Article |
2 Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, UK
Address correspondence to Robin C. Allshire, Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, 6.34 Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK. Tel.: 44-131-650-7117. Fax: 44-131-650-7778. E-mail: robin.allshire{at}ed.ac.uk
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Abstract |
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Key Words: kinetochore; chromatin; centromere; silencing; chromosome segregation
* Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; IP, immunoprecipitate; MNase, micrococcal nuclease; MT, microtubule; TBZ, thiabendazole.
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Introduction |
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Understanding these kinetochore properties requires the identification of its protein components. The kinetochore consists of a very large complex of many proteins even in simple organisms (He et al., 2001; Cheeseman et al., 2002). The fission yeast, Schizosaccharomyces pombe, provides an excellent system for the investigation of centromerekinetochore function because it combines genetic tractability with structurally complex centromeres (Pidoux and Allshire, 2000b). The three fission yeast centromeres share the same structural organization (Fig. 1 A; Hahnenberger et al., 1991; Takahashi et al., 1992; Pidoux and Allshire, 2000b). At each centromere, a central core region (cnt) is surrounded by inverted repeat elements (innermost repeats; imr), which are specific to each centromere. These are flanked by the outer repeat elements (otr), the organization of which differs between the three centromeres. The central cores of cen1 and cen3 share a 4-kb element, cnt1/cnt3, which is partially conserved in cen2 (Wood et al., 2002).
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Nucleosomes of the outer repeats, like other heterochromatin, are underacetylated on histone H3 and H4 tails (Ekwall et al., 1997) and methylated on lysine 9 of H3. Recent observations suggest a model in which Clr4, guided by RNAi activity, methylates histone H3, promoting Swi6 binding and the assembly of transcriptionally silent heterochromatin (Bannister et al., 2001; Nakayama et al., 2001; Partridge et al., 2002; Volpe et al., 2002, 2003), which is required for the recruitment of a high density of Rad21-cohesin to the centromere (Bernard et al., 2001; Nonaka et al., 2002).
Central core chromatin is unusual; limited digestion with micrococcal nuclease (MNase) generates a smear rather than a nucleosomal ladder typical of most chromatin (Polizzi and Clarke, 1991; Takahashi et al., 1992). Mutants in central coreassociated proteins disrupt this central corespecific chromatin structure (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002). The composition of central core chromatin is also distinct, as evidenced by the specific association of the H3 variant Cnp1 (fission yeast CENP-A; Takahashi et al., 2000). Mis6 is required for the incorporation of newly synthesized Cnp1GFP at centromeres (Takahashi et al., 2000). It is likely that the "kinetochore proper" assembles at the central core, and the outer repeats provide an important but auxiliary role, possibly affecting kinetochore conformation in addition to centromeric cohesion. In support of such a model, the MT-associated protein Dis1 is associated with the central core region in a mitosis-specific manner (Nakaseko et al., 2001). However, another MAP, Alp14/Mtc1, associates with otr and imr in mitosis, but not with cnt (Garcia et al., 2001; Nakaseko et al., 2001). Each fission yeast kinetochore, like those of many metazoa, contains multiple MT-binding sites (Ding et al., 1993). In contrast, budding yeast centromeres associate with one MT (Winey et al., 1995). An additional level of organization must operate at the more complex kinetochores so that all MT attachment sites are oriented in a concerted fashion toward the same pole.
Metazoan kinetochores are not easily dissected by genetic screens, and much knowledge has stemmed from the use of autoimmune sera (Pluta et al., 1995). An alternative route to the identification of mammalian kinetochore proteins is by homology to proteins discovered in genetically tractable organisms (Wigge and Kilmartin, 2001; Nishihashi et al., 2002). Screens based on minichromosome stability performed in budding and fission yeasts have garnered mutants affecting cohesion, replication, as well as kinetochore function (for review see Pidoux and Allshire, 2000b).
The phenomenon of silencing at fission yeast centromeres sets them apart from those of budding yeast and provides an effective tool for the direct identification of kinetochore components. We have exploited transcriptional silencing at fission yeast centromeres to isolate mutants that affect centromere function, chromatin structure, and chromosome segregation. The identification of sim2+ as Cnp1CENP-A validates this approach. A novel kinetochore protein, Sim4, has also been identified and shown to be associated with the centromere central core region, and to form a complex with the central core component Mis6.
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Results |
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Isolation of mutants that alleviate central core silencing
Wild-type strains were mutagenized (Fig. 1 B) and fast-growing arg+ colonies picked (Fig. 1 C). Because genes encoding central coreassociated proteins are essential, we screened for conditional lethality: 55 of 180 mutants were thermo- or cryosensitive and/or sensitive to the MT-disrupting drug thiabendazole (TBZ). 17 of the conditional lethal mutants were placed into four complementation groups, sim1, 2, 3, and 4, for silencing in the middle of the centromere (see Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200212110/DC1). These mutants alleviated silencing at cnt1:arg3 but maintained silencing at otr and the telomere (Fig. 1 C). None of the sim mutants are allelic to mis6+, mis12+, or mal2+, which are known, or predicted, to alleviate central core silencing (Saitoh et al., 1997; Goshima et al., 1999; Partridge et al., 2000; Jin et al., 2002). sim2+ is allelic to cnp1CENP-A (Takahashi et al., 2000; Mellone, B.G., personal communication).
