RESEARCH ARTICLE 653 Differential regulation of CENP-A and histone H3 phosphorylation in G2/M

Samantha G. Zeitlin1,*, Cynthia M. Barber2,*, C. David Allis2and 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 both pericentric initiation and genome-wide stages in order to segregate them during mitosis. The of histone H3 phosphorylation. Quantitative condensation process as well as subsequent segregation immunocytochemistry reveals that CENP-A requires phosphorylation of histone H3 at serine 10. phosphorylation begins in prophase and reaches maximal Histone H3 phosphorylation initiates during G2 in levels in prometaphase. CENP-A phosphoepitope reactivity pericentric foci prior to H3 phosphorylation in the is lost during anaphase and becomes undetectable in chromosome arms. Centromere protein A (CENP-A), a telophase cells. Duplication of prekinetochores, detected as histone H3-like protein found uniquely at centromeres, the doubling of CENP-A foci, occurs prior to complete contains a sequence motif similar to that around H3 Ser10, histone H3 phosphorylation in G2. Mitotic phosphorylation suggesting that CENP-A phosphorylation might be linked of histone H3-family proteins shows tight spatial and to pericentric initiation of histone H3 phosphorylation. To temporal control, occurring in three phases: (1) pericentric test this hypothesis, we generated peptide antibodies H3 phosphorylation, (2) chromosome arm H3 against the putative phosphorylation site of CENP-A. phosphorylation and (3) CENP-A phosphorylation at ELISA, western blot and immunocytochemical analyses kinetochores. These observations reveal new cytological show that CENP-A is phosphorylated at the shared landmarks characteristic of G2 progression. motif. Simultaneous co-detection demonstrates that phosphorylation of CENP-A and histone H3 are separate Key words: CENP-A, Histone, Phosphorylation, Kinetochore, events in G2/M. CENP-A phosphorylation occurs after Mitosis

INTRODUCTION terminal tails of each of the four core histones (reviewed by Luger and Richmond, 1998; Grunstein, 1998; Strahl and Allis, During the G2 phase of the cell cycle the replicated 2000). The chemical diversity of modifications, including chromosomes are extensively modified in preparation for lysine-N-acetylation, lysine-N-methylation, arginine mitosis. This process culminates in 20-100 fold chromosome methylation, ADP-ribosylation, ubiquitination and condensation and refolding into the familiar compact mitotic phosphorylation, has led to the ‘histone code hypothesis’, in configuration which facilitates efficient segregation as cells which each site of modification can serve a distinct function divide (Trask et al., 1993; Heck, 1997). Mitotic chromosome (Strahl and Allis, 2000). For example, acetylation of histone condensation involves both assembly and disassembly of H4 lysines 5 and 12 is correlated with deposition (Verrault et chromatin associated proteins as the chromosome switches al., 1996), and acetylation at residues 8 and 16 are associated from transcriptional activities to its transport form. During with transcriptional activation (Kuo et al., 1996), while G2, many transcription-associated proteins dissociate or hypoacetylation of histone H4 is associated with redistribute within the chromosomes (Platero et al., 1998), and heterochromatic domains (Turner et al., 1992). According specific mitosis-associated protein complexes are assembled or to the histone code model, modifications (singly or in activated (Hirano et al., 1997). One of these mitosis-specific combination) create changes in the overall charge density of complexes is the kinetochore, a tri-laminar plate-like structure histone tails, which in turn modulate histone interactions with that forms on the centromeric locus of each chromosome in DNA, with non-histone proteins and with other histones. By mitosis (reviewed by Rieder and Salmon, 1998). The this mechanism, histone modifications both receive and complexity of kinetochore structure exemplifies the unique transmit changes in protein-protein interactions within functional configuration of mitotic chromosomes which results chromatin to and from higher-level signaling pathways. For from the G2 remodeling process. example, histone H3 phosphorylation levels are increased in The histones play a central role in modulating protein response to mitogen stimulation, raising levels of