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CENP-V is required for centromere organization, chromosome alignment and cytokinesis

2008; Springer Nature; Volume: 27; Issue: 19 Linguagem: Inglês

10.1038/emboj.2008.175

1460-2075

Ana Tadeu, Susana A. Ribeiro, Josiah Johnston, I. Goldberg, Dietlind L. Gerloff, William C. Earnshaw,

Genomics and Chromatin Dynamics

Article4 September 2008Open Access CENP-V is required for centromere organization, chromosome alignment and cytokinesis Ana Mafalda Baptista Tadeu Ana Mafalda Baptista Tadeu Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Department of Biochemistry, PDBEB, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Susana Ribeiro Susana Ribeiro Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Josiah Johnston Josiah Johnston Image Informatics and Computational Biology Unit, NIH, National Institute on Aging, Baltimore, MD, USA Search for more papers by this author Ilya Goldberg Ilya Goldberg Image Informatics and Computational Biology Unit, NIH, National Institute on Aging, Baltimore, MD, USA Search for more papers by this author Dietlind Gerloff Dietlind Gerloff Department of Cellular and Molecular Pharmacology, Baskin School of Engineering, University of California, Santa Cruz, CA, USA Search for more papers by this author William C Earnshaw Corresponding Author William C Earnshaw Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ana Mafalda Baptista Tadeu Ana Mafalda Baptista Tadeu Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Department of Biochemistry, PDBEB, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal Search for more papers by this author Susana Ribeiro Susana Ribeiro Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Josiah Johnston Josiah Johnston Image Informatics and Computational Biology Unit, NIH, National Institute on Aging, Baltimore, MD, USA Search for more papers by this author Ilya Goldberg Ilya Goldberg Image Informatics and Computational Biology Unit, NIH, National Institute on Aging, Baltimore, MD, USA Search for more papers by this author Dietlind Gerloff Dietlind Gerloff Department of Cellular and Molecular Pharmacology, Baskin School of Engineering, University of California, Santa Cruz, CA, USA Search for more papers by this author William C Earnshaw Corresponding Author William C Earnshaw Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Ana Mafalda Baptista Tadeu1,2, Susana Ribeiro1, Josiah Johnston3, Ilya Goldberg3, Dietlind Gerloff4 and William C Earnshaw 1 1Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, UK 2Department of Biochemistry, PDBEB, Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal 3Image Informatics and Computational Biology Unit, NIH, National Institute on Aging, Baltimore, MD, USA 4Department of Cellular and Molecular Pharmacology, Baskin School of Engineering, University of California, Santa Cruz, CA, USA *Corresponding author. Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Mayfield Road, Edinburgh, EH9 3JR, UK. Tel.: +44 0131 650 7101; Fax: +44 0132 650 7100; E-mail: [email protected] The EMBO Journal (2008)27:2510-2522https://doi.org/10.1038/emboj.2008.175 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info The mechanism of mitotic chromosome condensation is poorly understood, but even less is known about the mechanism of formation of the primary constriction, or centromere. A proteomic analysis of mitotic chromosome scaffolds led to the identification of CENP-V, a novel kinetochore protein related to a bacterial enzyme that detoxifies formaldehyde, a by-product of histone demethylation in eukaryotic cells. Overexpression of CENP-V leads to hypercondensation of pericentromeric heterochromatin, a phenotype that is abolished by mutations in the putative catalytic site. CENP-V depletion in HeLa cells leads to abnormal expansion of the primary constriction of mitotic chromosomes, mislocalization and destabilization of the chromosomal passenger complex (CPC) and alterations in the distribution of H3K9me3 in interphase nucleoplasm. CENP-V-depleted cells suffer defects in chromosome alignment in metaphase, lagging chromosomes in anaphase, failure of cytokinesis