not DNA at the spindle poles.
Haspin is a nuclear protein during interphase and associates with the chromosomes and spindle apparatus during mitosis.
Dai et al.
474 GENES & DEVELOPMENT
Haspin is a histone H3 kinase
We next examined the in vitro kinase activity of haspin.
For these experiments we prepared a control haspin pro-
tein (myc-haspin-KD) containing a mutation of a single
conserved lysine residue (K511A) that is critical for ac-
tivity in essentially all protein kinases. Immunoprecipi-
tates from vector-alone-, myc-haspin-, and myc-haspin-
KD-transfected HEK293 cell lysates were subjected to in
vitro kinase assays in the presence of ?32P-ATP (Fig. 2B).
While no phosphorylated proteins were generated in as-
says of vector-transfected cells, a radiolabeled band of
∼85 kDa was visible in myc-haspin immunoprecipitates.
This band coincided with the position of myc-haspin de-
tected by immunoblotting, suggesting that myc-haspin
had undergone autophosphorylation (Fig. 2B). No such
phosphorylation was apparent when myc-haspin-KD im-
munoprecipitates were examined, providing evidence
that the kinase activity observed is intrinsic to the
haspin kinase domain.
Importantly, in the in vitro kinase assays with myc-
haspin immunoprecipitates from both HEK293 (Fig. 2B)
and HeLa cells (data not shown), we observed an addi-
tional phosphorylated band of ∼17 kDa. One possibility
was that this was a protein directly immunoprecipitated
by the antihaspin antibody due to cross-reactivity. Simi-
lar results were obtained, however, when myc-haspin
was immunoprecipitated with an anti-myc tag mono-
clonal antibody (data not shown), indicating that this
was not the case. We reasoned that the 17-kDa protein
might be a phosphorylation substrate that coimmuno-
precipitates with haspin. Recognizing the chromosomal
location of haspin during mitosis, and that the core his-
tones are major cellular components that have molecular
masses in the range of 14–17 kDa, we sought to deter-
mine if histones could serve as exogenous substrates for
haspin. Strikingly, when a purified mixture of histones
H1, H3, H2B, H2A, and H4 was tested, haspin showed
exquisite selectivity for a single band of ∼17 kDa, re-
markably similar in size to the endogenous phosphory-
lated band (Fig. 2C). No such band was obtained from in
vitro kinase assays of myc-haspin-KD or vector only
transfected cells. Based on an overlay of the autoradio-
gram with the Coomassie blue-stained SDS-PAGE gel,
the phosphorylated band appeared to coincide with his-
tone H3. When purified histones were tested separately
as targets for haspin kinase activity, we confirmed that
histone H3 was the most efficiently phosphorylated, al-
though in this situation weaker phosphorylation of other
histones was also observed (Supplementary Fig. 1B). In
intact nucleosomes, haspin phosphorylated only histone
richia coli and lacking post-translational modifications
(Luger et al. 1997) was also an efficient substrate for
haspin activity (Fig. 2E), indicating that pre-existing his-
tone modifications are not a requirement for haspin ac-
tion in vitro. A recombinant form of histone H3 lacking
the N-terminal 26 “tail” residues (gH3) was not phos-
phorylated by haspin (Fig. 2E), suggesting that the phos-
phorylation site resides within this region. Recombinant
histones H2B and H4 were relatively poor substrates of
haspin (Fig. 2E). We conclude that histone H3 can asso-
HEK293 cells were transiently transfected with a vector
encoding human haspin or vector alone. Fractions en-
riched for cytoplasmic (C) and nuclear (N) components
were obtained by hypotonic lysis and analyzed by immu-
noblotting with affinity-purified rabbit antihaspin 329–
44 antibodies. Appropriate fractionation was verified by
immunoblotting for the endoplasmic reticulum protein
calnexin and the nuclear protein histone H3. (B) HEK293
cells were transiently transfected with vectors encoding
myc-haspin or myc-haspin-KD or with vector alone. Rab-
bit antihaspin immunoprecipitates from lysed cells were
subjected to in vitro kinase reactions with ?32P-ATP as
described in Materials and Methods. That equivalent
amounts of myc-haspin and myc-haspin-KD were pres-
ent in the immunoprecipitates was verified by immu-
noblotting with rabbit antihaspin antibodies. (C) In vitro
kinase reactions of myc-haspin or myc-haspin-KD with
or without the addition of purified calf thymus histones
as an exogenous substrate were carried out as described
in B. The positions of the histones in the gel were deter-
mined by Coomassie blue staining. (D) In vitro kinase
reactions of rabbit antihaspin immunoprecipitates from
myc-haspin- or vector-alone-transfected HeLa cells using
intact polynucleosomes (with or without the linker his-
tone H1) as exogenous substrates were carried out as de-
scribed in B. (E) In vitro kinase reactions of rabbit anti-
haspin immunoprecipitates from myc-haspin- or vector-
Haspin is a histone H3 kinase in vitro. (A)
alone-transfected HEK293 cells with recombinant Xenopus histones purified from E. coli as exogenous substrates were carried out as
described in B. (gH3) Tailless histone H3.
Haspin is a histone H3 kinase
GENES & DEVELOPMENT475
ciate with and serve as a substrate for the haspin kinase,
at least in vitro.
Haspin phosphorylates histone H3 at Thr 3
The N-terminal tail regions of the histones are exposed
in nucleosomal oligomers and are the major targets of
the histone modifications so far analyzed (Jenuwein and
Allis 2001; Turner 2002). To determine if haspin targets
a residue in the N-terminal tail of H3, we generated a
protein (H3–GST) containing the first 45 residues of H3
fused to the N terminus of GST to preserve the normal
orientation of the tail. Immunoprecipitates of myc-
haspin from transfected cells were able to phosphorylate
H3–GST but not GST alone or H2B–GST or H4–GST
(Fig. 3A). To identify residues within the tail that are
required for phosphorylation by haspin, we generated
H3–GST proteins containing mutations to alanine of
each of the seven serine and threonine residues present
(T3A, T6A, S10A, T11A, T22A, S28A, T32A). The mu-
tation T3A abolished phosphorylation by haspin, while
the other mutant H3–GSTs behaved as wild type (Fig.
3A), suggesting that Thr 3 is the target of phosphoryla-
tion by haspin or that it is required for association of
haspin with H3–GST.
To examine the interaction of haspin with H3, we uti-
lized the H3–GST fusion proteins in “pulldown” assays
from lysates of myc-haspin-transfected cells (Fig. 3B). In
the conditions of the assay, myc-haspin bound to H3–
myc-haspin or vector alone. Immunoprecipitates from cell lysates with rabbit antihaspin antibodies (haspin) or with nonimmune
rabbit IgG (neg) were subjected to in vitro kinase assays with ?32P-ATP and histone tail–GST proteins as exogenous substrates.
Equivalent loading of the GST proteins was verified by Coomassie blue staining. (B) Lysates of HEK293 cells transiently transfected
with a vector encoding myc-haspin were incubated with glutathione-Sepharose beads coated with histone tail–GST proteins. After
washing, the binding of myc-haspin, cyclin A, and PCNA to the beads was assessed by immunoblotting. The lane marked lysate was
loaded with 1/20 volume of the input lysate, and equivalent loading of the GST proteins was verified by Coomassie blue staining of
the blot. (C) Peptides representing histone H3 amino acids 1–8 either without [H3(1–8)] or with phosphorylated Thr 3 [H3(1–8)pT3] or
peptides containing phosphorylated Thr 11 [H3(9–16)pT11] or Thr 22 [H3(20–27)pT22] were immobilized by slot blotting and probed
with rabbit anti-phospho-histone H3 (Thr-3) antibody B8634. Similar results were obtained with the commercial polyclonal anti-
phospho-histone H3 (Thr-3) antibody (Upstate), although some lower cross-reactivity with the phospho-Thr 22 peptide was observed
in this case. (D) Purified recombinant wild-type 6His-haspin kinase domain (kinase) or 6His-kinase containing the mutation K511A
(kinase-KD) was examined by SDS-PAGE and Coomassie blue staining. (E) In vitro kinase reactions were carried out using 15 ng
recombinant haspin 6His-kinase domain (kinase) or 6His-kinase-KD (kinase-KD) with 0.5 µg purified recombinant H3, H3–GST,
H3-T3A–GST, and GST alone as substrates. The products were analyzed by immunoblotting with rabbit anti-phospho-H3 (Thr-3),
followed by staining with Coomassie blue to verify similar loading of substrate proteins.
Haspin associates with histone H3 and phosphorylates it at Thr 3. (A) HeLa cells were transfected with vectors encoding
Dai et al.
476GENES & DEVELOPMENT
GST, whereas the binding of myc-haspin to GST alone,
H2B–GST, and H4–GST was not detected. Importantly,
the binding of H3-T3A–GST to myc-haspin was indis-
tinguishable from wild type, indicating that the failure of
haspin to phosphorylate this mutant is not due to a fail-
ure of the two proteins to associate. Mutation of residues
adjacent to Thr 3 (R2A and K4A) abolished detectable
association of haspin with H3–GST (Fig. 3B) and substan-
tially reduced phosphorylation of H3–GST by haspin
(Fig. 3A), indicating that these residues are involved in
the interaction of haspin and histone H3.