immediate- assembly on the chromatin fiber. This is regulated by post- early gene expression (Mahadevan et al., 1991; Thomson et al., translational modifications that occur on the flexible N- 1999; Chadee et al., 1999; Sassone-Corsi et al., 1999; Cheung 654 JOURNAL OF CELL SCIENCE 114 (4) et al., 2000). Understanding how histone modifications are performed as described (Muller et al., 1987). All antisera were diluted targeted to specific chromosomal loci and how regulation is 1:1000. For peptide competition experiments, 100 µl of antiserum was achieved in different nuclear compartments will be key steps incubated with 100 µg of peptide for 1 hour at room temperature prior toward unraveling chromatin-directed signaling pathways. to ELISA. The bound enzyme conjugate was quantitated by turnover Histone H3 phosphorylation at Ser10 in particular plays a of p-nitrophenyl phosphate substrate (Sigma), as detected by key role in mitotic chromosome condensation (Wei et al., 1998; absorbance at 405 nm. Wei et al., 1999; Hsu et al., 2000). In the absence of histone Acid extraction of histones H3 phosphorylation, chromosome condensation is incomplete HeLa cells grown to a density of ~1.5×105 cells/cm2 were treated with and anaphase chromosome separation is highly defective (Wei 15 µg/ml nocodazole and incubated for 18 hours, followed by et al., 1999; Hsu et al., 2000). Histone H3 phosphorylation is agitation to release loosely associated mitotic cells. Cells were highly regulated in G2, initiating specifically in pericentric collected by centrifugation and cell pellets were resuspended in one- heterochromatin in characteristic rings around the centromeres half volume nuclear isolation buffer (PBS, 0.1% Triton X-100, 1 mM prior to general H3 phosphorylation that occurs in chromatin MgCl2, 1 mM PMSF and 100 mg/ml DNase 1). Nuclei were spun throughout the chromosome arms (Hendzel et al., 1997). A down and stored frozen at –80°C. Resulting nuclei were resuspended unique histone H3-related protein, CENP-A, is found in 0.4 N sulfuric acid, incubated on ice for 30 minutes and acid- specifically at centromeres throughout the cell cycle where it insoluble proteins were pelleted by centrifugation at 12,000 g for 10 minutes. The soluble proteins were TCA precipitated on ice for 10 is thought to substitute for histone H3 in the nucleosomes minutes, spun at 12,000 g, washed with acetone/0.1% HCl, and of kinetochore-associated chromatin (Brenner et al., 1981; washed twice more with acetone, and resuspended in sterile water. Earnshaw and Rothfield, 1985; Sullivan et al., 1994; Yoda et al., 2000). In addition to a conserved H3-like histone fold Electrophoresis and immunoblotting domain, CENP-A contains an N-terminal tail that shares very Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- little sequence identity with that of histone H3. However, PAGE) was performed as described (Laemmli, 1970). The specificity several amino acids surrounding the histone H3 Ser10 are of antibodies used in this report was analyzed by immunoblotting as conserved in CENP-A, suggesting that the mitotic described previously (Hendzel et al., 1997). Protein representing 104 phosphorylation motif might be shared between these two HeLa cell equivalents were run on each lane. Blots were blocked for proteins. If this were the case, it might help clarify the 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, mechanisms through which histone H3 phosphorylation is affinity purified anti-CENP-A-Ser7P was used at 1:500. After regulated during G2/M. In this work we have demonstrated that washing, blots were probed with horseradish peroxidase-conjugated phosphorylation of CENP-A occurs at Ser7 within the shared secondary antibody and developed by ECL (Pierce, Rockford, IL) mitotic phosphorylation motif. Using immunocytochemistry, according to the manufacturer’s instructions. For alkaline phosphatase we compared the kinetic and spatial organization of CENP-A treatment, 104 HeLa cell equivalents were incubated with 10 units of and histone H3 phosphorylation in G2/M. Our results show that E. coli alkaline phosphatase (Sigma) at 37°C for 30 minutes prior to CENP-A phosphorylation is unlikely to play a role in the electrophoresis. initiation of histone H3 phosphorylation in pericentric Immunofluorescence regions. Instead, H3 phosphorylation precedes CENP-A phosphorylation, which occurs as a distinct reaction in prophase. 