and rapid cell death. CENP-V provides a novel link between centromeric chromatin, the primary constriction and the CPC. Introduction Chromosome behaviour in mitosis is directed by kinetochores, which regulate the attachment and movement of chromosomes on the mitotic spindle, and ensure the fidelity of chromosome segregation (Rieder and Maiato, 2004; Musacchio and Salmon, 2007). The outer kinetochore is a complex proteinaceous structure, with over 80 components (Maiato et al, 2004; Chan et al, 2005; Cheeseman and Desai, 2008). The inner kinetochore is composed of DNA plus proteins that package it into chromatin. In humans, kinetochores are found on the surface of a domain composed of highly repetitive α-satellite sequences (Hyman and Sorger, 1995; Bjerling and Ekwall, 2002) packaged into condensed heterochromatin. Heterochromatin was first defined (Heitz, 1928) on morphological grounds, but now it is defined in terms of a 'histone code' of post-translational modifications that influence transitions between chromatin states and the regulation of transcriptional activity (Strahl and Allis, 2000; Jenuwein, 2001; Nightingale et al, 2006; Kouzarides, 2007). For example, histone H4 methylated on lysine 20 (K20) and H3 methylated on lysines 9 or 27 are generally associated with genes the transcription of which is repressed (Bannister et al, 2001; Cao et al, 2002; Peters et al, 2003; Rice et al, 2003; Schotta et al, 2004). Kinetochores also have their own particular chromatin mark, the histone H3 variant CENP-A, which is conserved in eukaryotes from yeast to man (Earnshaw and Rothfield, 1985; Carroll and Straight, 2006; Schueler and Sullivan, 2006). CENP-A-containing chromatin is usually embedded within a large domain of pericentric heterochromatin, which may also be required for proper sister chromatid cohesion and chromosome segregation during mitosis (Nonaka et al, 2002; Dunleavy et al, 2005). Pericentric heterochromatin is rich in H3K9me2/H3K9me3 and hypoacetylated histones H3 and H4 (Dunleavy et al, 2005). Surprisingly, the chromatin domain immediately adjacent to CENP-A has been reported to contain H3K4me2, a mark of 'open' chromatin (Sullivan and Karpen, 2004). This has led to the suggestion that the inner kinetochore may possess a specialized form of 'centromere chromatin' rather than euchromatin or heterochromatin (Schueler and Sullivan, 2006). In early mitosis, the heterochromatin beneath the kinetochore is associated with the chromosomal passenger complex (CPC), a widely studied regulator of mitosis with functions ranging from the correction of kinetochore attachment errors to the completion of cytokinesis (Vagnarelli and Earnshaw, 2004; Vader et al, 2006; Ruchaud et al, 2007). To accomplish its diverse functions, the CPC occupies characteristic compartments during mitotic progression—the inner centromeres in early mitosis, and the central spindle and equatorial cortex during mitotic exit (Earnshaw and Cooke, 1991). The mechanism of CPC targeting to these compartments remains a major unanswered question—the checkpoint kinase Bub1 is involved (Boyarchuk et al, 2007), but kinetochore assembly itself does not appear to be required (Oegema et al, 2001). Here, we describe CENP-V, a putative formaldehyde-detoxifying enzyme that is required for normal compaction of heterochromatin and formation of a primary constriction. CENP-V is required for correct localization of the CPC and the centromeric cohesion protector Sgo1. Depletion of CENP-V causes a strong CPC phenotype (difficulties in chromosome bi-orientation and a failure to complete cytokinesis) that is rapidly followed by apoptotic cell death. Results CENP-V is conserved among vertebrates and resembles GFA of Paracoccus denitrificans A proteomic screen for novel human proteins associated with histone-depleted mitotic chromosomes (Gassmann et al, 2005) identified p30, a protein also found in a previous proteomic screen for novel nuclear pore complex components (Cronshaw et al, 2002). p30 was not a nuclear pore component and remained otherwise uncharacterized. As shown below, p30 is a component of the kinetochore of mitotic chromosomes, and for simplicity, we