To directly confirm that haspin phosphorylates Thr 3,
we generated anti-phospho-histone H3 (Thr 3) antibodies
(see Materials and Methods). First, to confirm the speci-
ficity of the antibodies, we demonstrated that they rec-
ognized a synthetic peptide representing residues 1–8 of
the H3 tail when phosphorylated at Thr 3 but failed to
bind to the equivalent nonphosphorylated peptide and to
H3 peptides phosphorylated at Thr 11 or Thr 22 (Fig. 3C).
Second, we produced a purified recombinant form of the
kinase domain of human haspin as a 6His-tagged fusion
protein in E. coli and an equivalent protein containing
the K511A mutation that is kinase deficient (Fig. 3D).
We then carried out in vitro kinase reactions with a va-
riety of purified substrates and used immunoblotting
with anti-phospho-histone H3 (Thr 3) antibodies to de-
tect phosphorylation of Thr 3 (Fig. 3E). The kinase do-
main of haspin, but not the kinase-KD form, was able to
phosphorylate Thr 3 in recombinant histone H3 and in
H3–GST. No such phosphorylation was detected on the
H3-T3A–GST mutant or on GST alone. Together, these
results reveal that both full-length haspin immunopre-
cipitated from transfected cells and the purified recom-
binant kinase domain of haspin specifically associate
with and phosphorylate a novel residue within the tail of
histone H3, Thr 3, at least in vitro.
Histone H3 is phosphorylated on Thr 3 during mitosis
We next wished to determine whether histone H3 is
phosphorylated on Thr 3 in cultured cells and, if so,
where and when. Prior to these studies, we further char-
acterized recognition by the anti-phospho-histone H3
(Thr 3) antibodies of a variety of H3 peptides carrying
other known modifications. This was important because
the binding of other phospho-specific H3 antibodies is
altered by the presence of flanking modifications (Turner
2002; Clayton and Mahadevan 2003). We tested biotinyl-
ated peptides representing residues 1–21 of H3 carrying
no modifications, asymmetric dimethylation on Arg 2,
dimethylation on Lys 4 or Lys 9, acetylation on Lys 9 and
Lys 14, or phosphorylation on Ser 10. All the peptides
were similarly phosphorylated by the recombinant
haspin kinase domain when the incorporation of radio-
labeled phosphate from ?32P-ATP was assessed (Fig. 4A,
right panel). In contrast, when the extent of peptide
phosphorylation was determined by immunoblotting
with the anti-phospho-histone H3 (Thr 3) antibody, it
was clear that, alone among the tested modifications,
methylation of Arg 2 substantially reduced, but did not
eliminate, the recognition of phosphorylated Thr 3 (Fig.
4A, left panel). Similar results were obtained with two
independent affinity-purified anti-phospho-histone H3
(Thr 3) antisera (see Materials and Methods).
To determine the timing of H3 Thr 3 phosphorylation,
we synchronized HeLa cells at the G1/S boundary by
double thymidine treatment and used the anti-phospho-
histone H3 (Thr 3) antibody in immunoblot analysis of
cell lysates at various times following release of the
block. DNA content analysis was used to follow cell
cycle progression, and the mitotic index was determined
by staining with the antibody MPM-2 as described (Tay-
lor and McKeon 1997). MPM-2 recognizes a group of pro-
teins that are phosphorylated in prophase, prometa-
phase, and metaphase and dephosphorylated during ana-
phosphorylation of H3 on Ser 10 and Ser 28 correlated
well with the number of mitotic cells (Fig. 4B). Phos-
phorylation of H3 on Thr 3 showed a very similar pat-
tern, suggesting that this modification, like that of Ser 10
and Ser 28, occurs primarily during mitosis.
We next examined the location and timing of H3 Thr
3 phosphorylation in HeLa cells by confocal immuno-
fluorescence microscopy and compared it to that of Ser
10 phosphorylation (Fig. 4C). Little staining with anti-
phospho-histone H3 (Thr 3) antibodies was observed in
the majority of interphase cells. Nevertheless, Thr 3
phosphorylation could be detected in a subset of cells
without clear chromosome condensation. These cells
were identical to those in which Ser 10 phosphorylation
could first be detected (Fig. 4C), presumably late G2 cells
as previously reported (Hendzel et al. 1997; Van Hooser
et al. 1998). In prophase, phosphorylated Thr 3 was de-
tected on condensing chromosomes, and prometaphase
and metaphase chromosomes were strongly reactive
with the antibody. The intensity of staining declined
substantially during anaphase and was absent on decon-
densing chromosomes in telophase. Overall, the timing
of Thr 3 phosphorylation and dephosphorylation was
very similar to that of Ser 10. In contrast, there were
differences in the location of the two modifications. In
late G2 cells, Thr 3 phosphorylation appeared in a speck-
led pattern, whereas diffuse patches of Ser 10 phosphory-
lation frequently originating at the nuclear periphery
were observed. The puncta of Thr 3 phosphorylation did
not coincide with centromeres, suggesting that the
modification originates at foci on chromosome arms. By
late prophase the two modifications were partially over-
lapping and had spread over the majority of each chro-
mosome, but the most intense staining for each modifi-
cation was in distinct locations. Costaining with centro-
mere antibodies in fixed cells and on spread metaphase
chromosomes indicated that, from late prophase on, Thr
3 phosphorylation was strongest at inner centromeric
regions, while that of Ser 10 was most intense at distinct
bands on the chromosome arms (Fig. 4C,D; Supplemen-
tary Fig. 2A). Similar results were obtained in U2OS
cells, although in this case phosphorylation of H3 at Ser
10 and Thr 3 was observed only after chromosome con-
Haspin is a histone H3 kinase
GENES & DEVELOPMENT477
various modifications were phosphorylated at Thr 3 in vitro using the haspin 6His-kinase domain (haspin) or were mock-treated in the
absence of the kinase (none). (Right panel) The extent of phosphorylation was assessed by incorporation of radiolabeled phosphate from
?32P-ATP. Peptides immobilized by slot blotting were probed with rabbit anti-phospho-histone H3 (Thr-3) (left panel), and equivalent
peptide loading was confirmed by streptavidin binding (center panel). Similar results were obtained with both anti-phospho-H3 (Thr-3)
antibodies. (B) HeLa cells were synchronized at G1/S by double thymidine block. At various times following removal of thymidine,
progression through the cell cycle was followed by propidium iodide staining for DNA content (G1 and G2/M) and MPM-2-FSE
staining to enumerate mitotic cells (M), and cell lysates were analyzed by immunoblotting as indicated. (C) HeLa cells or metaphase
spreads were fixed and stained with mouse anti-phospho-H3 (Ser-10) followed by anti-mouse IgG-Cy3 (red) and rabbit anti-phospho-H3
(Thr-3) followed by anti-rabbit IgG-Alexa488 (green). Cells were examined by confocal fluorescence microscopy, and the stages of
mitosis were determined by the distinctive pattern of DNA visualized by DRAQ5 staining. (D) HeLa cells or metaphase spreads were
stained as described in C with human centromeric autoantibodies followed by antihuman IgG-Cy3 (red) and rabbit anti-phospho-H3
(Thr-3) followed by anti-rabbit IgG-Alexa488 (green).
Histone H3 is phosphorylated at Thr 3 during mitosis. (A) Biotinylated peptides representing histone H3 residues 1–21 with
Dai et al.
478 GENES & DEVELOPMENT
densation was apparent (Supplementary Fig. 2B,C). It is
possible that changes in other histone modifications,
particularly at Arg 2, influence the staining pattern ob-
served with phospho-H3 (Thr 3) antibodies. Neverthe-
less, the most straightforward interpretation is that his-
tone H3 is phosphorylated on Thr 3 during the initial
stages of mitosis and dephosphorylated during anaphase.
Haspin overexpression causes increased H3 Thr 3
phosphorylation and a delay during mitosis
As a means to determine its function, we attempted to
generate stable HeLa cell lines overexpressing haspin.
Despite the ease with which we obtained stable trans-
fectants with vector alone, stable clones from cells trans-
fected with haspin all contained undetectable or aber-
rantly sized haspin proteins. This suggested that high-
level haspin expression is incompatible with cell growth.
To circumvent this problem, we generated stable trans-
fectants of HeLa Tet-On cells (BD Clontech) with myc-
haspin cDNA in the vector pTRE2pur and with vector
alone as a control. In this system, the haspin gene is
under an inducible promoter and is not expressed unless
doxycycline is added. We obtained stable lines that ex-
press levels of myc-haspin undetectable by immunoblot-
ting prior to induction, and maximal levels after 24-h
treatment with 1 µg/mL doxycycline (Fig. 5A).