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 MATERIALS AND METHODS with PBS-TX (PBS + 0.1% Triton X-100) and incubated in blocking solution (PBS-TX + 1% BSA) at room temperature for 15 minutes. Cell culture Primary antibodies were diluted as follows: 1:25 of affinity-purified HeLa (ATCC CCL3) and U2OS (ATCC HTB96) cells were anti-CENP-A-Ser7 or –Ser7P, and 1:500 of rabbit anti-lamin A (a gift maintained in DMEM with 10% FCS (Gibco-BRL, Gaithersburg, from Larry Gerace). Coverslips were incubated with primaries for 30 MD) at 37°C in a 5% CO2 atmosphere. minutes at 37°C, and then washed two times in PBS-TX for 10 minutes. Secondary antibodies were used at 1:100 dilutions (Jackson Peptide synthesis, conjugation and injection Laboratories, West Grove, PA), incubated on coverslips with DAPI Unmodified CENP-A peptide was synthesized corresponding to for 30 minutes at 37 degrees, washed once in PBS-TX for 10 minutes, residues 4-17 of human CENP-A (RRRSRKPEAPRRRS). washed once in detergent-free PBS for 10 minutes, and finally rinsed Phosphorylated CENP-A peptide was synthesized with a single once in distilled water before drying. Coverslips were mounted using phosphorylated serine residue at position 7 (RRRSpRKEAPRRRS). SloFade Lite (Molecular Probes, Eugene, OR) and visualized on a Both peptides contained an artificial cysteine residue at position 18 DeltaVision wide field optical sectioning microscope system based on for coupling to Keyhole limpet hemocyanin (Sigma, St Louis, MO) an Olympus IX70 epifluorescence microscope (Applied Precision, using standard protocols. Rabbits were immunized as previously Issaquah, WA). A ×100 1.35 NA NeofluAr oil immersion lens was described (Hendzel et al., 1997). used for all images. Images were processed using a constrained iterative deconvolution algorithm. Projection images were prepared Affinity purification of peptide antibodies and ELISA assay from 3-dimensional images stacks and composite images shown were Antibodies were purified using SulfoLink columns (Pierce, Rockford, assembled using Adobe Photoshop 5.5 (Adobe, Mountain View, CA). IL). Peptide coupling and antibody binding were performed according For quantitation of fluorescence intensities, images were collected to the manufacturer instructions. Anti-CENP-A-Ser7 was eluted in at 0.3 µm intervals spanning the entire chromatin volume as defined 100 mM triethylamine, pH 11.5, after washes with 1 M NaCl and 100 by DAPI staining. An exposure time was selected such that pixel mM glycine, pH 3. Anti-CENP-A-Ser7 was eluted in 100 mM intensities recorded from maximally labeled mitotic cells did not glycine, pH 2. Eluted protein was neutralized with 1 M Tris, pH 9.5, exceed the optimal response range of the CCD camera. This exposure and stored with 1% BSA at −20°C. ELISA procedures were time was used to image 21 cells on a single coverslip in a single data CENP-A phosphorylation in mitosis 655 collection session. After deconvolution, Softworx analysis software CENP-A Arg5-Arg15; Fig. 1A). This shared motif contains the (Applied Precision) was used to integrate CENP-A-Ser7P signal mitotic kinase substrate residue Ser10 in histone H3, raising within the volume of each cell using an automated thresholding the possibility that the analogous Ser7 in CENP-A may also be algorithm to define antibody signals. Visual inspection demonstrated a mitotic kinase substrate. This motif does not match any other that all labeled centromeres were accurately defined by this procedure. known phosphorylation motif (Kreegipuu et al., 1999). Integrated fluorescence intensity data were imported into a In order to test whether CENP-A is phosphorylated, we spreadsheet (Excel, Microsoft, Redmond, WA) for analysis and display. Chi-squared analysis demonstrated that independent samples raised rabbit antisera against two synthetic phosphorylation- from each stage of the cell cycle were significantly different between specific peptides (Fig. 1B). The first peptide, CENP-A-Ser7, the stages (P=0), and error bars report the standard error in the contains residues 4-17 of CENP-A. The second peptide, distribution. CENP-A-Ser7P, contains the same sequence with a phosphorylated serine 7. To begin to evaluate the specificity of Codetection of H3P and CENP-A these rabbit sera, ELISA assays were performed (Fig. 2A). The For co-detection of phosphorylated histone