Download Histone H3 phosphorylation is required for the initiation, but not

3497
Journal of Cell Science 111, 3497-3506 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS7316
Histone H3 phosphorylation is required for the initiation, but not
maintenance, of mammalian chromosome condensation
Aaron Van Hooser1, David W. Goodrich2, C. David Allis3, B. R. Brinkley1 and Michael A. Mancini1,*
1Department
2Department
3Department
of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
of Tumor Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA
of Biology, University of Rochester, Rochester, NY 14627, USA
*Author for correspondence (e-mail: [email protected])
Accepted 1 October; published on WWW 12 November 1998
SUMMARY
The temporal and spatial patterns of histone H3
phosphorylation implicate a specific role for this
modification in mammalian chromosome condensation.
Cells arrest in late G2 when H3 phosphorylation is
competitively inhibited by microinjecting excess substrate
at mid-S-phase, suggesting a requirement for activity of the
kinase that phosphorylates H3 during the initiation of
chromosome condensation and entry into mitosis. Basal
levels of phosphorylated H3 increase primarily in latereplicating/early-condensing heterochromatin both during
G2 and when premature chromosome condensation is
induced. The prematurely condensed state induced by
okadaic acid treatment during S-phase culminates with H3
phosphorylation throughout the chromatin, but in an
absence of mitotic chromosome morphology, indicating
that the phosphorylation of H3 is not sufficient for complete
condensation. Mild hypotonic treatment of cells arrested in
mitosis results in the dephosphorylation of H3 without a
cytological loss of chromosome compaction. Hypotonictreated cells, however, complete mitosis only when H3 is
phosphorylated. These observations suggest that H3
phosphorylation is required for cell cycle progression and
specifically for the changes in chromatin structure incurred
during chromosome condensation.
INTRODUCTION
of chromatin to condensation factors, transcription factors, and
proteases activated during apoptotic cell death (Roth and Allis,
1992; Hendzel et al., 1997; Waring et al., 1997). Additionally,
the amino-termini of core histones might be involved in
protein-protein interactions and the recruitment of trans-acting
factors to specific regions of chromatin (Hendzel et al., 1997).
Site-specific phosphorylation of histone H3 at serine 10
(Ser10) initiates during G2, becomes maximal during
metaphase, and diminishes during late anaphase and early
telophase (Gurley et al., 1978; Paulson and Taylor, 1982;
Hendzel et al., 1997). In Tetrahymena, H3 phosphorylation
occurs only in the mitotic micronucleus; H3 is not
phosphorylated in the macronucleus, which divides
amitotically without obvious chromosome condensation (Allis
and Gorovsky, 1981). When mammalian interphase cells are
fused with mitotic cells, premature chromosome condensation
(PCC) is accompanied by significantly increased levels of H3
phosphorylation (Johnson and Rao, 1970; Hanks et al., 1983).
Similarly, rapid and stable increases in H3 phosphorylation at
Ser10 accompany PCC induced by the protein phosphatase
inhibitors okadaic acid (OA) and fostriecin (Ajiro et al., 1996;
Guo et al., 1995).
In this report, we correlate the initiation of mammalian
chromosome condensation with H3 phosphorylation
The relaxed chromatin of interphase is condensed during cell
division to allow its segregation on the spindle apparatus.
Cytologically, condensation initiates after S-phase and reaches
its maximum during early mitosis (Adlakha and Rao, 1986). It
is generally thought that the condensation of chromatin occurs
through its folding into loops, compaction of the loops, and
coiling of the axis upon which the loops are anchored
(reviewed by Manuelidis and Chen, 1990; Koshland and
Strunnikov, 1996). Additionally, tangles are resolved that arise
during DNA replication and by simple diffusion (Sundin and
Varshavsky, 1981; Weaver et al., 1985).
Several histones are phosphorylated with chromosome
condensation, but a causal role for these modifications in
chromatin modeling has not been clearly established (reviewed
by Bradbury, 1992). Histone phosphorylations have been
implicated in both the initiation and maintenance of mitotic
chromosome condensation (Bradbury et al., 1973; Marks et al.,
1973; Gurley et al., 1975, 1978), as well as in the silencing of
transcription during mitosis (Paulson and Taylor, 1982). More
recently it has been proposed that the phosphorylation of
histone amino-terminal tails might reduce their affinity for
DNA and facilitate the movement of nucleosomes and access
Key words: Centromere, Heterochromatin, Histone, Mitosis, Okadaic
acid, Phosphorylation
3498 A. Van Hooser and others
specifically in late-replicating/early-condensing chromatin
during normal division cycles and when premature
condensation is induced. Activity of the kinase that
phosphorylates H3 is required for entry into mitosis, but is not
required for maintaining the condensation of chromosomes in
hypotonically-swollen cells. This modification, then, is
associated specifically with cell cycle progression and the
dynamics of chromatin modeling in mammalian cells, but may
not be required for maintaining the condensed state.
MATERIALS AND METHODS
Cell culture and synchronization
CHO-K1 (Chinese hamster ovary), HeLa, and Indian muntjac
(Muntiacus muntjac vaginalis) (IM) cells were cultured in Opti-MEM
I (Gibco Laboratories, Grand Island, NY), supplemented with 4%
FBS and 1% penicillin/streptomycin. Cultures were maintained in a
humidified 37°C incubator with a 5% CO2 atmosphere. Cell cultures
were synchronized in G0 by growing them to confluency in serumdepleted medium, replating to a density of 1×106 cells/28 mm2, and
incubating in serum-free medium for 66 hours at 37°C, at which time
they lacked rounded mitotic cells when examined under the phase
contrast microscope (Rao and Engleberg, 1966; Ashihara and
Baserga, 1979). The cells were then incubated in serum-containing
medium for 4 hours to allow their entry into G1 before adding 2 mM
hydroxyurea (HU; Sigma Chemical Co., St Louis, MO) to inhibit
DNA synthesis and arrest cells at the G1/S-phase boundary.