After induction, cells expressing myc-haspin showed a
deficit in proliferation compared with uninduced cells
(cell number reduced to 58% and 60% of uninduced con-
trol after 8 d in two separate experiments). Doxycycline
had no such effect on the growth of cells transfected with
vector alone (cell numbers were 104% and 115% of un-
induced control after 8 d in two experiments). We then
used DNA content analysis and MPM-2 staining to com-
pare progression through the cell cycle of synchronized
populations of myc-haspin and vector-transfected cells
in inducing conditions. After release from a double thy-
midine block at G1/S, both myc-haspin and control cells
progressed through S phase and entered G2 with similar
kinetics (Fig. 5B). Although entry into mitosis as defined
by MPM-2 staining was similar in the two cell lines, the
disappearance of the MPM-2 epitope was markedly de-
layed in myc-haspin-expressing cells (Fig. 5B), indicating
a delay prior to anaphase. A similar effect was seen when
induced and uninduced myc-haspin transfected cells
were compared, while doxycycline treatment of vector-
transfected cells had no effect on cell cycle progression
(Supplementary Fig. 3A). It is unlikely that the kinase
activity of haspin directly induces the MPM-2 phospho-
epitope because no increase in the intensity of MPM-2
staining was observed in haspin-transfected cells (data
not shown), and the extended period of mitosis was also
reflected in a delay in exit from G2/M and entry into G1
mitosis, and its overexpression delays mi-
totic progression. (A) HeLa Tet-On/myc-
haspin- or vector-alone-stably-transfected
cells were induced with 1 µg/mL doxycy-
cline for the times indicated, and myc-
haspin expression was assessed by rabbit
antihaspin immunoblotting. (B) HeLa Tet-
transfected cells were induced with 1 µg/
mL doxycycline for 24 h before synchroni-
zation at G1/S and analysis as described for
Figure 4B. (C) Synchronized HeLa Tet-On/
myc-haspin cells as shown in B were lysed
andanalyzed by immunoblotting
haspin in vitro kinase assay with ?32P-ATP
using H3–GST or H3-T3A–GST as a con-
trol. (D) Synchronized HeLa Tet-On/vector
cells as shown in B were analyzed as de-
scribed in C. (E) Induced HeLa Tet-On/myc-
haspin cells were fractionated into loosely-
adherent mitotic (mitosis) and adherent in-
terphase-enriched (interphase) populations
or treated with colcemid prior to lysis. Rab-
bit antihaspin or rabbit IgG control immu-
noprecipitates were analyzed by rabbit an-
tihaspin immunoblotting. (F) Induced HeLa
Tet-On/myc-haspin cells were treated col-
cemid for 12 h prior to release. Whole-cell
lysates made at the times indicated were
analyzed by antihaspin immunoblotting.
(G) Induced HeLa Tet-On/myc-haspin cells
Haspin is phosphorylated during
were treated colcemid for 12 h or untreated prior to cell lysis. Antihaspin immunoprecipitates were incubated with or without ?
phosphatase as described in Materials and Methods and analyzed by antihaspin immunoblotting.
Haspin is a histone H3 kinase
GENES & DEVELOPMENT479
as determined by DNA content (Fig. 5B; Supplementary
Fig. 3A). Furthermore, in a separate experiment, enu-
meration of mitotic cells at 13 h following release re-
vealed an accumulation in prophase/prometaphase and a
corresponding decrease in the number of anaphase/telo-
phase cells upon overexpression of myc-haspin (Supple-
mentary Fig. 3B).
Immunoblotting of lysates from synchronized cells
showed that myc-haspin protein was present at similar
levels throughout the cell cycle (Fig. 5C). Interestingly
however, the mobility of myc-haspin was significantly
retarded at time points during which cells were under-
going mitosis, particularly at 11–14 h post-release (Fig.
5C). In fact, myc-haspin in lysates of mitotic cells iso-
lated by selective detachment, or after colcemid treat-
ment, was almost entirely in this larger form (Fig. 5E).
Myc-haspin returned to a size of ∼85 kDa after release of
cells from the mitotic block (Fig. 5F), and only this lower
form was detected in interphase cells (Fig. 5C,E). Treat-
ment of myc-haspin immunoprecipitated from col-
cemid-blocked cells with ? phosphatase showed that the
increase size during mitosis could be ascribed to phos-
phorylation (Fig. 5G). We conclude that haspin is
strongly phosphorylated during mitosis.
To examine the kinase activity of overexpressed
haspin through the cell cycle, we immunoprecipitated
myc-haspin from the synchronized cell lysates and con-
ducted in vitro kinase assays using H3–GST as a sub-
strate or H3-T3A–GST as a negative control. Despite the
clear phosphorylation of myc-haspin during mitosis, no
change in its kinase activity was seen during the cell
cycle. Consistent with this, we saw no difference in the
activity of myc-haspin immunoprecipitated from mi-
totic cells obtained by selective detachment or colcemid
block and interphase or asynchronous cells (data not
To determine the effect of haspin overexpression on
histone phosphorylation, we examined the synchronized
cell lysates by immunoblotting with anti-phospho-his-
tone H3 antibodies. In control cells, the phosphorylation
of histone H3 on Thr 3, Ser 10, and Ser 28 correlated well
with the number of mitotic cells, as expected (Fig. 5D).
In myc-haspin-expressing cells, however, phosphoryla-
tion on Thr 3 was dramatically increased and was pres-
ent throughout the cell cycle (Fig. 5C). In contrast, the
intensity of Ser 10 and Ser 28 phosphorylation on H3 was
not significantly altered by myc-haspin overexpression.
These findings provide strong support for the hypothesis
that haspin acts as a histone H3 Thr 3 kinase in vivo.
Endogenous haspin is responsible for histone
H3 Thr 3 phosphorylation during mitosis
We wished to determine the role of endogenous haspin
during mitosis. Although the antihaspin antibody was
unable to detect endogenous haspin in HeLa or HEK293
cells by immunoblotting, our previous Northern analy-
ses suggested that all proliferating cell lines express
haspin mRNA (Higgins 2001a). As a more sensitive ap-
proach to detect endogenous haspin activity, we ana-
lyzed antihaspin immunoprecipitates from untrans-
fected HeLa cells by in vitro kinase assay. This revealed
a kinase activity that could phosphorylate H3–GST but
not H3-T3A–GST (Fig. 6A), suggesting the presence of an
H3 Thr 3 kinase, most likely endogenous haspin. No
such activity was found in negative control immunopre-
cipitates (Fig. 6A). Note that longer autoradiographic ex-
posures of gels were required to visualize endogenous
kinase activity than those shown in Figures 2, 3, and 5
for overexpressed myc-haspin. The pattern of substrate
specificity clearly differed from that of aurora B immu-
noprecipitated from mitotic HeLa cells, which effi-
ciently phosphorylated H3–GST and H3-T3A–GST but
not H3-S10A–GST, as expected (Fig. 6B).
When nocodazole-blocked cells or mitotic cells ob-
tained by selective detachment were compared with an
asynchronous or interphase population, no increase in
the kinase activity of immunoprecipitated endogenous
haspin was seen (Fig. 6B; data not shown). Indeed, when
we examined endogenous haspin from cells synchro-
nized at G1/S and then released, little change in kinase
activity was seen during the cell cycle (Fig. 6C), confirm-
ing the results with overexpressed haspin (Fig. 5C). In
contrast, the activity (Fig. 6B) and protein level (Fig. 4B)
of aurora B increase in G2/M as previously reported (Bis-
choff et al. 1998). It should be noted, however, that we
have been unable to directly determine the amount of
endogenous haspin protein in cells and that our results
do not rule out regulation of haspin activity during mi-
tosis in vivo (see Discussion).
To determine whether endogenous haspin is required
for phosphorylation of H3 on Thr 3, we conducted RNA
interference. At 100 nM, transfection of small interfering
RNA (siRNA) ID 1093-specific for human haspin reduces
haspin mRNA levels in HeLa cells by 89% ± 1% (Am-
bion). We confirmed that transfection of 20 or 100 nM of
this siRNA, but not of a negative control siRNA, reduced
endogenous haspin kinase activity in both asynchronous
and nocodazole-blocked mitotic HeLa cells (Fig. 6D).
The treatment had little effect on the aurora B kinase
activity detected in the same cell lysates. Strikingly,
haspin siRNA dramatically reduced the phosphorylation
of H3 on Thr 3 seen in mitotic cells (Fig. 6D). In contrast,
no change was seen in the level of H3 phosphorylation
on Ser 10. Haspin siRNA also caused a similar reduction
in H3 Thr 3 phosphorylation in U2OS cells (see Fig. 7A;
data not shown) and, using a different murine haspin
siRNA, in NIH3T3 cells (Fig. 6E). Inhibition of aurora B
activity by ZM447439 treatment dramatically reduced
histone H3 Ser 10 phosphorylation as reported previ-
ously (Ditchfield et al. 2003) but did not prevent Thr 3
phosphorylation (Supplementary Fig. 4). Therefore en-
dogenous haspin, but not aurora B, activity is required
for H3 phosphorylation on Thr 3 in mitotic cells.
