RESEARCH ARTICLE
653
Differential regulation of CENP-A and histone H3
phosphorylation in G2/M
Samantha G. Zeitlin1,*, Cynthia M. Barber2,*, C. David Allis2 and Kevin E. Sullivan1,‡
1Department of Cell Biology, The Scripps Research Institute, La Jolla, CA, USA
2Departments of Biochemistry and Molecular Genetics and Microbiology, University
of Virginia, Charlottesville, VA, USA
*These authors contributed equally to this work
‡Author for correspondence (e-mail: [email protected])
Accepted 28 November 2000
Journal of Cell Science 114, 653-661 © The Company of Biologists Ltd
SUMMARY
After DNA replication, cells condense their chromosomes
in order to segregate them during mitosis. The
condensation process as well as subsequent segregation
requires phosphorylation of histone H3 at serine 10.
Histone H3 phosphorylation initiates during G2 in
pericentric foci prior to H3 phosphorylation in the
chromosome arms. Centromere protein A (CENP-A), a
histone H3-like protein found uniquely at centromeres,
contains a sequence motif similar to that around H3 Ser10,
suggesting that CENP-A phosphorylation might be linked
to pericentric initiation of histone H3 phosphorylation. To
test this hypothesis, we generated peptide antibodies
against the putative phosphorylation site of CENP-A.
ELISA, western blot and immunocytochemical analyses
show that CENP-A is phosphorylated at the shared
motif. Simultaneous co-detection demonstrates that
phosphorylation of CENP-A and histone H3 are separate
events in G2/M. CENP-A phosphorylation occurs after
both pericentric initiation and genome-wide stages
of
histone
H3
phosphorylation.
Quantitative
immunocytochemistry
reveals
that
CENP-A
phosphorylation begins in prophase and reaches maximal
levels in prometaphase. CENP-A phosphoepitope reactivity
is lost during anaphase and becomes undetectable in
telophase cells. Duplication of prekinetochores, detected as
the doubling of CENP-A foci, occurs prior to complete
histone H3 phosphorylation in G2. Mitotic phosphorylation
of histone H3-family proteins shows tight spatial and
temporal control, occurring in three phases: (1) pericentric
H3 phosphorylation, (2) chromosome arm H3
phosphorylation and (3) CENP-A phosphorylation at
kinetochores. These observations reveal new cytological
landmarks characteristic of G2 progression.
INTRODUCTION
terminal tails of each of the four core histones (reviewed by
Luger and Richmond, 1998; Grunstein, 1998; Strahl and Allis,
2000). The chemical diversity of modifications, including
lysine-N-acetylation,
lysine-N-methylation,
arginine
methylation,
ADP-ribosylation,
ubiquitination
and
phosphorylation, has led to the ‘histone code hypothesis’, in
which each site of modification can serve a distinct function
(Strahl and Allis, 2000). For example, acetylation of histone
H4 lysines 5 and 12 is correlated with deposition (Verrault et
al., 1996), and acetylation at residues 8 and 16 are associated
with transcriptional activation (Kuo et al., 1996), while
hypoacetylation of histone H4 is associated with
heterochromatic domains (Turner et al., 1992). According
to the histone code model, modifications (singly or in
combination) create changes in the overall charge density of
histone tails, which in turn modulate histone interactions with
DNA, with non-histone proteins and with other histones. By
this mechanism, histone modifications both receive and
transmit changes in protein-protein interactions within
chromatin to and from higher-level signaling pathways. For
example, histone H3 phosphorylation levels are increased in
response to mitogen stimulation, raising levels of immediateearly gene expression (Mahadevan et al., 1991; Thomson et al.,
1999; Chadee et al., 1999; Sassone-Corsi et al., 1999; Cheung
During the G2 phase of the cell cycle the replicated
chromosomes are extensively modified in preparation for
mitosis. This process culminates in 20-100 fold chromosome
condensation and refolding into the familiar compact mitotic
configuration which facilitates efficient segregation as cells
divide (Trask et al., 1993; Heck, 1997). Mitotic chromosome
condensation involves both assembly and disassembly of
chromatin associated proteins as the chromosome switches
from transcriptional activities to its transport form. During
G2, many transcription-associated proteins dissociate or
redistribute within the chromosomes (Platero et al., 1998), and
specific mitosis-associated protein complexes are assembled or
activated (Hirano et al., 1997). One of these mitosis-specific
complexes is the kinetochore, a tri-laminar plate-like structure
that forms on the centromeric locus of each chromosome in
mitosis (reviewed by Rieder and Salmon, 1998). The
complexity of kinetochore structure exemplifies the unique
functional configuration of mitotic chromosomes which results
from the G2 remodeling process.