Synchronized cultures were released after 16 to 20 hours of block by
washing twice with PBS (0.14 M NaCl, 2.5 mM KCl, 8 mM
Na2HPO4, 1.6 mM KH2PO4, pH 7.4) at 37°C and adding fresh
medium with serum. Replicating DNA was labeled with the thymidine
analog 5-bromo-2′-deoxyuridine (BrdU; Sigma Chemical Co.), added
to the medium of cell cultures to a final concentration of 20 µM.
Incorporation was detected using mouse monoclonal anti-BrdU
antibody with nuclease (Amersham Life Science, Inc., Piscataway,
NJ) and the immunofluorescence procedure given below. PCC was
induced in CHO, HeLa, and IM cells with 0.25 to 0.5 µM OA (Gibco
BRL, Gaithersburg, MD) for 1.25 to 5 hours. Cells that became
detached from the substratum with prolonged treatment were
cytospun onto coverslips prior to immunostaining, as described below.
Mitotic cell preparations were obtained by exposing asynchronous
cultures to the microtubule-depolymerizing drug colcemid (Gibco
Laboratories) at 0.1 µg/ml for 4 (HeLa) to 16 (IM) hours. Hypotonic
treatment consisted of a 10 minute incubation of cells at 37°C in 75
mM KCl containing multiple protease inhibitors (0.1 mM
phenylmethylsulfonyl fluoride (PMSF) and 1 µg/ml each of aprotinin,
leupeptin, antipain, and pepstatin).
Immunofluorescence microscopy
Cells were grown on poly-amino acid-coated glass coverslips, or,
alternatively, mitotic cells were selectively detached by mitotic shakeoff, pelleted at 1,000 g for 5 minutes, and cytocentrifuged (1,000 rpm,
2 minutes) onto coverslips using a Cytospin 3 (Shandon Instruments,
Inc., Pittsburgh, PA). Cells were extracted with 0.5% Triton X-100 in
PEM (80 mM K-Pipes, pH 6.8, 5 mM EGTA, pH 7.0, 2 mM MgCl2)
for 2 minutes at 4°C. Samples were fixed in 4% formaldehyde
(Polysciences, Inc., Warrington, PA) in PEM for 20 minutes at 4°C
and permeabilized with 0.5% Triton X-100 in PEM for 30 minutes at
RT. Non-specific binding of antibodies was blocked overnight at 4°C
with 5% milk in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl,
0.1% Tween-20). Auto-antiserum from an individual with CREST
(calcinosis, Raynaud’s phenomenon, esophageal dysmotility,
sclerodactyly, and telangiectasia) variant scleroderma (designated SH)
was obtained from the Comprehensive Arthritis Center at the
University of Alabama at Birmingham and has been characterized
previously (Moroi et al., 1980; Valdivia and Brinkley, 1985). CREST,
anti-phosphorylated H3 (Upstate Biotechnology, Lake Placid, NY),
and anti-acetylated H3 primary antisera were diluted 1:1,000 in TBST and incubated on preparations for 1 hour at 37°C. The samples were
then rinsed in TBS-T and blocked again in 5% milk. Fluorophorelabeled goat anti-mouse, anti-rabbit, and anti-human IgG (H+L)
secondary antibodies (Jackson Immunoresearch Laboratories, Inc.,
West Grove, PA) were diluted in TBS-T and incubated with samples
for 45 minutes at 37°C. The preparations were then rinsed in TBS-T,
and DNA was counterstained with 0.1 µg/ml 4′,6-diamidino-2phenylindole (DAPI) in TBS-T. Coverslips were mounted onto glass
slides using Vectashield anti-fade medium (Vector Laboratories, Inc.,
Burlingame, CA). Figs 1I,J and 7 are composite images obtained with
a Deltavision, deconvolution-based optical workstation (Applied
Precision, Issaquah, WA). Z-series stacks of multiple focal planes
were deconvolved by a constrained iterative algorithm, giving rise to
high-resolution optical sections used in rendering 3-D volumes. Fig.
8 was acquired using a Molecular Dynamics (Sunnyvale, CA) Multi
Probe 2001 laser scanning confocal microscope with a KrAr dual-line
laser and digitally processed using Imagespace software. All other
images were collected using a Zeiss Axiophot fluorescence
microscope with a Hamamatsu high-resolution/high-sensitivity three
chip CCD video camera and digitally processed using Adobe
Photoshop.
Microinjections
Unmodified H3 peptide was synthesized corresponding to the
histone H3 amino-terminal amino acids 1-20 (A1RTKQTARKSTGGKAPRKQL20C). Phosphorylated H3 peptide was
synthesized such that it contained a single phosphorylated serine
residue at position 10 (A1RTKQTARKpSTGGKAPRKQL20C). All
peptides contained an artificial cysteine residue at position 21 for
coupling purposes. Peptides were diluted in injection buffer (20 mM
Tris, 50 mM KCl, pH 7.4) with 1:5 biotinylated anti-goat IgG (H+L)
included as a marker, detected by avidin-TXRD (Molecular Probes,
Inc., Eugene, OR), and injected into the cytoplasm using a gas-driven
microinjector (Eppendorf). Results from repeated measurements were
statistically analyzed using Student’s t-test. Statistical significance
was determined with a probability of less than 0.05.
RESULTS
Late-replicating/early-condensing chromatin is the
first to undergo H3 phosphorylation during G2
Late-replicating chromatin condenses maximally during G2
and early prophase, followed by the condensation of earlyreplicating chromatin through late metaphase (Kuroiwa, 1971;
Francke and Oliver, 1978; Yunis, 1980; Drouin et al., 1991).