Depletion of haspin prevents normal
metaphase chromosome alignment
To examine the effect of haspin RNA interference on
mitosis, we transfected U2OS and HeLa cells with
Dai et al.
480 GENES & DEVELOPMENT
haspin or control siRNAs and assessed the distribution
of chromosomes in mitotic cells by immunofluores-
cence. Among haspin siRNA-transfected U2OS cells, we
noted an increased number with a late prometaphase
configuration in which a partial metaphase plate was
“stranded” near the spindle poles (Fig. 7A). Staining with
anticentromere antibodies revealed doublets on most of
the unaligned chromosomes indicating that they were
mono-orientated sister chromatid pairs. The centro-
meres of chromosomes that were present at the meta-
phase plate often appeared poorly aligned (Fig. 7A; data
not shown). Enumeration of mitotic cells showed that
haspin siRNA caused an accumulation of cells in pro-
metaphase and a corresponding decrease in anaphase and
telophase cells compared with controls (Fig. 7B; Supple-
mentary Fig. 5A). We noted that within the haspin
siRNA-treated population, H3 Thr 3 phosphorylation
was reduced to varying extents in different cells. When
we examined only those cells with low levels of Thr 3
phosphorylation (“low pT3”), the increased ratio of pro-
metaphase over metaphase cells was particularly dra-
matic (Fig. 7C). In cells with moderate to high levels of
pT3, or in control transfectants, there was a 50%:50%
split between cells classified as prometaphase and meta-
phase, and <10% had a partial metaphase configuration.
In contrast, among haspin siRNA transfected cells with
low pT3 >80% were in prometaphase and <20% in meta-
phase. More than 40% had a partial metaphase arrange-
ment similar to that shown in Figure 7A. Similar results
were obtained with an independent haspin siRNA re-
agent (Fig. 7B,C) and in HeLa cells, although these cells
apparently required more complete haspin depletion to
disrupt mitosis (Supplementary Fig. 5B). We conclude
that haspin is required for normal alignment of chromo-
somes at metaphase.
Haspin functions as a kinase during mitosis
We show that, by a number of criteria, haspin is a kinase
that functions in mitosis. First and most importantly,
haspin overexpression or depletion results in defective
phorylation of histone H3 on Thr 3. (A) Immunopre-
cipitates with rabbit antihaspin or rabbit IgG nega-
tive control (neg) antibodies from HeLa cell lysates
were subjected to in vitro kinase assays with ?32P-
ATP and H3–GST or H3-T3A–GST as substrates.
Coomassie blue staining was used to confirm
equivalent levels of GST proteins. (B) Antihaspin
and anti-aurora B immunoprecipitates from HeLa
cell lysates were subjected to in vitro kinase reac-
tions with ?32P-ATP and H3–GST, H3-T3A–GST, or
H3-S10A–GST proteins as substrates. Coomassie
blue staining was used to confirm equivalent levels
of GST proteins. (C) The synchronized HeLa Tet-
On/vector cells shown in Figure 5B were analyzed
by haspin in vitro kinase assay described for Figure
5C. Note that this is a longer exposure of the auto-
radiogram shown in Figure 5D, carried out in order
to visualize endogenous haspin activity. Similar re-
sults were obtained with HeLa cells (data not
shown). (D) HeLa cells were transfected with haspin
siRNA (Ambion ID #1093), control siRNA (Ambion
4611), or no siRNA. Approximately 30 h after trans-
fection, the cells were incubated with nocodazole
for 16 h (mitotic) or left untreated (asynchronous)
prior to lysis. Antihaspin and anti-aurora B immu-
noprecipitates were subjected to in vitro kinase re-
actions with ?32P-ATP and H3–GST, H3-T3A–GST,
or H3-S10A–GST as substrates, and lysates were im-
munoblotted with anti-phospho-H3 (Thr-3) or anti-
phospho-H3 (Ser-10) antibodies. The anti-phospho-
H3 (Thr-3) blot was stripped and reprobed with an-
tihistone H3 antibodies. The increased haspin
activity in nocodazole-treated cells was not a repro-
ducible finding, as shown in B and C. (E) NIH3T3
cells were transfected with mouse haspin (Ambion
ID 67120) or negative control (Ambion 4611)
siRNAs and analyzed as described for D.
Endogenous haspin is required for phos-
Haspin is a histone H3 kinase
GENES & DEVELOPMENT481
mitosis. Second, haspin has a unique pattern of associa-
tion with critical components of the mitotic machinery.
Third, phosphorylation of haspin and haspin-dependent
phosphorylation of histone H3 occur specifically during
Haspin localizes to condensed chromosomes through-
out mitosis, to the centrosomes following nuclear enve-
lope breakdown (NEBD), to spindle microtubules during
metaphase and to the midbody in telophase. This local-
ization is similar to that of aurora A and Polo-like kinase
1 (Plk1) except that these proteins are found at the cen-
trosome prior to NEBD and, although Plk1 is found at
centromeres, neither show prominent association with
mitotic chromosome arms (Carmena and Earnshaw
2003; Barr et al. 2004). Like aurora B, haspin can associ-
ate with condensing chromosomes, particularly at cen-
tromeric regions, and with spindle components. We
found no evidence, however, that haspin undergoes the
sudden transfer from chromosomes to the spindle typical
of chromosome passenger proteins as anaphase begins
(Carmena and Earnshaw 2003).
Interestingly, we could not detect a change in haspin
kinase activity during the cell cycle, consistent with the
lack of residues that can be phosphorylated in the poten-
tial activation loop of its kinase domain (Higgins 2001b).
Despite this, we suspect that mitotic phosphorylation
controls haspin function in vivo by modulating binding
to protein(s) that regulate its activity or by regulating
haspin association with proteins that target it to chro-
matin and the spindle. This type of targeting of aurora B
ID #1093) or control siRNA (Ambion #4613) were fixed and stained with DRAQ5 to visualize DNA and with human centromeric
autoantibodies followed by antihuman IgG-Cy3 (red), mouse anti-?-tubulin mAb followed by anti-mouse IgG-Alexa488 (green), and
rabbit anti-phospho-H3 (Thr-3) followed by anti-rabbit IgG-Alexa488 (green) or anti-rabbit IgG-Cy3 (red) as indicated. (B) U2OS cells
were transfected with haspin siRNA A (Ambion ID #1093), haspin siRNA B (Dharmacon SMARTpool), control siRNA A (Ambion
#4613), or control siRNA B (Dharmacon SMARTpool), or without siRNA. After 48 h, the cells were fixed and stained with propidium
iodide, and ∼3000 cells were counted on each of three coverslips for each condition and classified as interphase, prophase, prometa-
phase, metaphase, anaphase/telophase, or partial metaphase (a metaphase plate and three or more unaligned chromosomes; a subset
of prometaphase) by the distinctive pattern of DNA staining. The percent of mitotic cells in each phase is shown. The increase in
prometaphase and partial metaphase cells and the decrease in anaphase/telophase cells have p < 0.0001 by two-tailed Student’s t-test
when comparing haspin siRNA A and B with control siRNA A, B, and no siRNA combined. (C) Haspin siRNA transfected U2OS cells
in prometaphase and metaphase from B stained with rabbit anti-phospho-H3 (Thr-3) followed by anti-rabbit IgG-Alexa488 were further
classified as containing low or high levels of phosphorylated H3 Thr 3 (pT3). The percentage of these cells that were in prometaphase,
metaphase, or partial metaphase is shown.
Depletion of haspin causes a failure of chromosome congression. (A) U2OS cells transfected with haspin siRNA (Ambion
Dai et al.
482 GENES & DEVELOPMENT
by INCENP and survivin has been well described (Car-
mena and Earnshaw 2003). Such associations may be dis-
rupted in the lysis conditions used in our experiments.
Haspin is required for normal mitosis
We find that haspin depletion by RNA interference pre-
vents normal chromosome alignment at metaphase,
while haspin overexpression results in a delay prior to
metaphase. In common with mitotic kinases such as
Plk1 and auroras A and B (Carmena and Earnshaw 2003;
Barr et al. 2004), it appears that haspin activity must be
maintained between certain limits and that either too
much or too little prevents normal mitosis. The failure
of chromosome congression upon reduction of haspin
activity is reminiscent of the effect of aurora B depletion
(Adams et al. 2001; Shannon and Salmon 2002; Andrews
et al. 2003). It is possible that haspin plays a role in
correcting syntelic chromosome attachments to the
spindle, a normal process during formation of the meta-
phase plate that is dependent on aurora B (Andrews et al.
2003). We note also that the effect of haspin depletion is
similar to that of disrupting the function of centromeric
kinesin-related proteins, particularly CENP-E, or of de-
pleting the kinetochore kinase Bub1 (Schaar et al. 1997;
Johnson et al. 2004). Haspin may therefore play a role in
regulating kinetochore assembly and spindle attachment
or in modulating the activity of microtubule motors re-
sponsible for chromosome movement.