The histones play a central role in modulating protein
assembly on the chromatin fiber. This is regulated by posttranslational modifications that occur on the flexible N-
Key words: CENP-A, Histone, Phosphorylation, Kinetochore,
Mitosis
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JOURNAL OF CELL SCIENCE 114 (4)
et al., 2000). Understanding how histone modifications are
targeted to specific chromosomal loci and how regulation is
achieved in different nuclear compartments will be key steps
toward unraveling chromatin-directed signaling pathways.
Histone H3 phosphorylation at Ser10 in particular plays a
key role in mitotic chromosome condensation (Wei et al., 1998;
Wei et al., 1999; Hsu et al., 2000). In the absence of histone
H3 phosphorylation, chromosome condensation is incomplete
and anaphase chromosome separation is highly defective (Wei
et al., 1999; Hsu et al., 2000). Histone H3 phosphorylation is
highly regulated in G2, initiating specifically in pericentric
heterochromatin in characteristic rings around the centromeres
prior to general H3 phosphorylation that occurs in chromatin
throughout the chromosome arms (Hendzel et al., 1997). A
unique histone H3-related protein, CENP-A, is found
specifically at centromeres throughout the cell cycle where it
is thought to substitute for histone H3 in the nucleosomes
of kinetochore-associated chromatin (Brenner et al., 1981;
Earnshaw and Rothfield, 1985; Sullivan et al., 1994; Yoda et
al., 2000). In addition to a conserved H3-like histone fold
domain, CENP-A contains an N-terminal tail that shares very
little sequence identity with that of histone H3. However,
several amino acids surrounding the histone H3 Ser10 are
conserved in CENP-A, suggesting that the mitotic
phosphorylation motif might be shared between these two
proteins. If this were the case, it might help clarify the
mechanisms through which histone H3 phosphorylation is
regulated during G2/M. In this work we have demonstrated that
phosphorylation of CENP-A occurs at Ser7 within the shared
mitotic phosphorylation motif. Using immunocytochemistry,
we compared the kinetic and spatial organization of CENP-A
and histone H3 phosphorylation in G2/M. Our results show that
CENP-A phosphorylation is unlikely to play a role in the
initiation of histone H3 phosphorylation in pericentric
regions. Instead, H3 phosphorylation precedes CENP-A
phosphorylation, which occurs as a distinct reaction in prophase.
MATERIALS AND METHODS
Cell culture
HeLa (ATCC CCL3) and U2OS (ATCC HTB96) cells were
maintained in DMEM with 10% FCS (Gibco-BRL, Gaithersburg,
MD) at 37°C in a 5% CO2 atmosphere.
Peptide synthesis, conjugation and injection
Unmodified CENP-A peptide was synthesized corresponding to
residues 4-17 of human CENP-A (RRRSRKPEAPRRRS).
Phosphorylated CENP-A peptide was synthesized with a single
phosphorylated serine residue at position 7 (RRRSpRKEAPRRRS).
Both peptides contained an artificial cysteine residue at position 18
for coupling to Keyhole limpet hemocyanin (Sigma, St Louis, MO)
using standard protocols. Rabbits were immunized as previously
described (Hendzel et al., 1997).
Affinity purification of peptide antibodies and ELISA assay
Antibodies were purified using SulfoLink columns (Pierce, Rockford,
IL). Peptide coupling and antibody binding were performed according
to the manufacturer instructions. Anti-CENP-A-Ser7 was eluted in
100 mM triethylamine, pH 11.5, after washes with 1 M NaCl and 100
mM glycine, pH 3. Anti-CENP-A-Ser7 was eluted in 100 mM
glycine, pH 2. Eluted protein was neutralized with 1 M Tris, pH 9.5,
and stored with 1% BSA at −20°C. ELISA procedures were
performed as described (Muller et al., 1987). All antisera were diluted
1:1000. For peptide competition experiments, 100 µl of antiserum was
incubated with 100 µg of peptide for 1 hour at room temperature prior
to ELISA. The bound enzyme conjugate was quantitated by turnover
of p-nitrophenyl phosphate substrate (Sigma), as detected by
absorbance at 405 nm.