To determine if late-replicating/early-condensing chromatin is
the first to undergo histone H3 phosphorylation, cells
synchronized in late S-phase of the cell cycle were labeled by
BrdU incorporation and in the subsequent G2 phase with an
antiserum specific for the phosphorylated amino terminus
(Ser10) of H3 (Hendzel et al., 1997). CHO cultures become
enriched for cells in late S-phase approximately 5 hours after
release from HU-block at the G1/S boundary (O’Keefe et al.,
1992). At this time, replicating DNA was labeled with BrdU
for 6 hours, approximately the time it takes CHO cells to reach
late G2 from late S-phase. Immunofluorescence revealed
extensive colocalization of anti-BrdU with anti-phosphorylated
H3 (anti-PH3) antibodies, indicating that H3 is phosphorylated
primarily in late-replicating chromatin during G2 (Fig. 1A-D).
H3 phosphorylation and chromosome condensation 3499
Fig. 1. H3 phosphorylation initiates primarily
in late-replicating chromatin during G2.
(A-D) CHO cultures were labeled in late Sphase with BrdU, then harvested in the
subsequent G2 and immunostained with antiBrdU (green) and anti-PH3 (red) antibodies.
DNA was counterstained with DAPI (blue).
Bar, 10 µm. (E-H) Early-replicating chromatin
was labeled with BrdU prior to harvesting in
G2 and immunostaining as above. Bar, 10 µm.
(I,J) CHO cells were labeled in late S-phase
with BrdU (green), then harvested in the
subsequent mitosis and immunostained as
above. A z-series of deconvolved optical
sections was rotated 180° around the y-axis
and projected as two composite views. Sister
chromatids separating at anaphase were
observed to have mirrored banding patterns of
late-replicating chromatin (arrows). The
dephosphorylation of H3 has begun by late
anaphase, resulting in intermediate levels of
anti-PH3 staining (red). Bar, 5 µm.
Colocalization was not observed between BrdU incorporated
during the first 5 hours of S-phase, immediately following
release from HU-block, and anti-PH3 staining 6 hours later in
the subsequent G2 (Fig. 1E-H). To additionally determine the
specificity of BrdU labeling in the above experiments, we
labeled late replicating chromatin and harvested the cells in
mitosis, 12 hours after release from HU-block. Sister
chromatids were observed to have identical banding patterns,
indicative of the semi-conservative incorporation of BrdU
during late S-phase (Fig. 1I,J).
H3 phosphorylation initiates in pericentric
heterochromatin when premature chromosome
condensation is induced
The centromere is a principal site for the initiation of
chromosome condensation in mammalian cells (He and
Brinkley, 1996). H3 phosphorylation concurrently increases in
pericentric heterochromatin during G2 and advances outward
into the chromosome arms (Hendzel et al., 1997). As shown in
Fig. 2A, chromatic regions surrounding prekinetochores are
the exclusive sites of H3 phosphorylation during G2 in IM
cells, since the number of initiation sites exactly matches the
diploid chromosome number (2N=7么). The centromeresprekinetochores appear as doublets, stained with CREST autoantiserum, indicating that the cell has reached G2 (Brenner et
al., 1981). To determine if the initiation of H3 phosphorylation
follows the same pattern when PCC is induced, asynchronous
cell cultures were treated with OA and immunostained with
CREST and anti-PH3 antisera. Treatment of mammalian cells
with OA, which is a specific inhibitor of the serine/threonine
protein phosphatases 1 and 2A, results in PCC and a rapid and
stable increase in cellular phosphorylation levels, including
that of histone H3 Ser10 (Bialojan and Takai, 1988; Haystead
et al., 1989; Cohen et al., 1990; Mahadevan et al., 1991; Ajiro
et al., 1996). When PCC is induced in IM cells with 0.25 µM
OA, the G2 pattern of pericentric H3 phosphorylation is
observed in most cells within 90 minutes (Fig. 2B). The
prekinetochores, however, appear as open arrays of CRESTstained subunits, indicating that they have not reached late Sphase/early G2 and are not fully replicated (Brenner et al.,
1981; He and Brinkley, 1996). H3 is similarly phosphorylated
in pericentric heterochromatin when PCC is induced in CHO
and HeLa cells with OA (data not shown). We also examined
H3 phosphorylation in cells that were induced to enter mitosis
prematurely from the G1/S boundary by caffeine treatment
(Schlegel and Pardee, 1986; Brinkley et al., 1988). Prior to
entering mitosis, these cells pass through a G2-like state, in
which H3 phosphorylation accumulates primarily in
pericentric heterochromatin (data not shown).
Fig. 2. H3 phosphorylation is first detected in pericentric
heterochromatin when premature chromosome condensation is
induced with OA. (A) IM cells were immunostained with CREST
(green) and anti-PH3 (red) antisera. DNA was counterstained with
DAPI (blue). Bar, 5 µm. (B) IM cells were treated with 0.25 µM OA
for 1.25 hours to induce PCC and immunostained as above. Bar, 5 µm.