H3 and CENP-A with binding of each antibody for its peptide was specific to within rabbit antisera, we utilized a successive labeling method with an 30ng/ml. The binding of the antisera to the opposing peptide additional blocking step to facilitate co-detection without cross- was negative to 90 ng/ml peptide. Western blots comparing reaction. It was necessary to use the CENP-A antibody first. After Nocodazole-blocked HeLa cells with asynchronous cultures washing, the secondary antibody was added (donkey anti-rabbit TRITC, Jackson Laboratories, West Grove, PA) at twice the usual illustrate antiserum against peptide CENP-A-Ser, anti-CENP- concentration (1:50 instead of 1:100). After washing, coverslips were A-Ser7, detects a 17 kDa antigen (Fig. 2B). In contrast, blocked for 15 minutes at 37°C with PBS-TX + 1% BSA and 5% antiserum elicited against CENP-A-Ser7P, anti-CENP-A- normal goat serum. Anti-H3-P (Hendzel et al., 1997) was added at a Ser7P, detected a slightly slower migrating species that is dilution of 1:3000 in PBS-TX + 1% BSA, incubated for 30 minutes greatly enriched in mitotically arrested cells. The signal seen at 37°C, coverslips were washed again, the secondary antibody with anti-CENP-A-Ser7P was removed when the samples were (donkey anti-rabbit cascade blue, Jackson Laboratories) was used at treated with alkaline phosphatase (Fig. 2C), demonstrating the usual concentration (1:100), and coverslips were washed again that the antibody specifically recognizes a CENP-A before mounting. phosphoepitope in mitotic HeLa nuclei. The cellular distribution of phosphorylated CENP-A RESULTS confirmed the mitotic nature of the modification. Immunofluorescence was performed using affinity purified anti- The N-terminal tail sequence of CENP-A is highly divergent CENP-A antibodies on a human osteosarcoma (U2OS) cell line from that of histone H3. Although CENP-A and histone H3 that constitutively expresses CENP-B-GFP as a marker for share 65% identity in their C-terminal histone fold domains, centromeres (Fig. 3). Interphase cells lacking detectable they diverge significantly in their N-terminal tail domains. The chromosome condensation (resolved with DAPI staining or anti- only direct sequence homology in the N-terminal tails is found H3P) showed no reactivity with anti-CENP-A-Ser7P (Fig. 3A). in an eleven amino acid segment (histone H3 Arg8-K18; 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 Fig. 1. Sequence similarity between CENP-A and histone H3 N- condensation (3H). In each experiment, some telophase termini is restricted to a motif surrounding histone H3 Ser10. cells were observed that lacked reactivity with either antibody (A) The N-terminal tail sequences of histone H3 (top) and CENP-A (Fig. 3O and P). Weakly reactive telophase cells are also (bottom) are aligned. Identical residues are highlighted in black observed with an antiserum against a similar N-terminal while similar residues are in grey. Serine 10 of histone H3 is unphosphorylated CENP-A peptide (data not shown; Figueroa phosphorylated in mitosis and the corresponding residue of CENP-A et al., 1998). Lack of detectable signal at this time could be due is Ser7. The position of the histone fold domain is indicated. The to masking of the epitope by association with other proteins, or motif of sequence similarity flanking histone H3 Ser10 is detailed additional N-terminal modifications of CENP-A. The long half- beneath the alignment. Note the basic amino acids flanking histone life of CENP-A-HA in HeLa cells rules out the idea that CENP- H3 Ser10 and CENP-A Ser7. (B) Peptide antigens used in antibody production. Two peptides were designed for immunization of rabbits. A is degraded at the end of mitosis (R. D. Shelby and K. F. CENP-A Ser7 peptide contains residues 4-17 of human CENP-A Sullivan, unpublished observations). while CENP-A-Ser7P spans the same residues and contains a Phosphorylation of histone H3 initiates in pericentromeric phosphorylated Ser7 residue. Each peptide contained a C-terminal regions prior to general chromatin phosphorylation (Hendzel cysteine residue for coupling to carrier. et al., 1997). To directly examine the relationship between 656 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 (Hendzel et al., 1997), three distinct phases of histone H3- performed simultaneous co-detection of the two proteins by family N-terminal phosphorylation