In the only other reported functional study of haspin,
Tanaka et al. (1999) found that transient transfection of
HEK293 cells with murine EGFP-haspin caused a pro-
found decrease in the proportion of cells with G2/M
DNA content and an accumulation in those with G1
DNA content after 4 d. A mutated form of haspin that
has 10 amino acids deleted from the kinase domain, and
lacks kinase activity, caused the same effect after 2 d
(Tanaka et al. 1999). The basis for the disparity with our
results is not known. HEK293 cells are reported to have
an ineffective spindle checkpoint (Kung et al. 1990), so it
is possible that haspin-induced mitotic defects lead to
subsequent activation of a G1 checkpoint in these cells.
Alternatively, haspin might have another role in control
of S-phase entry. To fully understand the function of
haspin, it will be necessary to identify its substrates. We
have identified one such substrate as Thr 3 of the core
Phosphorylation of histone H3 at Thr 3
We show that during mitosis the core histone H3 is
phosphorylated at Thr 3. Polioudaki et al. (2004) recently
reported comparable results, although we extend these
findings in two important ways. First, we find that the
timing of onset of Ser 10 and Thr 3 phosphorylation is
similar and can be detected prior to clear chromatin con-
densation in HeLa cells, likely late in G2 (Hendzel et al.
1997; Van Hooser et al. 1998). Second, we carried out
coimmunostaining of phosphorylated Ser 10 and Thr 3,
and show directly that they have distinct localizations
during mitosis. Thr 3 phosphorylation appears to origi-
nate at foci on the chromosome arms. By metaphase, it is
most intense at centromeric chromatin and is also pres-
ent along the chromosome arms, reflecting the distribu-
tion of haspin. In contrast, Ser 10 phosphorylation was
found primarily on chromosome arms in late prophase
through metaphase. The two modifications are removed
contemporaneously prior to chromosome decondensa-
tion in telophase.
The existence of a histone H3 Thr 3 kinase was first
described 25 years ago. Shoemaker and Chalkley (1980)
characterized a kinase activity associated with bovine
thymus chromatin that displayed “extraordinary sub-
strate specificity for histone H3” and phosphorylated
Thr 3. More recently, an H3 Thr 3 kinase from avian
nuclear envelope-associated peripheral heterochromatin
that was able to form a complex with HP1?–GST protein
was described (Polioudaki et al. 2004). The identity of
the kinase, however, was not determined in either study.
Four lines of evidence lead us to conclude that haspin is
the major kinase responsible for mitotic histone H3 Thr
3 phosphorylation, at least in HeLa, U2OS, and NIH3T3
cells. First, overexpression of myc-haspin leads to in-
creased phosphorylation of H3 specifically at Thr 3. Sec-
ond, small inhibitory RNAs that deplete endogenous
haspin activity dramatically reduce mitotic phosphory-
lation of H3 at Thr 3. Third, haspin associates with mi-
totic chromosomes at the time that H3 is phosphory-
lated on Thr 3. Fourth, haspin specifically associates
with histone H3 and phosphorylates Thr 3 in vitro. The
effects of overexpression and RNAi suggest that haspin
is a component of the machinery required for H3 Thr 3
phosphorylation during mitosis. When coupled with the
finding that haspin associates with and phosphorylates
H3 in vitro, these experiments provide persuasive evi-
dence that haspin directly phosphorylates H3 in vivo.
The function of mitotic histone
H3 Thr 3 phosphorylation
What might be the consequence of haspin-induced H3
Thr 3 phosphorylation? Although haspin siRNA did not
prevent chromosome condensation (see Fig. 7A), our re-
sults do not rule out a more subtle effect on chromatin
structure. The timing of Thr 3 phosphorylation suggests
that it could play a role in facilitating condensation and/
or resolution of sister chromatids in late G2 and pro-
phase. This might occur through alterations in the re-
cruitment or function of condensins, cohesins, or topo-
isomerases (Swedlow and Hirano 2003). Defects in chro-
matin structure caused by inappropriate Thr 3 phos-
phorylation might hinder chromosome alignment later
in mitosis, particularly given the importance of cohesion
for bi-orientation (Tanaka 2002). Alternatively, its pres-
ence at inner centromere regions might reflect a more
direct role for Thr 3 phosphorylation in regulating ki-
netochore assembly or function and the attachment or
activity of spindle microtubules. Tension across paired
kinetochores is critical to stabilize attachment of bi-ori-
Haspin is a histone H3 kinase
GENES & DEVELOPMENT483
entated chromosomes to the spindle. It has been pro-
posed that tension pulls kinetochores away from aurora
B at the inner centromere, thereby regulating kinase ac-
cess to its substrates and selectively stabilizing bi-orien-
tated attachments (Tanaka 2002; Andrews et al. 2003). It
is possible that phosphorylation of centromeric nucleo-
somes on Thr 3 of H3 influences transmission of tension
between kinetochores and centromeric chromatin. This
could affect separation of sister chromatids and impact
chromosome bi-orientation, perhaps by altering aurora B
At the molecular level, Thr 3 phosphorylation might
directly influence internucleosomal contacts or could
generate a binding site for regulatory proteins during mi-
tosis, in much the same way that Lys 9 methylation
facilitates HP1 binding (Lachner and Jenuwein 2002). Al-
ternatively, it has been hypothesized that Thr 3 serves as
a component of a “binary switch.” Phosphorylation of
Thr 3 could serve to eject as yet undefined proteins
bound to the adjacent methylated Lys 4 residue (Fischle
et al. 2003a). Dimethylation of Lys 4 in centromeric H3
has been reported (Sullivan and Karpen 2004). Lys 4 tri-
or dimethylation is associated with an active or compe-
tent transcriptional state and a more “open” chromatin
structure (Schneider et al. 2004), which might therefore
be counteracted by Thr 3 phosphorylation during mito-
sis. Arg 2 in histone H3 can be methylated, too (Schurter
et al. 2001), and similar interplay between Thr 3 and this
residue could take place.
Cross-talk between nonadjacent histone modifications
also occurs (Fischle et al. 2003b), and phosphorylation of
Thr 3 might influence the binding or activity of other
histone-modifying enzymes. Prior modifications might
also impinge upon the ability of haspin to phosphorylate
H3. We find that haspin phosphorylates recombinant H3
and chemically synthesized H3 peptides that lack amino
acid modifications, and associates with and phosphory-
lates recombinant H3–GST. In addition, H3 peptides
containing a variety of modifications are equally good
substrates for the haspin kinase domain (Fig. 4A). To-
gether, these results suggest that haspin activity is not
influenced by pre-existing H3 modifications in vitro. We
cannot rule out, however, the possibility that haspin ac-
tivity toward certain modified forms of H3 might be in-
creased or decreased in vivo. This might occur because of
the presence of combinations of histone modifications
not tested in our in vitro study, the existence of other
proteins in vivo that might compete for binding to modi-
fied H3, and the influence of the N-domain of haspin
that was not present in the recombinant haspin protein
we used. Indeed, the association of full-length haspin
with H3–GST is reduced by mutations at Arg 2 and Lys
4 (Fig. 3).
Nonhistone targets of haspin activity
It should be noted that it is unlikely that the sole target
of haspin activity is histone H3, and therefore we cannot
ascribe the effects of manipulating haspin activity only
to its influence on H3 Thr 3 phosphorylation. The pres-
ence of haspin at centrosomes and the spindle during
mitosis strongly indicates that substrates will be found
at these locations too. In fact, we have identified mitotic
spindle and centrosomal proteins as potential haspin-
binding proteins in a yeast two-hybrid screen, and we
have noted spindle disruptions in mitosis following
haspin siRNA treatment (J. Dai and J.M.G. Higgins, un-
publ.). Haspin therefore has features in common with
members of the Aurora, Nek, and Polo families that
regulate the activity of both chromatin and spindle pro-
teins at multiple stages of mitosis (Nigg 2001). Indeed,
the overlap in haspin, Aurora, and Polo functions and
localization suggest that it will be productive to inves-
tigate interactions between haspin and these other ki-
To the best of our knowledge, an equivalent of Thr 3 is
found in histone H3 of all eukaryotes, suggesting a
highly conserved and critical function for this residue.
Furthermore, the presence of haspin genes in diverse eu-
karyotes is suggestive of a crucial function in eukaryotic
life (Higgins 2003). The limited information available re-
garding the two haspin homologs in budding yeast is
consistent with the function of haspin that we describe.