will therefore designate it as CENP-V. The human CENP-V gene located on chromosome 17 encodes a basic protein (pI 9.78) of 275 amino acids (Uniprot Q7Z7K6∣PRR6_HUMAN). Sequence similarity searches in publicly available databases using BLAST (Altschul et al, 1990) revealed proteins related to CENP-V in all vertebrates as well as in plants and nematodes (Figure 1A). Besides a poly-alanine-rich and a proline-rich region near the N terminus of the protein, a conserved domain annotated as glutathione-dependent formaldehyde-activating enzyme (Gfa) in PFAM (Sonnhammer et al, 1997) (pfam04828, formerly designated duf636) was also identified (yellow bar in Figure 1A). This domain is defined by the presence of five strongly conserved cysteines. No Gfa domain-containing protein is found in Saccharomyces cerevisiae and Drosophila melanogaster, although family members are present in bacteria, Xenopus, Caenorhabditis elegans and in Schizosaccharomyces pombe (Supplementary Figure 1). Figure 1.CENP-V is conserved among vertebrates and nematodes and is related to GFA-Pn. (A) Multiple sequence alignment and secondary structure prediction of CENP-V and its closest homologues. The alignment was automatically generated by the program T-Coffee (Notredame et al, 2000) and was visualized using Jalview (Clamp et al, 2004). The consensus secondary structure prediction (H α-helix; E β-strand) shown is based on predictions obtained using the Genesilico metaserver (http://www.genesilico.pl/meta2). NCBI accession codes: Homo sapiens, NP_859067.1 (nuclear protein p30, chromosome17); Pan troglodytes, XP_511783.2 (predicted protein: similar to nuclear protein p30, chromosome 17); Gallus gallus, XP_415846.2 (predicted protein: hypothetical protein, chromosome 19); Mus musculus, XP_921840 (proline-rich protein 6, chromosome 11); Caenorhabditis elegans, NP_741541.1 (F25B4.8b, chromosome 5) and Arabidopsis thaliana, NP_197196.1 (carbon sulphur lyase). The cysteines in the putative catalytic site are represented by stars, the cysteines from the putative structural zinc (II)-binding site by triangles and the extra cysteine conserved only in the most closely related homologues of CENP-V by a diamond. The Gfa domain is indicated by a bright yellow bar—the total region modelled encompasses that plus the striped bar. (B) Human CENP-V Gfa domain structural model (green tube) superimposed onto the homologous GFA-Pn crystal structure (PDB: 1xa8; purple ribbon) that served as a template in the modelling. Cysteine positions conserved in higher eukaryotes are depicted as yellow balls. Model coordinates and figures were generated with SYBYL (Tripos Inc.). (C, D) Hydrophobicity mapped onto the solvent-accessible surfaces of the GFA-Pn structure (C, with co-crystallized glutathione shown in stick representation) and the CENP-V Gfa domain structural model (D) using SYBYL-MOLCAD (Tripos Inc.). Download figure Download PowerPoint Only prokaryotic enzymes containing the Gfa domain have been characterized functionally. GFA enzymes (EC no. 4.4.1.22) catalyse the first step of the glutathione-linked oxidation pathway for the conversion of formaldehyde—the condensation of formaldehyde and glutathione to S-hydroxymethylglutathione. In methylotrophic bacteria such as Paracoccus denitrificans and Rhodobacter sphaeroides, GFA is involved in the complete oxidation of methanol to carbon dioxide. In non-methylotrophic bacteria, such as Escherichia coli and in higher organisms, glutathione-linked oxidation serves to detoxify formaldehyde. The crystallographic structure of GFA from P. denitrificans (GFA-Pn) has been solved (Neculai et al, 2005). We used this structure together with multiple alignment information to predict a structure of the conserved region of CENP-V containing the Gfa domain (amino acids 126–260). To produce a three-dimensional (3D) model of this structure, we first generated a target-to-template sequence alignment of CENP-V plus its closest homologues with the GFA subfamily containing the P. denitrificans enzyme sequence. The automated alignment from PFAM required only minor adjustments, and local ambiguities were resolved by considering tertiary structural