Acid extraction of histones
HeLa cells grown to a density of ~1.5×105 cells/cm2 were treated with
15 µg/ml nocodazole and incubated for 18 hours, followed by
agitation to release loosely associated mitotic cells. Cells were
collected by centrifugation and cell pellets were resuspended in onehalf volume nuclear isolation buffer (PBS, 0.1% Triton X-100, 1 mM
MgCl2, 1 mM PMSF and 100 mg/ml DNase 1). Nuclei were spun
down and stored frozen at –80°C. Resulting nuclei were resuspended
in 0.4 N sulfuric acid, incubated on ice for 30 minutes and acidinsoluble proteins were pelleted by centrifugation at 12,000 g for 10
minutes. The soluble proteins were TCA precipitated on ice for 10
minutes, spun at 12,000 g, washed with acetone/0.1% HCl, and
washed twice more with acetone, and resuspended in sterile water.
Electrophoresis and immunoblotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was performed as described (Laemmli, 1970). The specificity
of antibodies used in this report was analyzed by immunoblotting as
described previously (Hendzel et al., 1997). Protein representing 104
HeLa cell equivalents were run on each lane. Blots were blocked for
1 hour with nonfat dry milk at 5% and incubated with antisera diluted
1:1000 in 5% nonfat dry milk. For the experiment shown in Fig. 2B,
affinity purified anti-CENP-A-Ser7P was used at 1:500. After
washing, blots were probed with horseradish peroxidase-conjugated
secondary antibody and developed by ECL (Pierce, Rockford, IL)
according to the manufacturer’s instructions. For alkaline phosphatase
treatment, 104 HeLa cell equivalents were incubated with 10 units of
E. coli alkaline phosphatase (Sigma) at 37°C for 30 minutes prior to
electrophoresis.
Immunofluorescence
Asynchronous human CENP-B-GFP U2OS cells were plated and
grown to confluency on acid-washed glass coverslips (Fisher) for 2
days. Cells were fixed in 1% paraformaldehyde in PBS containing 140
mM NaF to inhibit phosphatases. Coverslips were then washed once
with PBS-TX (PBS + 0.1% Triton X-100) and incubated in blocking
solution (PBS-TX + 1% BSA) at room temperature for 15 minutes.
Primary antibodies were diluted as follows: 1:25 of affinity-purified
anti-CENP-A-Ser7 or –Ser7P, and 1:500 of rabbit anti-lamin A (a gift
from Larry Gerace). Coverslips were incubated with primaries for 30
minutes at 37°C, and then washed two times in PBS-TX for 10
minutes. Secondary antibodies were used at 1:100 dilutions (Jackson
Laboratories, West Grove, PA), incubated on coverslips with DAPI
for 30 minutes at 37 degrees, washed once in PBS-TX for 10 minutes,
washed once in detergent-free PBS for 10 minutes, and finally rinsed
once in distilled water before drying. Coverslips were mounted using
SloFade Lite (Molecular Probes, Eugene, OR) and visualized on a
DeltaVision wide field optical sectioning microscope system based on
an Olympus IX70 epifluorescence microscope (Applied Precision,
Issaquah, WA). A ×100 1.35 NA NeofluAr oil immersion lens was
used for all images. Images were processed using a constrained
iterative deconvolution algorithm. Projection images were prepared
from 3-dimensional images stacks and composite images shown were
assembled using Adobe Photoshop 5.5 (Adobe, Mountain View, CA).
For quantitation of fluorescence intensities, images were collected
at 0.3 µm intervals spanning the entire chromatin volume as defined
by DAPI staining. An exposure time was selected such that pixel
intensities recorded from maximally labeled mitotic cells did not
exceed the optimal response range of the CCD camera. This exposure
time was used to image 21 cells on a single coverslip in a single data
CENP-A phosphorylation in mitosis
collection session. After deconvolution, Softworx analysis software
(Applied Precision) was used to integrate CENP-A-Ser7P signal
within the volume of each cell using an automated thresholding
algorithm to define antibody signals. Visual inspection demonstrated
that all labeled centromeres were accurately defined by this procedure.
Integrated fluorescence intensity data were imported into a
spreadsheet (Excel, Microsoft, Redmond, WA) for analysis and
display. Chi-squared analysis demonstrated that independent samples
from each stage of the cell cycle were significantly different between
the stages (P=0), and error bars report the standard error in the
distribution.
Codetection of H3P and CENP-A
For co-detection of phosphorylated histone H3 and CENP-A with
rabbit antisera, we utilized a successive labeling method with an
additional blocking step to facilitate co-detection without crossreaction. It was necessary to use the CENP-A antibody first. After
washing, the secondary antibody was added (donkey anti-rabbit
TRITC, Jackson Laboratories, West Grove, PA) at twice the usual
concentration (1:50 instead of 1:100). After washing, coverslips were
blocked for 15 minutes at 37°C with PBS-TX + 1% BSA and 5%
normal goat serum. Anti-H3-P (Hendzel et al., 1997) was added at a
dilution of 1:3000 in PBS-TX + 1% BSA, incubated for 30 minutes
at 37°C, coverslips were washed again, the secondary antibody
(donkey anti-rabbit cascade blue, Jackson Laboratories) was used at
the usual concentration (1:100), and coverslips were washed again
before mounting.