3500 A. Van Hooser and others
H3 phosphorylation is not sufficient for
chromosome condensation
The effects of OA treatment vary among cell type and cell
cycle stage. Exposure of most mammalian interphase cells to
the drug induces a mitosis-like state, characterized by PCC,
increased cdc2/H1 kinase activity, dispersion of nuclear
lamins, and the appearance of mitotic asters (Haystead et al.,
1989; Picard et al., 1989; Rime and Ozen, 1990; Yamashita
et al., 1990; Zheng et al., 1991). Morphologically normal
mitotic chromosome condensation with OA treatment,
however, is only observed in a small fraction of
unsynchronized cells and has been most clearly demonstrated
using mouse cells arrested at G2 (Guo et al., 1995; Ajiro et
al., 1996). Similarly, OA-treatment of mitotic cells results in
the overcondensation of chromosomes, as well as disruption
of the mitotic spindle and metaphase plate formation (Ghosh
Fig. 3. H3 phosphorylation is not sufficient for chromosome
condensation. (A,B) Asynchronous HeLa cells were treated with 0.5
µM OA for 2.5 hours prior to immunostaining. Rudimentary
chromosome morphology was observed in less than 10% of the cells
(arrow). Bar, 25 µm. (C,D) HeLa cells were released from HU-block
into early S-phase and treated with 0.5 µM OA for 2.5 hours prior to
immunostaining. Bar, 25 µm. (E,F) HeLa cells were released from
HU-block into early S-phase and treated with 0.5 µM OA for 5 hours
prior to immunostaining. High degrees of nuclear invagination were
common (arrowheads), though chromosome morphology was absent.
Bar, 25 µm. (G,H) In the absence of OA-treatment, phosphorylated
H3 is not observed in HeLa cells during early S-phase. Bar, 25 µm.
et al., 1992; Gliksman et al., 1992; Vandré and Wills, 1992).
To correlate the extent of H3 phosphorylation with OAinduced PCC, we exposed asynchronous cell populations to
0.5 µM OA for 2.5 hours. Under these conditions, over 70%
of HeLa and IM cells appeared rounded up with compact
nuclei characteristic of S-phase PCC. The nuclei of these
cells displayed high levels of H3 phosphorylation throughout
the chromatin (Fig. 3A,B), particularly at the nuclear
periphery, resembling prophase cells in untreated cultures
(Hendzel et al., 1997). Rudimentary chromosome
organization developed in less than 10% of the
unsynchronized cells treated under these conditions,
indicating that H3 phosphorylation throughout interphase
chromatin is not sufficient for mitotic chromosome
condensation. The number of cells demonstrating mitotic
chromosome organization was reduced to less than 1% when
cultures were arrested in early S-phase prior to treatment with
OA for 2.5 to 5 hours (Fig. 3C-F). CHO cells synchronized
in early S-phase demonstrated PCC and high levels of H3
phosphorylation only after 5 hours of treatment with 0.5 µM
OA (data not shown). Similarly, mitotic chromosome
morphology was observed in less than 1% of the cells.
In the absence of OA treatment, H3 phosphorylation was
undetectable during early interphase in HeLa cells (Fig. 3G,H).
Untreated CHO and IM cells, however, display nuclear foci of
anti-PH3 immunoreactivity during early interphase (Fig. 4).
The small size and number of these nuclear foci make them
distinct from the pericentric pattern of phosphorylation
observed during G2. To determine the temporal and spatial
occurrences of these foci in interphase nuclei, discrete phases
of DNA replication were visualized by pulse-labeling with
BrdU at progressive time points through S-phase (O’Keefe et
al., 1992). Thirty-two per cent of CHO cells blocked at the
G1/S boundary had anti-PH3 foci. This number increased upon
release and reached a maximum in late S-phase, when the
number of cells with foci was near 70%. The foci vary in
number from 1 to 5 at each stage and therefore do not appear
to be sequentially replicated. They do not correspond
exclusively to early-replicating (Fig. 4A-C) or late-replicating
regions of chromatin (Fig. 4D-F). All anti-PH3
immunoreactivity is abolished when the antiserum is incubated
with peptides corresponding to the phosphorylated form of the
H3 amino-terminal tail (Hendzel et al., 1997, and data not
shown).
Activity of the kinase that phosphorylates H3 is
required for entry into mitosis
The kinase responsible for histone H3 phosphorylation in vivo
has not been conclusively identified. It has been shown in a
cell-free system that cyclic AMP-dependent protein kinase can
phosphorylate H3 on Ser10 (Taylor, 1982; Shibata et al., 1990).
Similarly, H3 phosphorylation is linked with elevated cAMP
levels during gliotoxin-induced apoptosis (Waring et al., 1997).
When H3 phosphorylation is inhibited by the protein kinase
inhibitor staurosporine, mammalian cells arrest in late G2
(Crissman et al., 1991; Abe et al., 1991; Th’ng et al., 1994).
However, staurosporine is known to inhibit a wide range of
kinases, including p34cdc2, which controls mitotic entry
(Langan et al., 1989; Herbert et al., 1990; Gadbois et al., 1992).
To more directly define the role of the kinase that
phosphorylates H3 in cell cycle progression, we microinjected
H3 phosphorylation and chromosome condensation 3501
proliferating cells with peptide corresponding to the
unmodified amino-terminal 20 amino acids of H3 to compete
for the kinase activity. CHO cells were injected at mid-S-phase,
4 to 5 hours after release from HU-block at the G1/S boundary.
Control cells were injected with the phosphorylated (Ser10)
form of H3 peptide or with marker only (biotinylated anti-goat
IgG). Injected cultures were harvested at a time-point when
uninjected cultures become enriched for cells passing or just
having passed through mitosis, 12 to 13 hours after release
from HU-block. All preparations were masked, and the number
of mitotic cells, identified by DAPI staining, was scored at least
three times. Increasing concentrations of the unmodified H3
peptide resulted in proportional decreases in mitotic indexes
(Fig. 5A). The number of mitotic cells was nearly abolished
with the highest concentration of unmodified H3 peptide used.
Injection of phosphorylated H3 peptide or marker alone did not
significantly reduce the number of cells passing through
mitosis when compared to uninjected cells within the same
population (Fig. 5A).