features in the crystal structure, subfamily-specific conservation in both subfamilies and the predicted secondary structure for the CENP-V subfamily. Using this alignment as input, atomic coordinates were produced with the SYBYL software package (Tripos Inc.), yielding a robust model for the region of CENP-V containing the Gfa domain (Figure 1D). The N- and C-terminal portions of CENP-V (aa1–126 and 261–275) were not modelled, as they appear to contain non-conserved secondary structure and are unlikely to be intrinsic to the function of the Gfa domain. The three cysteines present in the catalytic site of GFA-Pn (Figure 1A, stars) are highly conserved, as are four cysteines forming a structural zinc (II)-binding site (two within the Gfa domain and two upstream of it—triangles in Figure 1A). Another cysteine found only in the most closely related homologues of CENP-V is buried inside the structure according to our model (Figure 1A, diamond). The conserved cysteine residues in GFA-Pn form part of a redox switch that regulates the activity of the protein (Neculai et al, 2005). We predict no notable differences between the surface charge characteristics of CENP-V and the GFA-Pn domains (data not shown). However, a hydrophobic pocket present in the catalytic site of the bacterial protein (Figure 1C) is not reproduced in our CENP-V model (Figure 1D). This pocket is important for glutathione binding in GFA-Pn. Its absence in the model suggests that if CENP-V is an enzyme, it is unlikely to utilize glutathione in a mechanism exactly similar to that of GFA-Pn. CENP-V localizes to kinetochores in early mitosis Affinity-purified rabbit polyclonal antibody Ra4552 raised against full-length CENP-V recognizes a protein of the expected size (30 kDa) on immunoblots of HeLa cell lysates (Figure 2A). Specificity of the antibody is supported by the fact that this band disappears when CENP-V is depleted by RNA interference (RNAi) (Figure 3A, Supplementary Figure 2B) as does the specific immunostaining pattern observed in mitotic cells, as described below (Supplementary Figure 3B–B′′). The purified antibody also recognizes a novel 63-kDa band in immunoblots of extracts from HeLa cells stably expressing CENP-V–GFP–TrAP (Figure 2A). Figure 2.CENP-V localizes to kinetochores throughout mitosis. (A) Immunoblot of HeLa cell lysate probed with serum Ra4552. T, lysate from HeLa cells expressing CENP-V–GFP–TrAP; U, lysate from untransfected HeLa cells. (B–B′′′) Localization of endogenous CENP-V (red) to kinetochores in (B, B′) prometaphase and metaphase, (B′′) to the mid-zone in anaphase and (B′′′) the mid-body in cytokinesis in HeLa cells (microtubules, green; DNA, blue). Right panels show CENP-V (red) co-stained with anticentromere antibody (ACA, green). (C) Localization of endogenous CENP-V (green) to kinetochores co-stained with Aurora B (red) and ACA (white), in cells treated with colcemid (2 h); (D–F) Localization of endogenous CENP-V (green (D, E), red (F)) co-stained with various centromere/kinetochore markers as follows: (D) CENP-A (red), (E) Hec1 (red), INCENP (white), (F) CENP-E (green) and the plus end MAP EB1 (white). Insets show higher magnification views of a single optical section of the same region. Bars, 5 μm. Download figure Download PowerPoint Figure 3.Depletion of CENP-V by RNA interference causes broadening of the primary constriction without affecting heterochromatin levels. (A) Levels of endogenous CENP-V decrease 48 h after transfection with a specific siRNA oligo. Levels of H3K9me3 are not affected when CENP-V is depleted. (B) At 48 h after transfection with control siRNA, chromosomes from cells show a prominent primary constriction. (C) At the same time point, cells transfected with a specific CENP-V siRNA show abnormal centromeric structure. Insets show higher magnification views of chromosomes with ACA, red. (D, E) Quantification of the distance between ACA spots (D) and the width of primary constriction of chromosomes (E). Both values are increased when CENP-V is depleted. Bar, 5 μm. Download figure Download PowerPoint Indirect immunofluorescence using this antibody revealed that CENP-V is localized to