can be defined during G2 immunofluorescence (Fig. 4). G2 cells exhibiting the pericentromeric pattern of early histone H3 phosphorylation were negative for CENP-A-Ser7P staining but reacted strongly Fig. 3. Detection of CENP-A N-terminal epitopes by with anti-CENP-A-Ser7 (Fig. 4A and E). Similarly, cells with immunofluorescence. Alpha satellite domains are detected with CENP-B-GFP stably expressed in U2OS cells (green; Shelby et al., anti-H3-P reactivity along chromosome arms reacted strongly 1996). DNA is stained with DAPI (blue) in A-N. Phosphorylated with anti-CENP-A-Ser7 but cells in a similar stage lacked histone H3 is detected with rabbit anti-H3-P antiserum and cascade reactivity with anti-CENP-A Ser7P (Fig. 4B and F). Cells with blue-coupled secondary antibody (blue) in O-P. (A-F) Anti-CENP-A- detectable anti-CENP-A-Ser7P staining exhibited significant Ser7P detected with rhodamine-coupled secondary antibody (red); chromosome condensation in addition to anti-H3-P reactivity (G-L) anti-CENP-A-Ser7 detected with rhodamine-coupled along chromosome arms (Fig. 4C and D). Little or no signal secondary antibody (red). CENP-A is unphosphorylated in most with anti-CENP-A-Ser7 was seen at this stage (4G and H). interphase cells (A and G). Phosphorylation begins in prophase (B These data indicate that CENP-A phosphorylation at Ser7 does and H) and continues through prometaphase (C and I) and metaphase not initiate until histone H3 phosphorylation has occurred (D and J). Phosphorylation drops in anaphase (E and K) and is throughout the chromosome arms. Examination of the nuclear completely gone by telophase (F and L). There is apparently a brief period of time in telophase when neither peptide antibody is able to lamina revealed that CENP-A phosphorylation is detected at detect CENP-A (O, anti-CENP-A-Ser7 in red; P, anti-CENP-A- all centromeres prior to nuclear envelope breakdown (Fig. 5). Ser7P in red). Characteristic kinetochore double dots are sometimes CENP-A phosphorylation thus constitutes a kinetically distinct detectable in interphase cells (G, inset enlarged 300% in N) and are phase in phosphorylation of the histone H3 family proteins in easily discerned in prophase cells (B, inset enlarged 300% in M). preparation of mitosis. Taken together with previous results Bars: 10 µm (A, K,P); 2 µm (N). CENP-A phosphorylation in mitosis 657 and M, prior to nuclear envelope breakdown: pericentromeric the morphological duplication of kinetochore chromatin is H3 phosphorylation, general chromatin H3 phosphorylation, complete in early G2 (Fig. 4E). Reactivity with anti-CENP-A- and kinetochore-specific CENP-A phosphorylation. Ser7P is absent at this stage (Fig. 4A). Flanking alpha satellite Paired spots of anti-CENP-A-Ser7 are first detectable during arrays, visualized with CENP-B-GFP (Shelby et al., 1996), G2, near the onset of H3 phosphorylation demonstrating that undergo morphological changes during the time when histones 658 JOURNAL OF CELL SCIENCE 114 (4)

Fig. 4. Simultaneous co- detection of CENP-A epitopes and phospho- histone H3. CENP-B-GFP (green), anti-H3P (cascade blue-coupled secondary antibody, blue), anti- CENP-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 and metaphase, the CENP-B-GFP-labeled alpha satellite arrays H3 phosphorylation, CENP-B-GFP spots appear as small, adopt characteristic stretched dumbbell shapes indicative of spherical dots. In cells in early G2, defined as having fewer microtubule attachment, as previously described (Shelby et al., than 10 pericentromeric sites of anti-H3-P signal, CENP-B- 1996). GFP foci are observed as larger, more irregular spots. These Histone H3 phosphorylation is known to begin in G2 irregular spots are seen associated with paired anti-CENP-A- and persist through mitosis. The period of CENP-A Ser7P-labeled kinetochores in prophase cells. In prometaphase 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 anti- CENP-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 Fig. 5. CENP-A is phosphorylated prior to nuclear envelope plotted in Fig. 6. Cells in prophase exhibit a wide range of breakdown. CENP-B-GFP (green), anti-CENP-A-Ser7P (red), fluorescence intensities and variable number of reactive DAPI (blue), and lamin A (purple) were visualized in U2OS cells. The cell on the left is in interphase, without detectable CENP-A centromeres. The peak of intensity occurs in late prophase or phosphoepitope and an intact nuclear envelope. The cell on the early prometaphase. Phosphorylation or epitope accessibility right is in prophase, showing CENP-A phosphorylation and decreases by ~30% in metaphase and then rapidly declines advanced chromosome condensation within an intact nuclear through anaphase to reach undetectable levels in late anaphase envelope. Bar, 10 µm. or early telophase. CENP-A phosphorylation in mitosis 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 Fig. 6. Quantitation of CENP-A phosphorylation by analysis of structurally and functionally distinct chromatin domains, fluorescence intensity. 