To characterize the phenotype of cells inhibited with the
H3 competitor peptide, as well as those progressing through
mitosis despite injection, we repeated the injections with an
intermediate concentration (0.42 µg/µl) of unmodified H3
peptide, empirically determined in the previous experiment.
Use of an intermediate dose also reduced the possibility that
we were observing effects of peptide toxicity. The number of
mitotic, daughter, and binucleate cells was scored
morphologically (Fig. 5B). Control preparations were
injected with approximately five-times the concentration
(2.26 µg/µl) of H3 peptide in the phosphorylated form or with
marker only. A statistically significant reduction in the
number of mitotic and daughter cells was observed in
preparations injected with an intermediate dose of the
unmodified H3 peptide when compared to populations
injected with the phosphorylated form of H3 peptide or with
marker alone (Fig. 5C). When cell populations injected with
H3 peptide are normalized against populations injected with
marker only, we observed a 38% reduction in the number of
mitotic cells and a 36% reduction in the number of daughter
cells with an intermediate dose of competitor peptide (Fig.
5D). The number of cells with the focal anti-PH3 staining
pattern characteristic of late G2 was not significantly reduced,
indicating with the above data that arrest occurred just prior
to mitosis. A unique pattern of anti-PH3 staining was
observed in 5.5% of the interphase cells injected with
unmodified H3 peptide, characterized by high levels of
diffuse, predominately nuclear anti-PH3 antibody reactivity
in the absence of chromosome condensation (Fig. 5E-G). The
percentage of cells with this anti-PH3 staining pattern (5.5%)
nearly equals the sum of differences between unmodified H3
and marker only populations in percentages of G2 (1.71.3=0.4%), mitotic (3.9-2.4=1.5%), and daughter cells (9.05.7=3.3%). Anti-PH3 staining was diffuse throughout the
cytoplasm and nucleus of cells injected with the
phosphorylated form of H3 peptide, with remarkably high
nuclear staining in 1.3% of the cells (data not shown).
Significantly, all mitotic cells identified by DAPI staining had
H3 highly phosphorylated despite competitive inhibition
(Fig. 5E-G), further suggesting that the phosphorylation of
H3 is essential for entry into mitosis.
H3 phosphorylation is not required for the structural
maintenance of isolated chromosomes
Individual metaphase chromosomes can be microscopically
identified and examined in much greater detail by incubating
cells in water or mild hypotonic solution (75 mM KCl) for 10
minutes (Hsu, 1952). We have found that, though most
chromosomes are swollen in hypotonic solution without a
detectable alteration in their pattern of anti-PH3 staining, a
significant number of cells (up to 40% in IM preparations and
20% in HeLa) have chromosomes within which H3 appears
completely unphosphorylated (Fig. 6A,B). No difference in the
level of condensation between phosphorylated and
unphosphorylated chromosomes within the same preparation
is observable by DAPI staining, indicating that H3
phosphorylation is not required for maintaining high levels of
chromosome condensation. To determine if the lack of antiPH3 antibody reactivity with hypotonic treatment was due to
alterations in nucleosome structure, we immunostained the
preparations with an antibody that reacts with the diacetylated
(9, 14) H3 tail throughout the mammalian cell cycle (Boggs et
al., 1996). The pattern of anti-diacetylated H3 immunostaining
was unaltered in all hypotonic-treated cells, indicating that the
Ser10 epitope was likely still accessible and the distribution of
H3 unchanged (Fig. 6C,D). All cells arrested in mitosis for
prolonged periods (overnight) with colcemid had
chromosomes in which H3 was highly phosphorylated,
indicating that H3 dephosphorylation did not occur with
prolonged mitotic arrest or in the absence of hypotonic
treatment (data not shown).
Pericentric heterochromatin is resistant to
hypotonic-induced H3 dephosphorylation
Intermediate levels of H3 dephosphorylation were observed in
a number (less than 10%) of hypotonically treated IM and
HeLa mitotic cells. In each case, pericentric regions of
chromatin remained highly phosphorylated, whereas the
chromosomal arms were completely dephosphorylated. This
retention of H3 phosphorylation is easily observed in the large
compound centromeres-kinetochores of IM chromosomes
(Fig. 7) that have been stretched by cytocentrifugation
following brief hypotonic treatment (Zinkowski et al., 1991).
The region of phosphorylated chromatin spans the pairing
domain of the centromere-kinetochore complex, where sister
chromatids are joined at metaphase, and extends into the
chromosomal arms, corresponding to the span of
heterochromatin in these chromosomes (Brinkley et al., 1992).
Both phosphorylated and dephosphorylated centromeres
remain constricted and can be highly elongated, suggesting that
H3 phosphorylation is not required for maintaining high levels
of chromosome condensation in pericentric heterochromatin
and that stretching occurred by a mechanism other than H3
dephosphorylation. Interestingly, the region of chromatin
immediately subjacent to the CREST-stained centromerekinetochore lacks anti-PH3 staining (Fig. 7), similar to results
obtained by immunogold EM (Hendzel et al., 1997).
H3 phosphorylation correlates with cell cycle
progression after hypotonic treatment
Hypotonic swelling of mammalian cells leads to the loss of
kinetochore plate morphology, as well as the disruption of
3502 A. Van Hooser and others
H3, then released them into tissue culture medium and
followed their progress by immunostaining. Congression of
IM and HeLa chromosomes to the metaphase plate and entry
into anaphase began within 60 minutes of their return to
tissue culture medium from hypotonic. Every cell passing
through early anaphase had H3 highly phosphorylated,
though the chromosomes retained a swollen appearance (Fig.
8). No intermediate stages of H3 phosphorylation were
observed after 30 minutes of release. If hypotonic-treated
cells were returned to tissue culture medium containing
colcemid, every mitotic cell was phosphorylated within 30
minutes. H3 phosphorylation, then, is correlated with cell
viability and progression through mitosis following
hypotonic treatment.