kinetochores from prometaphase to metaphase (Figure 2B and B′). CENP-V staining is external to CENP-A and CENP-B (Figure 2D, data not shown), internal to CENP-E and EB1 (Figure 2F) and shows most overlap with Hec1 (Figure 2E). Thus, CENP-V is localized to the outer kinetochore. This specific localization is further supported by the pattern of kinetochore staining in cells treated with colcemid (Figure 2C) and the absence of centromere staining in interphase cells (Supplementary Figure 1B). At anaphase onset, CENP-V transfers to the spindle mid-zone and then the mid-body in telophase and cytokinesis (Figure 2B′′ and B′′′). Of known chromosomal proteins, the distribution of CENP-V most closely resembles that of CENP-E (Yen et al, 1991). CENP-V is required for a normal primary constriction of mitotic chromosomes To analyse the role of CENP-V in chromosome structure and function, we depleted the protein by RNAi in HeLa cells. Immunoblotting showed that by 48 h exposure of cells to a specific siRNA, CENP-V was significantly depleted (Figure 3A). At this time, >90% of cells showed an abnormal morphology when compared with cells exposed to control siRNA (Supplementary Figure 2A and A′). By 60 h, most cells exposed to CENP-V siRNA were dead (data not shown). Thus, CENP-V is essential for viability. Unfortunately, it was not possible to use siRNA rescue experiments to exclude possible off-target phenotypes, as all CENP-V constructs tested killed HeLa cells by 60 h after transfection. CENP-V depletion caused a marked change in the structure of mitotic chromosomes in spreads prepared at 48 and 60 h after exposure to specific siRNA. Mitotic chromosomes from CENP-V-depleted cells appeared less compact overall than chromosomes from cells transfected with control siRNA (Figure 3B and C). In addition, the arms of CENP-V-depleted chromosomes frequently appeared to be less distinct from one another than the arms of chromosomes from control cells. CENP-V depletion also had a remarkable effect on centromere structure in chromosome spreads. In CENP-V-depleted cells, the primary constriction of mitotic chromosomes appeared wider and less focused than that of chromosomes from control cells (Figure 3B and C). Measurement of DAPI-stained chromosomes revealed that the width of the primary constriction in chromosomes from cells exposed to control siRNA was ∼0.6 μm. After depletion of CENP-V, this distance increased to ∼1.5 μm (Figure 3E). Staining with anticentromere autoantibodies (ACA) revealed an increase in the distance between sister kinetochores to ∼0.8 μm, about twice the distance measured in cells exposed to control siRNA (0.45 μm; Figure 3D). This could not be explained by changes in tension, as these measurements were performed on spreads of chromosomes from nocodazole-blocked cells where tension is absent. These changes in chromosomal morphology were not due to changes in the distribution of SMC2. Chromosome arms are slightly decondensed in cells lacking this key condensin subunit (Hudson et al, 2003). However, with the exception of the loss of a focus at the primary constriction, no differences in the distribution of SMC2 were observed in metaphase chromosomes from CENP-V-depleted cells (Figure 4A and A′). Figure 4.CENP-V depletion by RNAi affects the centromeric localization of chromosomal passenger proteins and Sgo1 but not SMC2. (A) At 48 h after transfection with control siRNA, HeLa cells show normal condensin and INCENP localization. (A′) At same time point, cells transfected with a specific-CENP-V siRNA show normal condensin localization but INCENP staining is decreased and appears delocalized to chromosome arms. (B, B′) Sgo1 and Aurora B localize to centromeres in cells exposed to a control siRNA (B), whereas the staining is reduced or absent in cells exposed to a specific CENP-V siRNA (B′′). Insets show higher magnification views of chromosomes. (C) Aurora B fluorescence intensity levels at centromeres (normalized relative to ACA staining for the same centromeres) are significantly decreased when CENP-V is depleted from cells. Average values are indicated for each condition. (D) Levels of CPC proteins Aurora B and