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