Fig. 4. Foci of phosphorylated H3 form in CHO nuclei during early
interphase. G1 (A) and G2 (not shown) phases of the cell cycle were
distinguished by a 5-hour BrdU-labeling of DNA replication. The
cells were then immunostained with anti-PH3 (red) and anti-BrdU
(green) antibodies. DNA was counterstained with DAPI (blue). To
visualize discrete phases of DNA replication, cells were arrested at
the G1/S boundary with HU, released for 0.5 (B), 2 (C), 5 (D), 7 (E),
or 9 hours (F) and pulse-labeled for 20 minutes with BrdU prior to
immunostaining. Bars, 5 µm.
microtubules, centrioles, and centrosomes (Brinkley et al.,
1980; Ris and Witt, 1981). However, if the period of
hypotonic swelling is brief (under 15 minutes), the damage
can be reversed by returning the cells to an isotonic tissue
culture environment (Brinkley et al., 1980). Under these
conditions, cell viability is not adversely affected in most
cell lines. To determine if H3 phosphorylation is required for
progression through mitosis, we exposed mitotic cells to
hypotonic conditions, resulting in the dephosphorylation of
DISCUSSION
The nucleosomal histones are wrapped by DNA as octamers,
consisting of two H2A-H2B dimers and a tetramer of 2 H3 and
2 H4 histones (D’Anna and Isenberg, 1974; Moss et al., 1976;
Kornberg, 1977). H3 and H4 amino-terminal tails are exposed
from the compact core of the nucleosome and may be involved
in regulatory interactions with DNA, histone H1, and other
proteins (Glotov et al., 1978; Hardison et al., 1977; Mazen et
al., 1987; Roth and Allis, 1992). Interactions of the H3 tail may
be modulated by post-translational modifications, including the
acetylation of lysine residues 9, 14, 23, and 27, methylation of
lysines 9 and 27, and serines 10 and 28 may be phosphorylated
(Dixon et al., 1975; Taylor, 1982; Shibata et al., 1990; Bradbury,
1992). The residues involved in the above interactions are
largely invariable (Wells and McBride, 1989) and reflect a very
high universality in the mechanisms by which chromatin is
modeled. The anti-PH3 antiserum used in these studies reacts
Fig. 5. Delayed entry into mitosis with the competitive
inhibition of the kinase that phosphorylates H3.
(A) Synchronized CHO cells were injected at late Sphase with increasing concentrations of peptide
corresponding to the unmodified (䉬) or
phosphorylated (䊐) H3 amino terminus. The mitotic
index of each injected population (~125 cells each)
was divided by the mitotic index of uninjected cells on
the same coverslip to derive normalized values on the
y-axis. (B) Injected populations were stained for
coinjected marker antibody, and the number of mitotic
(filled arrow), recently divided daughter (arrowhead),
and binucleate cells (open arrow) was scored. G2 cells
were scored by their characteristic, focal pattern of
anti-PH3 immunostaining (see Figs 1, 2). Bar, 50 µm.
(C) Cells were injected with marker only (n=4), with
0.42 µg/µl unmodified H3 competitor peptide (n=6),
or with 2.26 µg/µl of the phosphorylated form of the
H3 peptide (n=2), where n is the number of separate
experiments consisting of ~200 injected cells each.
(D) Values from C were normalized by dividing the
number of cells injected with unmodified (black bars)
or phosphorylated (hatched bars) H3 peptide by the
number of cells injected with marker only (white bars)
at the same stage within the same preparation. Injected cells were immunostained for coinjected marker antibody (E), anti-PH3 antibody (F),
and DAPI (G). All mitotic chromosomes had H3 phosphorylated (arrow). A number of cells injected with unmodified H3 peptide had high
levels of diffuse, predominately nuclear anti-PH3 staining in the absence of chromosome condensation (arrowhead). Bar, 25 µm.
H3 phosphorylation and chromosome condensation 3503
Fig. 6. H3 phosphorylation is not required for the maintenance of
condensed chromosomes. (A,B) IM cells were arrested in mitosis
and incubated in hypotonic solution. Preparations were
immunostained with anti-PH3 antibody (red), and DNA was
counterstained with DAPI (blue). Unphosphorylated chromosomes
appeared condensed to the same degree as phosphorylated
chromosomes within the same preparation, as resolved by DAPI
staining (asterisks). Bar, 50 µm. (C) Anti-diacetylated H3
immunoreactivity (red) was observed in all hypotonically swollen
IM chromosomes (m). Interphase cells (i) stain more intensely
with the antibody, and low levels of cytoplasmic staining are
observed regardless of hypotonic treatment. Bar, 25 µm. (D) A
higher magnification view of hypotonically-swollen IM
chromosomes immunostained with anti-diacetylated H3 antibody
(red). Bar, 10 µm.
with the N-terminal phosphorylated form of Ser/Thr 10 in all
eukaryotes examined, staining with the highest intensity during
mitotic and, in Tetrahymena, meiotic chromosome
condensation (Hendzel et al., 1997; Wei et al., 1998).
Heterochromatic regions of chromosomes, including
centromeres and telomeres, remain highly condensed
throughout the cell cycle and likely contain elements that direct
chromosome condensation (Koshland and Strunnikov, 1996).
The centromere is a principal site for the initiation of
chromosome condensation at the onset of mitosis in
mammalian cells (He and Brinkley, 1996). Consistent with a
role in the initiation of mammalian cell chromosome
condensation, H3 phosphorylation dramatically increases
during G2 exclusively in the late-replicating/early-condensing
heterochromatin surrounding centromeres. The same pattern of
H3 phosphorylation is observed when PCC is induced by the
phosphatase inhibitor OA and in cells induced to enter mitosis
prematurely. These results provide additional evidence of the
tight coupling between H3 phosphorylation and the initiation of
chromosome condensation near centromeres-prekinetochores.