5-6 cells were analyzed for each stage of each with characteristic protein composition (reviewed by mitosis (as judged by DAPI staining) with detectable CENP-A-Ser7P Partridge et al., 2000). The discrete regulation of CENP-A signal. No signal was detected in telophase. Mean integrated phosphorylation underscores the unique character of fluorescence intensity is displayed as a histogram with error bars kinetochore chromatin, separate from both pericentric reflecting the standard error for each stage of the cell cycle. On heterochromatin and the chromosome arms. average, the peak of phosphorylation is in prometaphase, drops 30% The identity of the mitotic histone H3 kinase and its in metaphase, and continues to decline until mid-anaphase. Chi- relationship with CENP-A kinase have not yet been determined squared analysis demonstrates that these phases of the cell cycle can be distinguished, with P=0 for comparisons among the phases. 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 DISCUSSION phosphorylation during mitosis in budding yeast and nematodes (Hsu et al., 2000). Based upon the sequences In this work we tested the hypothesis that mitotic immediately surrounding Ser7 in CENP-A, it seems likely that phosphorylation is a general property of histone H3-related a basic-directed kinase is involved. In keeping with this proteins by raising antibodies against a synthetic N-terminal hypothesis, recombinant yeast Ipl1 phosphorylates unmodified peptide CENP-A peptide phosphorylated at serine 7. These CENP-A peptide as well as H3 peptide in vitro (Z. W. Sun and antibodies detected a phosphorylated CENP-A epitope in C. D. Allis, unpublished observations). However, histone H3 mitotic cells, allowing us to characterize the spatial can also be phosphorylated by Rsk-2 and MSK1 in vitro, and organization and kinetics of CENP-A phosphorylation and by JIL-1 in Drosophila (Sassone-Corsi et al., 1999; Thomson dephosphorylation in mitosis. Previously, antibodies against et al., 1999; Jin et al., 1999). Studies in Aspergillus implicate histone H3 Ser10P were used to demonstrate two sequential NimA kinase in phosphorylation of histone H3 (DeSouza et phases of phosphorylation of histone H3 in G2, a pericentric al., 2000). The kinetic relationship between H3 and CENP-A phase in which centromere-associated heterochromatic regions phosphorylation rule out a simple model in which a common are phosphorylated followed by general histone H3 kinase is first recruited to prekinetochores and then phosphorylation (Hendzel et al., 1997). Based on the shared progressively moves in cis to phosphorylate first pericentric sequence context surrounding the mitotic phosphorylation H3, and then chromosome arm H3. Our data imply that either substrate residues in histone H3 and CENP-A, we multiple kinases are activated sequentially during G2/M, hypothesized that the two proteins may share a common similar to differential temporal regulation of Cdk1 and 2 mitotic kinase or regulatory pathway. Activation of histone (Furuno et al., 1999; Hagting et al., 1999), or alternatively a family phosphorylation at kinetochores might then account for single kinase could be subject to differential regulation during the observed pericentric initiation of chromosomal H3 G2 progression. phosphorylation. Direct evaluation of this hypothesis by co- Accumulation of CENP-A phosphorylation displays a detection of histone H3 and CENP-A phosphorylation rule out kinetic pattern similar to that of the mitotic checkpoint-related a single activation pathway for phosphorylation of these 3F3/2 phosphoepitope (Gorbsky and Ricketts, 1993), however two proteins. Our experiments reveal a sequence of the kinetics of dephosphorylation are very different. 