INCENP decrease when cells are transfected with a CENP-V-specific siRNA. (E) Sgo1 fluorescence intensity levels at centromeres (normalized relative to ACA staining for the same centromeres) are significantly decreased when CENP-V is depleted from cells. Average values are indicated for each condition. Bar, 5 μm. Download figure Download PowerPoint CENP-V depletion reduces the centromeric localization of chromosomal passenger proteins and Sgo1 The pronounced effects on the morphology of the primary constriction caused by CENP-V depletion were accompanied by a dramatic change in the distribution of several key centromeric components. In chromosome spreads prepared after 48 h exposure to CENP-V siRNA, INCENP was no longer concentrated in the inner centromere as in control cells, but instead was decreased in intensity and distributed all along the axis between the chromosome arms (Figure 4A′). A similar effect was observed on the distribution of Aurora B, the active kinase constituent of the CPC (Figure 4B and B′). In fixed samples, Aurora B is able to transfer to the central spindle and mid-body of CENP-V-depleted cells. However, its localization pattern was abnormal (Supplementary Figure 3B–B′′) when compared to the control experiment (Supplementary Figure 3A-A′′). The apparent depletion of the CPC at inner centromeres was confirmed by quantification of the fluorescence intensity levels for Aurora B. In each case, the level of Aurora B fluorescence was normalized to the level of staining seen for the corresponding centromere using ACA (Figure 4C). Although there was a marked variability in the levels of Aurora B associated with individual centromeres, depletion of CENP-V caused a clear and dramatic decline in these levels. We further confirmed the decline of cellular Aurora B and INCENP by immunoblotting (Figure 4D). To determine whether this decline in the levels of centromere-associated CPC proteins was functionally significant, we also stained chromosome spreads for the shugoshin protein Sgo1. The CPC is required for the stable localization of the Drosophila shugoshin MEI-S332 to centromeres in meiosis and mitosis (Resnick et al, 2006), and interactions between these proteins have been observed in a variety of cell types (Dai et al, 2006; Huang et al, 2007; Kawashima et al, 2007). Indeed, depletion of the CPC at centromeres was accompanied by a decrease in Sgo1 after 48 h exposure to CENP-V siRNA (Figure 4B′). In contrast, no loss of centromeric Sgo1 was seen in cells exposed to control siRNA (Figure 4B). Quantification of the fluorescence intensity values confirmed the decline in centromere-associated Sgo1 in CENP-V-depleted cells (Figure 4E). This drop in Sgo1, though significant, was not sufficient to cause premature sister chromatid separation. Thus, CENP-V is required for the normal structure of the inner centromere in mitotic chromosomes and for the proper centromeric localization of Aurora B, INCENP and Sgo1. CENP-V is required for proper alignment of metaphase chromosomes and for correct cytokinesis If CENP-V is required for CPC function as well as localization during mitosis, we would expect its depletion to produce strong phenotypes. To analyse the consequences of CENP-V depletion, we performed multi-site imaging by DIC and fluorescence microscopy of the chromosomes over 48 h with a 5 min time lapse, starting 24 h after transfection with fluorofore-labelled oligonucleotides. The analysis employed both control and siRNA oligonucleotides in three independent experiments, and more than 60 cells were assessed for each condition. This analysis enabled us to recognize three phenotypes following CENP-V depletion. Approximately 30% of cells died without achieving a metaphase chromosome alignment or entering anaphase (Figure 5A and C; for a control cell, see Supplementary Figure 4). Another ∼20% of cells either failed to complete cytokinesis or appeared to divide, but the furrow later regressed. Chromosome segregation in these cells was frequently characterized by lagging chromosomes. These cells generally underwent apoptosis soon after failing to complete cytokinesis (Figure 5B). This rapid cell death explains