RESULTS
The N-terminal tail sequence of CENP-A is highly divergent
from that of histone H3. Although CENP-A and histone H3
share 65% identity in their C-terminal histone fold domains,
they diverge significantly in their N-terminal tail domains. The
only direct sequence homology in the N-terminal tails is found
in an eleven amino acid segment (histone H3 Arg8-K18;
Fig. 1. Sequence similarity between CENP-A and histone H3 Ntermini is restricted to a motif surrounding histone H3 Ser10.
(A) The N-terminal tail sequences of histone H3 (top) and CENP-A
(bottom) are aligned. Identical residues are highlighted in black
while similar residues are in grey. Serine 10 of histone H3 is
phosphorylated in mitosis and the corresponding residue of CENP-A
is Ser7. The position of the histone fold domain is indicated. The
motif of sequence similarity flanking histone H3 Ser10 is detailed
beneath the alignment. Note the basic amino acids flanking histone
H3 Ser10 and CENP-A Ser7. (B) Peptide antigens used in antibody
production. Two peptides were designed for immunization of rabbits.
CENP-A Ser7 peptide contains residues 4-17 of human CENP-A
while CENP-A-Ser7P spans the same residues and contains a
phosphorylated Ser7 residue. Each peptide contained a C-terminal
cysteine residue for coupling to carrier.
655
CENP-A Arg5-Arg15; Fig. 1A). This shared motif contains the
mitotic kinase substrate residue Ser10 in histone H3, raising
the possibility that the analogous Ser7 in CENP-A may also be
a mitotic kinase substrate. This motif does not match any other
known phosphorylation motif (Kreegipuu et al., 1999).
In order to test whether CENP-A is phosphorylated, we
raised rabbit antisera against two synthetic phosphorylationspecific peptides (Fig. 1B). The first peptide, CENP-A-Ser7,
contains residues 4-17 of CENP-A. The second peptide,
CENP-A-Ser7P, contains the same sequence with a
phosphorylated serine 7. To begin to evaluate the specificity of
these rabbit sera, ELISA assays were performed (Fig. 2A). The
binding of each antibody for its peptide was specific to within
30ng/ml. The binding of the antisera to the opposing peptide
was negative to 90 ng/ml peptide. Western blots comparing
Nocodazole-blocked HeLa cells with asynchronous cultures
illustrate antiserum against peptide CENP-A-Ser, anti-CENPA-Ser7, detects a 17 kDa antigen (Fig. 2B). In contrast,
antiserum elicited against CENP-A-Ser7P, anti-CENP-ASer7P, detected a slightly slower migrating species that is
greatly enriched in mitotically arrested cells. The signal seen
with anti-CENP-A-Ser7P was removed when the samples were
treated with alkaline phosphatase (Fig. 2C), demonstrating
that the antibody specifically recognizes a CENP-A
phosphoepitope in mitotic HeLa nuclei.
The cellular distribution of phosphorylated CENP-A
confirmed the mitotic nature of the modification.
Immunofluorescence was performed using affinity purified antiCENP-A antibodies on a human osteosarcoma (U2OS) cell line
that constitutively expresses CENP-B-GFP as a marker for
centromeres (Fig. 3). Interphase cells lacking detectable
chromosome condensation (resolved with DAPI staining or antiH3P) showed no reactivity with anti-CENP-A-Ser7P (Fig. 3A).
Reactivity with anti-CENP-A-Ser7P is first detectable in
prophase cells that exhibit visible chromosome condensation
(Fig. 3B). Phosphoepitope staining exhibits characteristic
kinetochore double dot morphology (Fig. 3B, inset 3M; Moroi
et al., 1980). Reactivity with anti-CENP-A-Ser7P is evident at
all centromeres in prometaphase cells and persists through
metaphase (Fig. 3C and D). Reactivity with anti-CENP-A-Ser7P
is lost beginning in anaphase (3E), becoming undetectable in
telophase cells (3F). The complementary staining pattern is
observed with anti-CENP-A-Ser7 antibody, which labels
centromeres brightly in all interphase cells (Fig. 3G and N), fails
to label mitotic cells (3I and J) and reappears in anaphase
cells (3K and L). Anti-CENP-A-Ser7 continues to recognize
centromeres in cells that exhibit moderate levels of chromosome
condensation (3H). In each experiment, some telophase
cells were observed that lacked reactivity with either antibody
(Fig. 3O and P). Weakly reactive telophase cells are also
observed with an antiserum against a similar N-terminal
unphosphorylated CENP-A peptide (data not shown; Figueroa
et al., 1998). Lack of detectable signal at this time could be due
to masking of the epitope by association with other proteins, or
additional N-terminal modifications of CENP-A. The long halflife of CENP-A-HA in HeLa cells rules out the idea that CENPA is degraded at the end of mitosis (R. D. Shelby and K. F.