H3 appears highly phosphorylated throughout the chromatin of
OA-treated cells, but mitotic chromosome morphology does not
develop. This result indicates that H3 phosphorylation is not
sufficient for chromosome condensation.
Though chromatin-associated H3 provides a significantly
Fig. 7. Pericentric heterochromatin is resistant to hypotonic-induced
H3 dephosphorylation. IM cells were arrested in mitosis and incubated
in hypotonic solution prior to immunostaining with anti-PH3 (A) and
CREST antisera (B). DNA was counterstained with DAPI (C).
(D) Intermediate levels of H3 phosphorylation were observed in some
cells in which pericentric heterochromatin remained highly
phosphorylated, whereas chromosome arms were dephosphorylated.
Both regions remained highly condensed. Bar, 5 µm.
better substrate, free H3 monomers are phosphorylated in vitro
by cyclic AMP-dependent protein kinase (Belyavsky et al., 1980;
Shibata et al., 1990). We therefore expected injected H3 aminoterminal peptides containing the unmodified Ser10 epitope to
compete with, but not necessarily abolish chromatin-associated
H3 phosphorylation. This competition would likely become less
significant with the onset of mitosis and increased kinase activity
and/or chromatin-associated substrate accessibility. Injection of
unmodified H3 peptide into cells at mid-S-phase resulted in a
marked reduction of cells entering mitosis when compared to
cells injected with the phosphorylated form of H3 peptide or with
marker only. Though reduced in number, all injected cells passing
through mitosis had H3 highly phosphorylated, suggesting that
the modification is essential for entry into mitosis. Since injection
of H3 tails in the phosphorylated form did not result in the same
reduction of cells entering mitosis, competition for histone
Fig. 8. Cells do not pass from metaphase to anaphase with H3
dephosphorylated. (A) Cells arrested in mitosis were incubated
briefly in hypotonic solution to induce the dephosphorylation of H3
and released into tissue culture medium. The cells were collected 60
minutes after release and immunostained with anti-PH3 antibody
(red) and CREST auto-antiserum (green). All cells entering anaphase
after hypotonic treatment had H3 phosphorylated throughout their
chromosomes, though they retained a swollen appearance relative to
untreated preparations (B). Bar, 10 µm.
3504 A. Van Hooser and others
acetylase activity did not result in cell cycle arrest under the
conditions of our experiment. It remains possible that the injected
N-terminal peptides were not suitable substrates for acetylation;
or, interestingly, phosphorylated H3 tails are not substrates for
acetylation.
Concomitant with a reduction in the number of cells entering
mitosis in populations injected with unmodified H3 peptide, there
was an equivalent accumulation of interphase cells with high
levels of anti-PH3 reactivity diffuse throughout uncondensed
nuclei. We postulate that this fraction of cells represents the cell
cycle stage at which H3 kinase activity is elevated, resulting in
the phosphorylation of competitor peptides in the nucleus.
Together with the fact that the number of cells in late G2 was not
significantly reduced in injected populations, this suggests that
the competitive inhibition of H3 phosphorylation prevented
chromosome condensation and entry into mitosis. Our
conclusions support earlier studies, in which the protein kinase
inhibitor staurosporine was found to block H3 phosphorylation
and arrest mammalian cells in late G2 (Crissman et al., 1991; Abe
et al., 1991; Th’ng et al., 1994). However, it remains possible that
inhibition of the kinase that phosphorylates H3 prevented the
phosphorylation of other targets essential for entry into mitosis.
Collectively, the above evidence implicates a role for H3
phosphorylation in the initiation of chromosome condensation
and entry into mitosis. This modification, however, may not have
a critical role in the maintenance of chromosome condensation,
as chromosomes can be hypotonically swollen with H3
remaining phosphorylated, and the dephosphorylation of H3
does not result in decondensation. It is important to note that
hypotonic treatment results in extensive physiological disruption
of cells, albeit reversible in most cases. The primary conclusion
that can be drawn from the above data, then, is that H3
phosphorylation is not required for maintaining the condensation
of isolated chromosomes. However, this result also confirms the
in vivo observations of Rao and colleagues, who found that the
fusion of mammalian mitotic cells with interphase cells results
in a large reduction of H1 and H3 phosphorylations without a
loss of chromosome condensation (Johnson and Rao, 1970;
Hanks et al., 1983). It also suggests that the decondensation of
metaphase chromosomes observed following treatment with the
protein kinase inhibitors staurosporine (Th’ng et al., 1994) and
N-6 dimethylaminopurine (Ajiro et al., 1996) may not have been
produced exclusively by H3 dephosphorylation. This is easily
conceived, as staurosporine is a potent inhibitor of all kinases
tested in vitro, though its action in vivo may be narrower
(Herbert et al., 1990; Th’ng et al., 1994); and N-6
dimethylaminopurine is a non-specific kinase inhibitor in vitro,
capable of affecting multiple phases of the cell cycle (Vesely et
al., 1994; Simili et al., 1997).
Though hypotonic treatment resulted in a large number of
cells with dephosphorylated chromosomal H3, when these cells
were returned to an isotonic tissue culture environment, they
were not observed to enter anaphase with H3 dephosphorylated.
It is not structural inhibition that prevents cells with
dephosphorylated chromosomes from progressing through
mitosis, as these chromosomes appear as compact as those with
H3 phosphorylated that undergo anaphase within the same
preparation. It is both possible that hypotonically-treated cells
rephosphorylate chromosomal H3 upon their return to an
isotonic environment, or simply that cells with dephosphorylated
chromosomes are not viable. Suggestive of the latter, IM cells
are particularly susceptible to H3 dephosphorylation and recover
poorly from hypotonic treatment relative to other mammalian
cell lines (B.R.B., unpublished observations).