why the number of multinucleated cells in fixed samples (Supplementary Figure 2C) was much lower than that usually seen after perturbation of CPC function (Carvalho et al, 2003; Lens and Medema, 2003; Gassmann et al, 2004). Finally, of the 40% of cells that appeared to complete mitosis normally, 80% underwent apoptosis without re-entering mitosis. Figure 5.Live imaging of CENP-V-depleted cells reveals defects in chromosome alignment and cytokinesis. (A, B) Selected fluorescence and DIC frames of live cell imaging experiments performed on a mitotic HeLa cell line stably expressing histone H2B–GFP treated with a specific CENP-V siRNA oligonucleotide. (A) Some cells are incapable of establishing a correct alignment in metaphase and fail to enter anaphase. (B) Other cells manage to enter anaphase with chromatin bridges and fail cytokinesis. (C) Quantification of the various phenotypes observed when CENP-V is depleted. Bar, 5 μm. Download figure Download PowerPoint Thus, not only does CENP-V depletion produce an apparent perturbation of CPC localization and stability, the subsequent cell death appears to reflect a loss of CPC function during both chromosome bi-orientation and cytokinesis. Mutations in the putative catalytic domain of CENP-V abolish its ability to induce hypercondensation of pericentromeric heterochromatin In cells overexpressing CENP-V–GFP, the transfected protein was found in regions of dense chromatin reminiscent of the chromocentres seen in murine cell lines (Figure 6B). Centromeres were frequently associated with the surface of these condensed domains (Gassmann et al, 2005), and the domains contained the heterochromatin marker H3K9me3 (Supplementary Figure 5A). The CENP-V-induced chromatin hypercondensation was particularly prominent in HeLa cells in early G1 (e.g. in cells joined by a mature mid-body structure; Supplementary Figure 5B, D). Thus, CENP-V overexpression might either promote the condensation of pericentromeric chromatin or interfere with its decondensation during mitotic exit. Figure 6.Mutations in the putative catalytic domain of CENP-V abolish the chromatin hypercondensation phenotype of the wild-type protein but do not affect levels of histone modification H3K9me3. (A) Diagram depicting the cysteine residues mutated to alanine in CENP-VC174A (red) and CENP-VCC172/177AA (blue) mutants. (B) Chromatin morphology of non-transfected cells (NT) and cells transiently over expressing CENP-V–GFP (CENP-V) and the C-A mutants (C174A–GFP and CC172/177AA–GFP). The panel shows deconvolved images of DAPI staining of the DNA. (C) Dendrogram based on image similarity of the overexpression phenotype of non-transfected cells and cells overexpressing wild-type or mutant CENP-V–GFP. (D) Quantification of percentages of HeLa cells that undergo mitosis or enter apoptosis in non-transfected cells and in cells overexpressing CENP-V–GFP. (E) Immunoblots reveal that the levels of H3K9me3 are not affected when CENP-V–GFP, CENP-VC174A–GFP or CENP-VCC172/177AA–GFP are transiently overexpressed in HeLa cells. To enrich for transfected cells expressing the fusion proteins, at 24 h after transfection, cells were incubated 10 h with puromycin and extracts were prepared 14 h thereafter (nt, non-transfected cells). Bar, 5 μm. Download figure Download PowerPoint CENP-V overexpression is highly toxic. Most cells transfected with CENP-V traverse mitosis apparently normally, but half of cells die in interphase 17–22 h later (Figure 6D). The mechanism of this death is not known, but it is unlikely to reflect interference with the function of the CPC as CENP-V overexpression had no obvious effect on the distribution of INCENP (Supplementary Figure 6) or Aurora B (Supplementary Figure 7). The timing of death suggests that CENP-V overexpression causes a fatal disruption in cell cycle progression in either S or G2. To test whether the chromatin-condensing activity of CENP-V requires its putative catalytic site, we used a site-directed mutagenesis approach to specifically inactivate the putative enzyme function of CENP-V. Mutant CENP-VC174A removes a cysteine involved in binding of the cofactor glutathione in GFA-Pn.