Sullivan, unpublished observations).
Phosphorylation of histone H3 initiates in pericentromeric
regions prior to general chromatin phosphorylation (Hendzel
et al., 1997). To directly examine the relationship between
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JOURNAL OF CELL SCIENCE 114 (4)
Fig. 2. Phosphorylated CENP-A is detectable in mitotic cells. (A) ELISA analysis of anti-peptide antisera. ELISA assays were performed using
CENP-A-Ser7P (top row), CENP-A-Ser7 (center row) and histone H3P (bottom) peptides immobilized on microtiter dishes. Sera (legend at
bottom) were diluted to 1:1000 prior to incubation. For peptide competition, sera were incubated with 1 mg/ml peptide prior to dilution and
ELISA. Assays in the left column included no peptide competitor, CENP-A-Ser7 peptide was used in the center column and CENP-A-Ser7P
was used in the right column. (B) Detection of CENP-A epitopes by western blot. Nuclei from asynchronous (A) or nocodozole-blocked
mitotic (M) HeLa cells were acid-extracted, and proteins precipitated and resolved by 12% SDS PAGE. Samples were examined by Coomassie
staining (left panel, Coomassie) or by immunoblotting using antibodies as follows: anti-H3-P, anti-CENP-A-Ser7 and anti-CENP-A-Ser7P.
Note the difference between phosphorylated H3 (15KDa), unmodified CENP-A (17KDa) and the phosphorylated, slower migrating CENP-A
band (>17KDa). (B) Alkaline phosphatase treatment abolishes detection of the CENP-A phosphoepitope. Acid soluble protein from extracts of
mitotic (M) or asynchronous HeLa cell cultures were incubated in the presence (+AP) or absence (−AP) of alkaline phosphatase for 30 minutes
prior to electrophoresis. Samples were then resolved by SDS-PAGE and blotted with anti-CENP-A antisera as indicated. Alkaline phosphatase
treatment had no effect on the reactivity of anti-CENP-A-Ser7 with unmodified CENP-A. In contrast, alkaline phosphatase removed essentially
all of the reactivity of anti-CENP-A-Ser7P with the slower migrating phosphorylated CENP-A band.
timing of histone H3 and CENP-A phosphorylation, we
performed simultaneous co-detection of the two proteins by
immunofluorescence (Fig. 4). G2 cells exhibiting the
pericentromeric pattern of early histone H3 phosphorylation
were negative for CENP-A-Ser7P staining but reacted strongly
with anti-CENP-A-Ser7 (Fig. 4A and E). Similarly, cells with
anti-H3-P reactivity along chromosome arms reacted strongly
with anti-CENP-A-Ser7 but cells in a similar stage lacked
reactivity with anti-CENP-A Ser7P (Fig. 4B and F). Cells with
detectable anti-CENP-A-Ser7P staining exhibited significant
chromosome condensation in addition to anti-H3-P reactivity
along chromosome arms (Fig. 4C and D). Little or no signal
with anti-CENP-A-Ser7 was seen at this stage (4G and H).
These data indicate that CENP-A phosphorylation at Ser7 does
not initiate until histone H3 phosphorylation has occurred
throughout the chromosome arms. Examination of the nuclear
lamina revealed that CENP-A phosphorylation is detected at
all centromeres prior to nuclear envelope breakdown (Fig. 5).