The mRNA levels of haspin homolog ALK1 are strik-
ingly periodic during the mitotic cell cycle, with a peak
in expression early in mitosis (Cho et al. 1998; Spellman
et al. 1998). The second homolog, YBL009W, is signifi-
cantly induced during sporulation (Chu et al. 1998). The
data suggest that Alk1p and Ybl009wp function during
mitosis and meiosis, respectively (Higgins 2003). We pro-
pose that haspin is a member of the select group of ki-
nases with a critical role in integrating the regulation of
chromosome and spindle function during mitosis and
probably meiosis. The high level of haspin in post-mei-
otic spermatids (Tanaka et al. 1999) also might suggest a
role in the dramatic reorganization and compaction of
chromatin that occurs during spermiogenesis. Further
study of haspin function will help decipher the histone
code and is likely to provide crucial insight into the
mechanisms that maintain genomic stability during mi-
totic and meiotic cell division.
Materials and methods
Antibodies, proteins, peptides, and cells
A rabbit anti-serum to a KLH-conjugated peptide corresponding
to residues 329–44 of human haspin ([C]DRLERTRSSRK-
SKHQE) was generated and affinity purified on the immunizing
peptide by Zymed Laboratories Inc. The rabbit polyclonal anti-
serum B8634 to phospho-histone H3 (Thr 3) was produced by
immunization with the peptide AR[pT]KQTAR(Ahx)C conju-
gated to BSA (Ahx indicates aminohexanoic acid), depleted us-
ing the equivalent non-phosphorylated peptide, and affinity pu-
rified on the phosphorylated peptide (Biosource). Alternatively,
rabbit polyclonal antibody to phospho-histone H3 (Thr 3) from
Upstate was used with similar results. Rabbit anti-phospho-
Dai et al.
484 GENES & DEVELOPMENT
histone H3 (Ser 28) was from Upstate, rabbit polyclonal and
mouse monoclonal anti-phospho-histone H3 (Ser 10) from Cell
Signaling Technology, and rabbit antihistone H3 from Abcam.
Sheep antihuman aurora B was as described (Ditchfield et al.
2003). Goat anti-B23/nucleophosmin and mouse 9E10 anti-
myc-FITC were from Santa Cruz Biotechnology, and human
autoantibody to centromeres was from Immunovision. Mouse
AF8 monoclonal antihuman calnexin (Hochstenbach et al.
1992) was a gift from Dr Michael Brenner (Brigham and Wom-
en’s Hospital, Boston, MA). Mouse monoclonal antibodies to
human cyclin A, cyclin B, and PCNA were from BD Transduc-
tion Laboratories, and to ?-tubulin were from Sigma. Purified
calf thymus histones were from Roche. Human polynucleo-
somes isolated from sonicated MCF-7 cell nuclei were provided
by Dr Gavin Schnitzler (Tufts University, Boston, MA). Human
histone H3 peptides H3(1–8), H3(1–8)pT3, H3(9–16)pT11, and
H3(20–27)pT22, each with an additional Ahx and cysteine resi-
due (≈95% pure), were synthesized by Biosource. Other peptides
were human H3 residues 1–21 followed by GGK-biotin and
asymmetrically dimethylated at Arg 2 (>95% pure, synthesized
by Abgent), dimethylated at Lys 4 or Lys 9, acetylated at Lys 9
and Lys 14, or phosphorylated at Ser 10 (>90% pure, Upstate).
Human HEK293, HeLa, U2OS, and DLD-1 cells were main-
tained in 10% FBS/DMEM.
Recombinant protein production
Recombinant Xenopus H3, tailless H3 (gH3), H2B, and H4 his-
tones, prepared according to Luger et al. (1997), were a gift from
Dr Robert Kingston (Massachusetts General Hospital, Boston,
MA). To generate plasmids encoding histone tail–GST proteins,
PCR productsencoding residues
(NM_003537) or 1–26 of human H4 (NM_003541) amplified
from the plasmids pBOS-H3-N-GFP and pBOS-H4-N-GFP
(kindly provided by Dr Hiroshi Kimura, Tokyo Medical and
Dental University, Tokyo, Japan) (Kimura and Cook 2001) and a
PCR product encoding residues 1–35 of H2B (NM_003526) am-
plified from human HeLa cell genomic DNA were inserted into
the NcoI site of pETGEX-CT (a gift from Dr A.D. Sharrocks,
University of Manchester, UK) (Sharrocks 1994). Site-directed
mutants of H3–GST were generated by PCR mutagenesis. A
construct encoding the 6His-tagged kinase domain was gener-
ated by insertion of a PCR product encoding residues 471–798 of
human haspin into the PshAI site of the vector pET45b(+) (No-
vagen). An equivalent construct containing the mutation
K511A was produced by PCR-based mutagenesis. All constructs
were confirmed by DNA sequencing and introduced into E. coli
strain BL21 (Novagen). GST and 6His fusion proteins were pu-
rified from IPTG-induced E. coli by standard procedures.
1–45 of humanH3
Haspin expression in mammalian cells
Using customized double stranded adapters, full-length human
haspin cDNA (amino acids 1–798) (Higgins 2001a) was ligated
into the HindIII–XbaI sites of the expression vector pcDNA3
(Invitrogen), and to generate myc-tagged haspin, cDNA encod-
ing residues 2–798 was inserted into the pcDNA3-derived plas-
mid pCANmyc1. To generate a plasmid encoding EGFP-haspin,
haspin cDNA encoding amino acids 2–798 was inserted into the
SacII–BamHI sites of pEGFP-C1 (Clontech). To generate induc-
ible vectors, cDNA encoding myc-haspin from pCANmyc-
haspin (HindIII–EcoRV) was blunt-ended and inserted into the
PvuII site of pTRE2/pur (BD Clontech), and NheI–XbaI frag-
ments encoding EGFP-haspin or EGFP alone from pEGFP-
haspin or pEGFP-C1, respectively, were ligated into the NheI
site of pTRE2pur. All constructs were verified by DNA sequenc-
ing. Transient transfection was carried out using Lipofectamine
2000 (Invitrogen). Stable transfection of HeLa Tet-On (BD Clon-
tech) cells was carried out using Lipofectin with Plus Reagent
(Invitrogen). After 24 h, cells were transferred to 96-well plates
and incubated in medium containing 1 µg/mL puromycin and
100 µg/mL G418. Clones expressing myc-haspin or EGFP-
haspin in the presence, but not absence, of 1 µg/mL doxycycline
were selected by anti-myc immunoblotting or flow cytometric
analysis of EGFP fluorescence, respectively.
Unless otherwise stated, cells or metaphase spreads were fixed
with 4% paraformaldehyde/PBS for 10 min, incubated in metha-
nol for 5 min at −20°C; washed three times with 5% FBS/PBS
over 30 min; then incubated with 2 µg/mL anti-myc 9E10-FITC,
1 µg/mL goat anti-B23/nucleophosmin, 0.1 µg/mL mouse anti-
?-tubulin, 1/4000 human anti-centromere, 1/1000 mouse anti-
phospho-histone H3 (Ser 10), or 0.2 µg/mL rabbit anti-phospho-
histone H3 (Thr 3) in 5% FBS/PBS for 2 h at 25°C followed by ∼1
µg/mL donkey anti-goat, mouse, rabbit, or human IgG-Cy3
(Jackson ImmunoResearch) or goat anti-rabbit or mouse IgG-
Alexa488 (Invitrogen). To detect DNA, 0.5 µg/mL Hoechst
33342 (Sigma) or 2.5 µM DRAQ5 (Alexis) was used. Fluores-
cence microscopy was carried out by using a Nikon TE2000
inverted confocal microscope and video microscopy using a Ni-
kon ECLIPSE E600 inverted fluorescence microscope, with
heated stages at 37°C for live cell imaging.
Where necessary, HeLa Tet-On stable transfectants were incu-
bated with 1 µg/mL doxycycline for 24–48 h prior to synchro-
nization. Cells were synchronized at the G1/S boundary by
double thymidine block (Spector et al. 1997) or in prometaphase
by treatment with 50 ng/mL (HeLa) or 150 ng/mL (NIH3T3)
nocodazole or 100 ng/mL colcemid for 12–16 h. For cell cycle
analysis, cells were permeabilized in 70% ice-cold ethanol,
blocked with 1% BSA/5% FBS, and stained with 10 µg/mL
mouse monoclonal MPM-2-FSE (Upstate) (Taylor and McKeon
1997) followed by incubation in 50 µg/mL propidium iodide,
100 U/mL RNAse A, and PBS for 1 h at 25°C to stain DNA.
Analysis was conducted on a FACSort flow cytometer (BD Bio-
Immunoprecipitation, GST pulldown, and immunoblotting
For immunoprecipitation, cells were suspended in 50 mM Tris/
0.5 M NaCl/1% Triton X-100/1% DOC/0.1% SDS/2 mM
EDTA at pH 7.4 (buffer L) with 1 µg/mL pepstatin/1 µg/mL
leupeptin/1 µg/mL antipain/1 µg/mL chymostatin/1 mM
phenylmethylsulphyl fluoride/1 mM NaF/0.1 µM okadaic acid
and lysed by shearing 15 times through a 21-gauge needle. In-
soluble material was removed by centrifugation for 15 min at
13,000 rpm, the lysates precleared with protein G-Sepharose,
and concentrations equalized based on Bradford assay (Bio-Rad
Laboratories). Lysates were incubated with antibodies for 1.5 h
at 4°C before addition of protein G-Sepharose for a further 1.5 h.