3F3/2 phosphorylation steps in G2 that point to a more complex phosphorylation mirrors the pattern of accumulation of mitotic compartmental regulation of histone H3-family protein checkpoint proteins Mad1p, Mad2p, Bub3p and BubR1p, with modification in G2/M. assembly onto kinetochores occurring in prophase or early Several lines of evidence point to the idea that the prometaphase, followed by a microtubule/tension dependent centromere and pericentric regions of chromosomes exhibit decrease in signal intensity in late prometaphase and distinctive structural and regulatory properties. metaphase (Hardwick and Murray, 1995; Chen et al., 1996; Pericentromeric histone H3 is resistant to dephosphorylation Waters et al., 1998; Martinez-Exposito et al., 1999; Jablonski induced by hypotonic treatment, suggesting that et al., 1998). Unlike 3F3/2 phosphorylation and checkpoint pericentromeric histone H3 may be in a different chromatin protein accumulation, CENP-A phosphorylation exhibits no context, or is differently modified, than bulk H3 (Van Hooser chromatid asymmetry and persists in metaphase. Thus, CENP- et al., 1998). The demonstration that Su(var)3-9 overexpression A phosphorylation kinetics are not consistent with a role in the suppresses the pericentric stage of histone H3 phosphorylation metaphase-anaphase spindle checkpoint. However, CENP-A promotes the hypothesis that a distinctive regulatory context 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 De Souza, C. P., Osmani, A. H., Wu, L. P., Spotts, J. L. and Osmani, S. A. et al., 1995). (2000). Mitotic histone H3 phosphorylation by the NIMA kinase in Histone H3 phosphorylation can be used to mark the Aspergillus nidulans. Cell 102, 293-302. 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Based on studies of H3 phosphorylation progression in G2 Freeman-Cook, L. L., Sherman, J. M., Brachmann, C. B., Allshire, R. C., (Hendzel et al., 1997), we estimate that 1% or less of the Boeke, J. D. and Pillus, L. (1999). The Schizosaccharomyces pombe hst4(+) gene is a SIR2 homologue with silencing and centromeric functions. population is in this early stage. Alpha-satellite DNA arrays, Mol. Biol. Cell 10, 3171-3186. visualized with CENP-B-GFP, appear as small symmetric spots Furuno, N., den Elzen, N. and Pines, J. (1999). Human cyclin A is required in late S phase/early G2 cells, but form larger, more irregular for mitosis until mid prophase. J. Cell Biol. 147, 295-306. spots later in G2 by the time H3 phosphorylation begins. Gorbsky, G. J. and Ricketts, W. A. (1993). Differential expression of a phosphoepitope at the kinetochores of moving chromosomes. J. Cell Biol. 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Biol. 9, 680-689. same time frame as the onset of H3 phosphorylation at Hardwick, K. G. and Murray, A. W. (1995). Mad1p, a phosphoprotein pericentromeric sites. Instead we propose that CENP-A component of the spindle assembly checkpoint in budding yeast. J. Cell Biol. 131, 709-720. phosphorylation may be involved in mitotic kinetochore Heck, M. M. (1997). Condensins, cohesins, and chromosome architecture: assembly or maturation. These observations extend the classic how to make and break a mitotic chromosome. Cell 91, 5-8. demonstration that centromere replication occurs in G2, Hendzel, M., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., defined by the appearance of paired spots with anti-centromere Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis- antibodies (Brenner et al., 1981), and provide a new set of specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered landmarks that resolve the physiological progression of the cell fashion coincident with mitotic chromosome condensation. Chromosoma through G2. 106, 348-360. Hirano, T., Kobayashi, R., Hirano, M. (1997). Condensins, chromosome The authors thank Rich Shelby for kindly donating the CENP-B- condensation protein complexes containing XCAP-C, XCAP-E and a GFP transfected U2OS cell line used in this work, Mike Blower for Xenopus homolog of the Drosophila Barren protein. Cell 89, 511-521. technical advice relating to co-detection of multiple rabbit antibodies, Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K., Grushcow, B., Caldwell, C. J., Hunt, J. A., Lin, D. F., Smith, M. M. and Larry Gerace for the gift of anti-Lamin A antiserum and Upstate Allis, C. D. (2000). 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