CENP-A phosphorylation thus constitutes a kinetically distinct
phase in phosphorylation of the histone H3 family proteins in
preparation of mitosis. Taken together with previous results
(Hendzel et al., 1997), three distinct phases of histone H3family N-terminal phosphorylation can be defined during G2
Fig. 3. Detection of CENP-A N-terminal epitopes by
immunofluorescence. Alpha satellite domains are detected with
CENP-B-GFP stably expressed in U2OS cells (green; Shelby et al.,
1996). DNA is stained with DAPI (blue) in A-N. Phosphorylated
histone H3 is detected with rabbit anti-H3-P antiserum and cascade
blue-coupled secondary antibody (blue) in O-P. (A-F) Anti-CENP-ASer7P detected with rhodamine-coupled secondary antibody (red);
(G-L) anti-CENP-A-Ser7 detected with rhodamine-coupled
secondary antibody (red). CENP-A is unphosphorylated in most
interphase cells (A and G). Phosphorylation begins in prophase (B
and H) and continues through prometaphase (C and I) and metaphase
(D and J). Phosphorylation drops in anaphase (E and K) and is
completely gone by telophase (F and L). There is apparently a brief
period of time in telophase when neither peptide antibody is able to
detect CENP-A (O, anti-CENP-A-Ser7 in red; P, anti-CENP-ASer7P in red). Characteristic kinetochore double dots are sometimes
detectable in interphase cells (G, inset enlarged 300% in N) and are
easily discerned in prophase cells (B, inset enlarged 300% in M).
Bars: 10 µm (A, K,P); 2 µm (N).
CENP-A phosphorylation in mitosis
and M, prior to nuclear envelope breakdown: pericentromeric
H3 phosphorylation, general chromatin H3 phosphorylation,
and kinetochore-specific CENP-A phosphorylation.
Paired spots of anti-CENP-A-Ser7 are first detectable during
G2, near the onset of H3 phosphorylation demonstrating that
657
the morphological duplication of kinetochore chromatin is
complete in early G2 (Fig. 4E). Reactivity with anti-CENP-ASer7P is absent at this stage (Fig. 4A). Flanking alpha satellite
arrays, visualized with CENP-B-GFP (Shelby et al., 1996),
undergo morphological changes during the time when histones
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JOURNAL OF CELL SCIENCE 114 (4)
Fig. 4. Simultaneous codetection of CENP-A
epitopes and phosphohistone H3. CENP-B-GFP
(green), anti-H3P (cascade
blue-coupled secondary
antibody, blue), antiCENP-A-Ser7-P (A-D,
rhodamine-coupled
secondary antibody, red)
and anti-CENP-A-Ser7
(E-H, rhodamine-coupled
secondary antibody, red)
were visualized in U2OS
cells. At the earliest stages
of H3 phosphorylation,
defined as a small number
of pericentric H3P signals,
CENP-A is
unphosphorylated (A and
E). When H3
phosphorylation has
extended throughout the
chromosomes, CENP-A
remains unphosphorylated
(B and F). CENP-A
phosphorylation is detected
only when chromosomes
are significantly condensed (C and D). The unphosphorylated epitope is undetectable in cells with extensive chromosome condensation (G and
H). Bar, 10 µm.
are being phosphorylated. In interphase cells prior to histone
H3 phosphorylation, CENP-B-GFP spots appear as small,
spherical dots. In cells in early G2, defined as having fewer
than 10 pericentromeric sites of anti-H3-P signal, CENP-BGFP foci are observed as larger, more irregular spots. These
irregular spots are seen associated with paired anti-CENP-ASer7P-labeled kinetochores in prophase cells. In prometaphase
Fig. 5. CENP-A is phosphorylated prior to nuclear envelope
breakdown. CENP-B-GFP (green), anti-CENP-A-Ser7P (red),
DAPI (blue), and lamin A (purple) were visualized in U2OS cells.
The cell on the left is in interphase, without detectable CENP-A
phosphoepitope and an intact nuclear envelope. The cell on the
right is in prophase, showing CENP-A phosphorylation and
advanced chromosome condensation within an intact nuclear
envelope. Bar, 10 µm.
and metaphase, the CENP-B-GFP-labeled alpha satellite arrays
adopt characteristic stretched dumbbell shapes indicative of
microtubule attachment, as previously described (Shelby et al.,
1996).
Histone H3 phosphorylation is known to begin in G2
and persist through mitosis. The period of CENP-A
phosphorylation from prophase to mid-anaphase is estimated
to be in the range of 30-60 minutes in cultured human cell lines.