Beads were washed four times with buffer L and thrice with
Hepes-buffered saline (HBS) at pH 7.4. For GST pulldown as-
says, cells were lysed in 20 mM Tris/1% Triton X-100/1 mM
EDTA/1 mM DTT at pH 7.4 (buffer T) containing 0.3 M NaCl
and protease and phosphatase inhibitors as above, clarified, and
precleared with glutathione-Sepharose. Cell lysates (200 µg)
were incubated in 200 µL buffer T containing 0.4 M NaCl for 1
h at 4°C with 2.5 µg GST fusion protein preabsorbed to 5 µL
Haspin is a histone H3 kinase
GENES & DEVELOPMENT485
glutathione-Sepharose, followed by three washes in the same
buffer. Whole-cell lysates for immunoblotting were prepared in
30 mM Tris/1.5% SDS/5% glycerol/0.1% bromophenol blue
(pH 6.8). Hypotonic lysis in 10 mM Hepes/0.5% Triton X-100/
1.5 mM MgCl2/10 mM KCl (pH 7.4) with protease inhibitors
was used to produce nuclear (pellet) and cytoplasm-enriched
(supernatant) cell fractions. SDS-PAGE and immunoblotting
were carried out using standard procedures (Coligan et al. 1994).
Peptide slot blots to 0.2 µm PVDF Immunblot membrane were
carried out with the Bio-Dot SF apparatus (Bio-Rad Laborato-
In vitro kinase assays
Haspin and aurora B kinase assays were conducted in 30 µL
HBS/10 mM MnCl2with 2.5 µM ATP and 2.5 µCi [?32P]-ATP
(3000 Ci/mmol) or with 100 µM ATP for 15 min at 37°C. Ex-
ogenous substrates were added at 0.5–1 µg per reaction for his-
tone and GST proteins and at 0.02–1 nmol for peptides. Bioti-
nylated peptides were quantified by HABA/avidin assay
(Sigma). Incorporation of32P into histone and GST proteins was
visualized by SDS-PAGE and autoradiography and into biotinyl-
ated peptides by immobilization on SAM2Biotin Capture Mem-
brane according the manufacturer’s recommendations (Promega
Corporation) and Cherenkov counting.
Myc-haspin immunoprecipitates from colcemid-treated or un-
treated induced HeLa Tet-On/myc-haspin cells were incubated
with 200 U ? phosphatase in ? phosphatase buffer (New England
Biolabs) or in buffer alone for 30 min at 30°C before analysis by
SDS-PAGE and immunoblotting.
Human haspin validated siRNA (ID #1093), murine haspin pre-
designed siRNA (ID #67120), and negative control 1 or 2 siRNAs
(#4611, #4613) were from Ambion, and human haspin and nega-
tive control SMARTpool siRNAs were from Dharmacon.
Haspin SMARTpool siRNA at 50 nM caused a 63% reduction in
haspin mRNA compared with control SMARTpool siRNA in
HeLa cells after 30 h as determined by real-time RT–PCR.
Transfection with siRNAs was carried out with siPORT lipid
according to the manufacturer’s recommendations (Ambion).
We are grateful to Yu Weng and Yuping Sun for technical assis-
tance in preparing recombinant proteins; Hui-Ya Gilbert for her
assistance with confocal microscopy; Dr. Mari Porcionatto for
help with video microscopy; Stuart Levine, Dr Robert Kingston,
and Dr Gavin Schnitzler for gifts of reagents; and Drs Michael
Brenner, David Pellman, and Beth Sullivan for useful discus-
sions. This work was funded by grants to J.M.G.H. from the
NIH (HD043833) and the William F. Milton Fund of Harvard
Adams, R.R., Maiato, H., Earnshaw, W.C., and Carmena, M.
2001. Essential roles of Drosophila inner centromere protein
(INCENP) and aurora B in histone H3 phosphorylation,
metaphase chromosome alignment, kinetochore disjunc-
tion, and chromosome segregation. J. Cell. Biol. 153: 865–
Aihara, H., Nakagawa, T., Yasui, K., Ohta, T., Hirose, S., Dho-
mae, N., Takio, K., Kaneko, M., Takeshima, Y., Muramatsu,
M., et al. 2004. Nucleosomal histone kinase-1 phosphory-
lates H2A Thr 119 during mitosis in the early Drosophila
embryo. Genes & Dev. 18: 877–888.
Andrews, P.D., Knatko, E., Moore, W.J., and Swedlow, J.R. 2003.
Mitotic mechanics: The auroras come into view. Curr. Opin.
Cell. Biol. 15: 672–683.
Barr, F.A., Sillje, H.H., and Nigg, E.A. 2004. Polo-like kinases
and the orchestration of cell division. Nat. Rev. Mol. Cell.
Biol. 5: 429–440.
Bischoff, J.R., Anderson, L., Zhu, Y., Mossie, K., Ng, L., Souza,
B., Schryver, B., Flanagan, P., Clairvoyant, F., Ginther, C., et
al. 1998. A homologue of Drosophila aurora kinase is onco-
genic and amplified in human colorectal cancers. EMBO J.
Carmena, M. and Earnshaw, W.C. 2003. The cellular geography
of aurora kinases. Nat. Rev. Mol. Cell. Biol. 4: 842–854.
Cho, R.J., Campbell, M.J., Winzeler, E.A., Steinmetz, L., Con-
way, A., Wodicka, L., Wolfsberg, T.G., Gabrielian, A.E.,
Landsman, D., Lockhart, D.J., et al. 1998. A genome-wide
transcriptional analysis of the mitotic cell cycle. Mol. Cell
Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D.,
Brown, P.O., and Herskowitz, I. 1998. The transcriptional
program of sporulation in budding yeast. Science 282: 699–
Clayton, A.L. and Mahadevan, L.C. 2003. MAP kinase-mediated
phosphoacetylation of histone H3 and inducible gene regu-
lation. FEBS Lett. 546: 51–58.
Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M.,
and Strober, W. 1994. Current protocols in immunology. In
Current protocols (ed. R. Coico), Chapter 8. John Wiley &
Sons, Inc., New York.
Crosio, C., Fimia, G.M., Loury, R., Kimura, M., Okano, Y.,
Zhou, H., Sen, S., Allis, C.D., and Sassone-Corsi, P. 2002.
Mitotic phosphorylation of histone H3: Spatio-temporal
regulation by mammalian Aurora kinases. Mol. Cell. Biol.
De Souza, C.P., Osmani, A.H., Wu, L.P., Spotts, J.L., and Os-
mani, S.A. 2000. Mitotic histone H3 phosphorylation by the
NIMA kinase in Aspergillus nidulans. Cell 102: 293–302.
Ditchfield, C., Johnson, V.L., Tighe, A., Ellston, R., Haworth,
C., Johnson, T., Mortlock, A., Keen, N., and Taylor, S.S.
2003. Aurora B couples chromosome alignment with ana-
phase by targeting BubR1, Mad2, and Cenp-E to kineto-
chores. J. Cell. Biol. 161: 267–280.
Fischle, W., Wang, Y., and Allis, C.D. 2003a. Binary switches
and modification cassettes in histone biology and beyond.
Nature 425: 475–479.
———. 2003b. Histone and chromatin cross-talk. Curr. Opin.
Cell. Biol. 15: 172–183.
Giet, R. and Glover, D.M. 2001. Drosophila aurora B kinase is
required for histone H3 phosphorylation and condensin re-
cruitment during chromosome condensation and to organize
the central spindle during cytokinesis. J. Cell. Biol. 152: 669–
Goto, H., Tomono, Y., Ajiro, K., Kosako, H., Fujita, M., Sakurai,
M., Okawa, K., Iwamatsu, A., Okigaki, T., Takahashi, T., et
al. 1999. Identification of a novel phosphorylation site on
histone H3 coupled with mitotic chromosome condensa-
tion. J. Biol. Chem. 274: 25543–25549.
Goto, H., Yasui, Y., Nigg, E.A., and Inagaki, M. 2002. Aurora-B
Dai et al.
486GENES & DEVELOPMENT
phosphorylates histone H3 at serine28 with regard to the
mitotic chromosome condensation. Genes Cells 7: 11–17.
Hauf, S., Cole, R.W., LaTerra, S., Zimmer, C., Schnapp, G., Wal-
ter, R., Heckel, A., van Meel, J., Rieder, C.L., and Peters, J.M.
2003. The small molecule Hesperadin reveals a role for Au-
rora B in correcting kinetochore-microtubule attachment
and in maintaining the spindle assembly checkpoint. J. Cell.
Biol. 161: 281–294.
Hendzel, M.J., Wei, Y., Mancini, M.A., Van Hooser, A., Ranalli,
T., Brinkley, B.R., Bazett-Jones, D.P., and Allis, C.D. 1997.