Intermediate levels of H3 phosphorylation were observed in
hypotonic-treated chromosomes of IM and HeLa cells, in which
pericentric heterochromatin remained phosphorylated and
chromosome arms were dephosphorylated. Since pericentric
heterochromatin is more resistant to hypotonic swelling than
other chromosomal regions (Brinkley et al., 1980), H3
phosphorylation may have a role in maintaining the highest
levels of chromosome condensation. Similarly, we found that
the chromosomes of cells arrested in mitosis with colcemid
were more resistant to hypotonic-induced dephosphorylation of
H3 than those of unarrested mitotic cells, perhaps related to the
varying degrees of hypercondensation observed in colcemid
treated preparations (see Rieder and Palazzo, 1992). H3
phosphorylation, then, correlates with the most condensed
regions of chromatin during hypotonic treatment, though its
removal does not result in decondensation.
The periodicity of interaction between DNA and nuclear
matrix proteins appears to hold interphase chromatin in loops of
approximately 60 kb, which may be folded to form the ~200 nm
wide chromatin fiber observed by electron microscopy (Marsden
and Laemmli, 1979; Rattner and Lin, 1985; Fey et al., 1986;
Nelson et al., 1986). Euchromatin is compacted from a ~200 nm
diameter interphase fiber to a 700 nm metaphase structure. H3
phosphorylation correlates best with the initial events of
condensation: specifically, compaction of the ~200 nm
chromatid fiber (Hendzel et al., 1997). However,
heterochromatin remains at an approximately equivalent level of
compaction (700 nm) throughout the cell cycle (reviewed by
Manuelidis and Chen, 1990). The fact that metaphase
chromosomes can have H3 hypotonically dephosphorylated
without a cytological loss of chromosome condensation is
consistent with the fact that heterochromatin is
dephosphorylated at the end of mitosis, yet maintains a high
level of compaction. Why then is heterochromatin
phosphorylated? One possibility is that H3 phosphorylation in
pericentric heterochromatin may be required for the release of
sister chromatid cohesion at the metaphase to anaphase
transition, a process interrelated with chromosome condensation
(Guacci et al., 1997). Additionally H3 phosphorylation may be
required throughout early mitosis for the initiation of
chromosome condensation, which continues until late
metaphase (Adlakha and Rao, 1986; Drouin et al., 1991).
Correlating morphological chromatin data with the
quantification of histone phosphorylations led to the suggestion
that H3 phosphorylation occurs during the final organization and
maintenance of chromosomes (Gurley et al., 1978). More
recently, however, the phosphorylation of H3 has been observed
to precede detectable chromosome condensation and its
dephosphorylation to precede detectable chromosome
decondensation (Hendzel et al., 1997). Additionally, H3
phosphorylation may be associated with the relatively relaxed
state of transcriptionally active chromatin coincident with the
mitogenic induction of early-response genes, as well as make
chromatin accessible to nucleases involved in apoptotic pathways
(Halegoua and Patrick, 1980; Mahadevan et al., 1988, 1991;
Barratt et al., 1994; Waring et al., 1997). An attractive hypothesis
that reconciles these apparently divergent properties is that the
phosphorylation of histone N-terminal tails reduces their affinity
H3 phosphorylation and chromosome condensation 3505
for DNA and facilitates the movement of nucleosomes and
targeting of factors involved in condensation, transcription, and
apoptosis (Roth and Allis, 1992; Hendzel et al., 1997; Waring et
al., 1997). In this way, H3 phosphorylation would work in
conjunction with other mechanisms involved in the modeling of
active chromatin, including the phosphorylation and depletion of
histone H1 and the enrichment of hyperacetylated histones H3
and H4 (Allegra et al., 1987; Kamakaka and Thomas, 1990; Roth
and Allis, 1992; Mizzen and Allis, 1998). Our data supports this
role for histone phosphorylation in chromatin modeling, in that
the phosphorylation of H3 appears to be required for dynamic
changes in chromatin structure, but not for their maintenance.
The molecular mechanisms by which heterochromatin is kept
refractory from the dynamic changes of other chromosomal
regions remain largely unknown, as do the mechanisms by
which centromeres-prekinetochores direct the initiation of
chromosome condensation in mammalian cells during G2, and
why. Seemingly disparate events coalesce at the nuclear
envelope. H3 phosphorylation initiates with chromosome
condensation in pericentric heterochromatin, often observed to
be at the nuclear periphery (Comings and Okada, 1970; Robbins
et al., 1970). Similarly, chromosome condensation initiates in
foci on the nuclear envelope during the early embryonic
divisions of Drosophila nuclei, though these regions are distinct
from centromeres and telomeres (Hiraoka et al., 1989). The
highest levels of H3 phosphorylation in mammalian prophase
nuclei are consistently observed at the periphery of the nucleus.
Just as the nuclear lamina is phosphorylated and disassembles,
kinetochore plates first appear. In this way, nuclear structures are
central components of the signaling cascades leading to mitosis.
The authors thank T. Goepfert, I. Ouspenski, and S. Sazer for
helpful comments regarding the manuscript; and M. G. Mancini, C.
P. Schultz, and L. Zhong for contributing their technical expertise.
These studies were supported by grants from the NIH to D. W.
Goodrich (CA70292), to C. D. Allis (GM40922), from the NIH/NCI
to B. R. Brinkley (CA41424 and CA64255), and a National Scientist
Development Grant (9630033N) from the American Heart
Association to M. A. Mancini. This work is dedicated to T. C. Hsu
on the occasion of his 82nd birthday.
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