However, within this short period, the appearance and
disappearance of phosphoepitope reactivity seemed gradual,
rather than sudden and complete. Simultaneous and complete
staining of every centromere at once might imply that CENP-A
phosphorylation could represent a discrete step in kinetochore
assembly. A more gradual modulation of phosphorylation
might imply involvement in a counting mechanism similar to
the spindle checkpoint proteins (e.g. MAD2; Waters et al.,
1998). Alternatively, gradual modulation could imply
involvement in a continuing process, such as chromosome
condensation, which is not complete until metaphase (Drouin
et al., 1991). To examine the CENP-A immunofluorescence
data quantitatively, we measured the total intensity of antiCENP-A-Ser7-P labeling in 21 cells sampled at 0.3 µm
intervals through the volume of the nucleus. Cells were staged
within mitosis on the basis of chromosome and centromere
morphology, and the corresponding fluorescence intensities are
plotted in Fig. 6. Cells in prophase exhibit a wide range of
fluorescence intensities and variable number of reactive
centromeres. The peak of intensity occurs in late prophase or
early prometaphase. Phosphorylation or epitope accessibility
decreases by ~30% in metaphase and then rapidly declines
through anaphase to reach undetectable levels in late anaphase
or early telophase.
CENP-A phosphorylation in mitosis
Fig. 6. Quantitation of CENP-A phosphorylation by analysis of
fluorescence intensity. 5-6 cells were analyzed for each stage of
mitosis (as judged by DAPI staining) with detectable CENP-A-Ser7P
signal. No signal was detected in telophase. Mean integrated
fluorescence intensity is displayed as a histogram with error bars
reflecting the standard error for each stage of the cell cycle. On
average, the peak of phosphorylation is in prometaphase, drops 30%
in metaphase, and continues to decline until mid-anaphase. Chisquared analysis demonstrates that these phases of the cell cycle can
be distinguished, with P=0 for comparisons among the phases.
DISCUSSION
In this work we tested the hypothesis that mitotic
phosphorylation is a general property of histone H3-related
proteins by raising antibodies against a synthetic N-terminal
peptide CENP-A peptide phosphorylated at serine 7. These
antibodies detected a phosphorylated CENP-A epitope in
mitotic cells, allowing us to characterize the spatial
organization and kinetics of CENP-A phosphorylation and
dephosphorylation in mitosis. Previously, antibodies against
histone H3 Ser10P were used to demonstrate two sequential
phases of phosphorylation of histone H3 in G2, a pericentric
phase in which centromere-associated heterochromatic regions
are phosphorylated followed by general histone H3
phosphorylation (Hendzel et al., 1997). Based on the shared
sequence context surrounding the mitotic phosphorylation
substrate residues in histone H3 and CENP-A, we
hypothesized that the two proteins may share a common
mitotic kinase or regulatory pathway. Activation of histone
family phosphorylation at kinetochores might then account for
the observed pericentric initiation of chromosomal H3
phosphorylation. Direct evaluation of this hypothesis by codetection of histone H3 and CENP-A phosphorylation rule out
a single activation pathway for phosphorylation of these
two proteins. Our experiments reveal a sequence of
phosphorylation steps in G2 that point to a more complex
compartmental regulation of histone H3-family protein
modification in G2/M.
Several lines of evidence point to the idea that the
centromere and pericentric regions of chromosomes exhibit
distinctive
structural
and
regulatory
properties.
Pericentromeric histone H3 is resistant to dephosphorylation
induced by hypotonic treatment, suggesting that
pericentromeric histone H3 may be in a different chromatin
context, or is differently modified, than bulk H3 (Van Hooser
et al., 1998). The demonstration that Su(var)3-9 overexpression
suppresses the pericentric stage of histone H3 phosphorylation
promotes the hypothesis that a distinctive regulatory context
659
for chromatin exists at pericentric regions (Melcher et al.,
2000). Evidence indicates that pericentric compartments
exhibit a dynamic composition during the cell cycle. GAGA
factor is shuttled into pericentric regions of D. melanogaster
in mitosis (Platero et al., 1998) while CBP/p300 is found in
pericentric regions during S phase (Tang and Lane, 1999).
Ikaros is concentrated in pericentric heterochromatin during G1
and G2 phases of the cell cycle, where it may play a role in
regional silencing of transcriptionally inactive genes in
lymphocytes (Brown et al., 1997). These data lead to a model
of the mammalian centromere that is reminiscent of the S.
Pombe centromere, which contains at least two types of
structurally and functionally distinct chromatin domains,
each with characteristic protein composition (reviewed by
Partridge et al., 2000). The discrete regulation of CENP-A
phosphorylation underscores the unique character of
kinetochore chromatin, separate from both pericentric
heterochromatin and the chromosome arms.