Mitosis-specific phosphorylation of histone H3 initiates pri-
marily within pericentromeric heterochromatin during G2
and spreads in an ordered fashion coincident with mitotic
chromosome condensation. Chromosoma 106: 348–360.
Higgins, J.M.G. 2001a. The Haspin gene: Location in an intron
of the Integrin ?E gene, associated transcription of an Inte-
grin ?E-derived RNA and expression in diploid as well as
haploid cells. Gene 267: 55–69.
———. 2001b. Haspin-like proteins: A new family of evolution-
arily conserved putative eukaryotic protein kinases. Protein
Sci. 10: 1677–1684.
———. 2003. Structure, function and evolution of haspin and
haspin-related proteins, a distinctive group of eukaryotic
protein kinases. Cell. Mol. Life Sci. 60: 446–462.
Hochstenbach, F., David, V., Watkins, S., and Brenner, M.B.
1992. Endoplasmic reticulum resident protein of 90 kilodal-
tons associates with the T- and B-cell antigen receptors and
major histocompatibility complex antigens during their as-
sembly. Proc. Natl. Acad. Sci. 89: 4734–4738.
Hsu, J.Y., Sun, Z.W., Li, X., Reuben, M., Tatchell, K., Bishop,
D.K., Grushcow, J.M., Brame, C.J., Caldwell, J.A., Hunt,
D.F., et al. 2000. Mitotic phosphorylation of histone H3 is
governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in
budding yeast and nematodes. Cell 102: 279–291.
Jenuwein, T. and Allis, C.D. 2001. Translating the histone code.
Science 293: 1074–1080.
Johnson, V.L., Scott, M.I., Holt, S.V., Hussein, D., and Taylor,
S.S. 2004. Bub1 is required for kinetochore localization of
BubR1, Cenp-E, Cenp-F and Mad2, and chromosome con-
gression. J. Cell. Sci. 117: 1577–1589.
Kimura, H. and Cook, P.R. 2001. Kinetics of core histones in
living human cells: Little exchange of H3 and H4 and some
rapid exchange of H2B. J. Cell. Biol. 153: 1341–1353.
Kung, A.L., Sherwood, S.W., and Schimke, R.T. 1990. Cell line-
specific differences in the control of cell cycle progression in
the absence of mitosis. Proc. Natl. Acad. Sci. 87: 9553–9557.
Lachner, M. and Jenuwein, T. 2002. The many faces of histone
lysine methylation. Curr. Opin. Cell. Biol. 14: 286–298.
Luger, K., Rechsteiner, T.J., Flaus, A.J., Waye, M.M., and Rich-
mond, T.J. 1997. Characterization of nucleosome core par-
ticles containing histone proteins made in bacteria. J. Mol.
Biol. 272: 301–311.
Nigg, E.A. 2001. Mitotic kinases as regulators of cell division
and its checkpoints. Nat. Rev. Mol. Cell. Biol. 2: 21–32.
Petersen, J., Paris, J., Willer, M., Philippe, M., and Hagan, I.M.
2001. The S. pombe aurora-related kinase Ark1 associates
with mitotic structures in a stage dependent manner and is
required for chromosome segregation. J. Cell. Sci. 114: 4371–
Polioudaki, H., Markaki, Y., Kourmouli, N., Dialynas, G., The-
odoropoulos, P.A., Singh, P.B., and Georgatos, S.D. 2004. Mi-
totic phosphorylation of histone H3 at threonine 3. FEBS
Lett. 560: 39–44.
Preuss, U., Landsberg, G., and Scheidtmann, K.H. 2003. Novel
mitosis-specific phosphorylation of histone H3 at Thr11 me-
diated by Dlk/ZIP kinase. Nucleic Acids Res. 31: 878–885.
Prigent, C. and Dimitrov, S. 2003. Phosphorylation of serine 10
in histone H3, what for? J. Cell. Sci. 116: 3677–3685.
Schaar, B.T., Chan, G.K., Maddox, P., Salmon, E.D., and Yen,
T.J. 1997. CENP-E function at kinetochores is essential for
chromosome alignment. J. Cell. Biol. 139: 1373–1382.
Schneider, R., Bannister, A.J., Myers, F.A., Thorne, A.W., Crane-
Robinson, C., and Kouzarides, T. 2004. Histone H3 lysine 4
methylation patterns in higher eukaryotic genes. Nat. Cell.
Biol. 6: 73–77.
Schurter, B.T., Koh, S.S., Chen, D., Bunick, G.J., Harp, J.M.,
Hanson, B.L., Henschen-Edman, A., Mackay, D.R., Stallcup,
M.R., and Aswad, D.W. 2001. Methylation of histone H3 by
coactivator-associated arginine methyltransferase 1. Bio-
chemistry 40: 5747–5756.
Shannon, K.B. and Salmon, E.D. 2002. Chromosome dynamics:
New light on Aurora B kinase function. Curr. Biol. 12: R458–
Sharrocks, A.D. 1994. A T7 expression vector for producing N-
and C-terminal fusion proteins with glutathione S-transfer-
ase. Gene 138: 105–108.
Shoemaker, C.B. and Chalkley, R. 1980. H3-specific nucleohis-
tone kinase of bovine thymus chromatin: Purification, char-
acterization, and specificity for threonine residue 3. J. Biol.
Chem. 255: 11048–11055.
Spector, D.L., Goldman, R.D., and Leinward, L.A. 1997. Cells: A
laboratory manual. Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY.
Spellman, P.T., Sherlock, G., Zhang, M.Q., Iyer, V.R., Anders,
K., Eisen, M.B., Brown, P.O., Botstein, D., and Futcher, B.
1998. Comprehensive identification of cell cycle-regulated
genes of the qeast Saccharomyces cerevisiae by microarray
hybridization. Mol. Biol. Cell. 9: 3273–3297.
Sullivan, B.A. and Karpen, G.H. 2004. Centromeric chromatin
exhibits a histone modification pattern that is distinct from
both euchromatin and heterochromatin. Nat. Struct. Mol.
Biol. 11: 1076–1083.
Swedlow, J.R. and Hirano, T. 2003. The making of the mitotic
chromosome: Modern insights into classical questions. Mol.
Cell 11: 557–569.
Tanaka, T.U. 2002. Bi-orienting chromosomes on the mitotic
spindle. Curr. Opin. Cell Biol. 14: 365–371.
Tanaka, H., Yoshimura, Y., Nishina, Y., Nozaki, M., Nojima,
H., and Nishimune, Y. 1994. Isolation and characterization
of cDNA clones specifically expressed in testicular germ
cells. FEBS Lett. 355: 4–10.
Tanaka, H., Yoshimura, Y., Nozaki, M., Yomogida, K., Tsu-
chida, J., Tosaka, Y., Habu, T., Nakanishi, T., Okada, M.,
Nojima, H., et al. 1999. Identification and characterization
of a haploid germ cell-specific nuclear protein kinase
(haspin) in spermatid nuclei and Its effects on somatic cdells.
J. Biol. Chem. 274: 17049–17057.
Tanaka, H., Iguchi, N., Nakamura, Y., Kohroki, J., Egydio de
Carvalho, C., and Nishimune, Y. 2001. Cloning and charac-
terization of human haspin gene encoding haploid germ cell-
specific nuclear protein kinase. Mol. Hum. Reprod. 7: 211–
Taylor, S.S. and McKeon, F. 1997. Kinetochore localization of
murine Bub1 is required for normal mitotic timing and
checkpoint response to spindle damage. Cell 89: 727–735.
Turner, B.M. 2002. Cellular memory and the histone code. Cell
Vandre, D.D. and Borisy, G.G. 1989. Anaphase onset and de-
phosphorylation of mitotic phosphoproteins occur concomi-
tantly. J. Cell Sci. 94: 245–258.
Van Hooser, A., Goodrich, D.W., Allis, C.D., Brinkley, B.R., and
Mancini, M.A. 1998. Histone H3 phosphorylation is re-
Haspin is a histone H3 kinase
GENES & DEVELOPMENT 487
quired for the initiation, but not maintenance, of mamma- Download full-text
lian chromosome condensation. J. Cell Sci. 111: 3497–3506.
Wei, Y., Yu, L., Bowen, J., Gorovsky, M.A., and Allis, C.D. 1999.
Phosphorylation of histone H3 is required for proper chro-
mosome condensation and segregation. Cell 97: 99–109.
Yoshimura, Y., Tanaka, H., Nozaki, M., Yomogida, K., Yasu-
naga, T., and Nishimune, Y. 2001. Nested structure of hap-
loid germ cell specifc haspin gene. Gene 267: 49–54.
Zeitlin, S.G., Shelby, R.D., and Sullivan, K.F. 2001. CENP-A is
phosphorylated by Aurora B kinase and plays an unexpected
role in completion of cytokinesis. J. Cell Biol. 155: 1147–
Dai et al.
488 GENES & DEVELOPMENT