The identity of the mitotic histone H3 kinase and its
relationship with CENP-A kinase have not yet been determined
in mammalian cells Recently, Ipl1/aurora kinase, and its
genetically interacting phosphatase, Glc7/PP1, have been
demonstrated to be responsible for the balance of H3 Ser10
phosphorylation during mitosis in budding yeast and
nematodes (Hsu et al., 2000). Based upon the sequences
immediately surrounding Ser7 in CENP-A, it seems likely that
a basic-directed kinase is involved. In keeping with this
hypothesis, recombinant yeast Ipl1 phosphorylates unmodified
CENP-A peptide as well as H3 peptide in vitro (Z. W. Sun and
C. D. Allis, unpublished observations). However, histone H3
can also be phosphorylated by Rsk-2 and MSK1 in vitro, and
by JIL-1 in Drosophila (Sassone-Corsi et al., 1999; Thomson
et al., 1999; Jin et al., 1999). Studies in Aspergillus implicate
NimA kinase in phosphorylation of histone H3 (DeSouza et
al., 2000). The kinetic relationship between H3 and CENP-A
phosphorylation rule out a simple model in which a common
kinase is first recruited to prekinetochores and then
progressively moves in cis to phosphorylate first pericentric
H3, and then chromosome arm H3. Our data imply that either
multiple kinases are activated sequentially during G2/M,
similar to differential temporal regulation of Cdk1 and 2
(Furuno et al., 1999; Hagting et al., 1999), or alternatively a
single kinase could be subject to differential regulation during
G2 progression.
Accumulation of CENP-A phosphorylation displays a
kinetic pattern similar to that of the mitotic checkpoint-related
3F3/2 phosphoepitope (Gorbsky and Ricketts, 1993), however
the kinetics of dephosphorylation are very different. 3F3/2
phosphorylation mirrors the pattern of accumulation of mitotic
checkpoint proteins Mad1p, Mad2p, Bub3p and BubR1p, with
assembly onto kinetochores occurring in prophase or early
prometaphase, followed by a microtubule/tension dependent
decrease in signal intensity in late prometaphase and
metaphase (Hardwick and Murray, 1995; Chen et al., 1996;
Waters et al., 1998; Martinez-Exposito et al., 1999; Jablonski
et al., 1998). Unlike 3F3/2 phosphorylation and checkpoint
protein accumulation, CENP-A phosphorylation exhibits no
chromatid asymmetry and persists in metaphase. Thus, CENPA phosphorylation kinetics are not consistent with a role in the
metaphase-anaphase spindle checkpoint. However, CENP-A
phosphorylation follows a pattern similar to that of CENP-F
660
JOURNAL OF CELL SCIENCE 114 (4)
accumulation at centromeres, which is also asynchronous (Liao
et al., 1995).
Histone H3 phosphorylation can be used to mark the
temporal progression of G2. Kinetochore duplication
characterized by paired spots of anti-CENP-A-Ser7 staining
begins early in G2, before H3 phosphorylation. We have noted
that at the time of onset of H3 phosphorylation, when 10 or
fewer pericentric H3 domains have been phosphorylated, the
anti-CENP-A-Ser7 antibody consistently yields detectable
nuclear staining not associated with centromeres (Fig. 4E).
Based on studies of H3 phosphorylation progression in G2
(Hendzel et al., 1997), we estimate that 1% or less of the
population is in this early stage. Alpha-satellite DNA arrays,
visualized with CENP-B-GFP, appear as small symmetric spots
in late S phase/early G2 cells, but form larger, more irregular
spots later in G2 by the time H3 phosphorylation begins.
CENP-A phosphorylation then occurs prior to the attachment
of spindle microtubules, which is associated with the
mechanical stretching of centromeres in mitosis. Thus, the
structural and biochemical maturation of the kinetochore and
associated supporting chromatin structures are seen to occur in
several steps that span the course of G2. We conclude that
CENP-A phosphorylation is not required for the morphological
duplication of the pre-kinetochore, which occurs during the
same time frame as the onset of H3 phosphorylation at
pericentromeric sites. Instead we propose that CENP-A
phosphorylation may be involved in mitotic kinetochore
assembly or maturation. These observations extend the classic
demonstration that centromere replication occurs in G2,
defined by the appearance of paired spots with anti-centromere
antibodies (Brenner et al., 1981), and provide a new set of
landmarks that resolve the physiological progression of the cell
through G2.
The authors thank Rich Shelby for kindly donating the CENP-BGFP transfected U2OS cell line used in this work, Mike Blower for
technical advice relating to co-detection of multiple rabbit antibodies,
Larry Gerace for the gift of anti-Lamin A antiserum and Upstate
Biotechnologies, Inc., for assistance in raising rabbit anti-peptide
antisera. During this work, S.G.Z was partly supported by a fellowship
from the ARCS Foundation. This work was supported by grants from
the National Institutes for General Medical Sciences to C.D.A
(GM40922) and K.F.S. (GM39068).
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