To quantify defective centromere silencing, sim mutant strains with the ura4+ gene inserted at either cnt1, otr1, or a random integrant euchromatic control locus (Rint) and a ura4 minigene control (ura4-DSE) at the endogenous ura4+ locus (Allshire et al., 1994; Partridge et al., 2000) were analyzed by RT-PCR. There was a significant increase in ura4+ message from the cnt1:ura4 site in sim mutants compared with wild type at permissive and restrictive temperatures (Fig. 1, D and E). Little or no alleviation of silencing was observed at otr1:ura4. These data confirm that the alleviation of silencing in sim mutants was specific to the central core region.
sim mutants display severe defects in chromosome segregation
sim mutants showed enhanced loss rates of a minichromosome (Fig. S1). sim1, sim3, and sim4 showed greater sensitivity to TBZ than wild type, but lower sensitivity than otr mutants such as rik1, clr4, and swi6 (Fig. 1 C; Ekwall et al., 1996). Neither sim2 nor mis6 showed supersensitivity to TBZ. To investigate the chromosome segregation defects, sim mutants were grown at permissive (25°C) or restrictive temperature (36°C for 6 h), fixed, and processed for immunofluorescence with -tubulin antibody. sim1, 3, and 4 displayed lagging chromosomes on late anaphase spindles (Fig. 2, BD) and uneven segregation of chromosomes. Short spindles with hypercondensed chromatin were common. Star or V-shaped spindles were seen frequently in sim3 mutants, which may indicate defects in the organization of a bipolar spindle.
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Central core chromatin structure is disrupted in the sim4 mutant
Chromatin was analyzed by limited MNase digestion and hybridization with a cnt1 probe. As shown in Fig. 3, the unique central core smear pattern seen in wild type (Polizzi and Clarke, 1991; Takahashi et al., 1992) was lost and replaced by a ladder in the sim4 mutant at permissive and restrictive temperatures. Similar observations have been made in other strains that have defects in central core proteins (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002).
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Antibodies were raised against GSTSim4 and affinity purified. Western analysis of total S. pombe protein extracts revealed a band of 30 kD (Fig. S2) that increased in intensity when Sim4 was expressed from a multicopy plasmid, and was replaced by an
60-kD band in a strain with a sim4GFP fusion gene at the sim4+ locus (see below). Thus, the polyclonal antibodies are specific for Sim4. Western analysis showed that there was no change in the amount of Sim4 present in a sim4 strain, indicating that its defect is not due to decreased protein stability (Fig. S2; unpublished data).
The sim4+ ORF was COOH-terminally tagged at its genomic locus with GFP (Bahler et al., 1998). A single GFP spot was seen in the nucleus of living cells in interphase, reminiscent of clustered centromeres (Fig. 4 A). Several spots were seen in early mitotic cells, likely to be individual (or replicated) centromeres (Fig. 4, A and B); in anaphase B cells, spots were observed at the leading edges of the daughter nuclei (Fig. 4 A). Cells expressing both Sim4GFP and Mis6HA (Saitoh et al., 1997) were fixed and stained with -GFP and
-HA antibodies, revealing colocalization of the two epitopes (Fig. 4 B). Colocalization of endogenous Sim4 and Mis6HA was also observed (Fig. 4 C). Thus, the Sim4 localization pattern and colocalization with the bona fide kinetochore protein Mis6 indicate that Sim4 is a novel centromere-associated protein.
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Sim4 coimmunoprecipitates with the central core kinetochore component, Mis6
The fission yeast kinetochore is likely to be a massive multiprotein complex made up of smaller subcomplexes. To investigate proteinprotein interactions at the fission yeast kinetochore, we asked whether Sim4 coimmunoprecipitates with other kinetochore components. Immunoprecipitation with -HA,
-GFP, or
-Sim4 antibodies was performed on extracts from a strain containing Mis6HA and Sim4GFP. Immunoprecipitates (IPs) were analyzed by Western blotting with complementary antibodies. Mis6HA and Sim4GFP clearly coimmunoprecipitated (Fig. 6 A). This complex appeared to be very tightly associated, as washing IPs with high concentrations of salt, urea, or nonionic detergents failed to disrupt it (unpublished data). This complex is specific, as Sim4 does not coimmunoprecipitate with other kinetochore proteins, such as Mis12 (unpublished data; Goshima et al., 1999).
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Genetic interactions between sim mutants
Genetic interactions were investigated between sim mutants and mutants in other known kinetochore proteins. Overexpression of Sim2/Cnp1CENP-A suppressed the temperature sensitivity of the other sim mutants to varying extents (Table I). Overexpression of Sim4 suppressed mis6 and sim3, but not sim1 or sim2. A genomic clone bearing the putative sim1+ ORF (unpublished data) also partially suppressed sim3 and sim4 and slightly suppressed mis6, but not sim2. Overexpression of Mis6 partially suppressed sim1, but not sim3 or sim4. In all cases tested, the bona fide sim+ gene complemented the temperature sensitivity and reimposed silencing at cnt1:arg3, whereas multicopy extragenic suppressors allowed only improved growth at the restrictive temperature but did not reimpose central core silencing (unpublished data).
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Discussion |
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Our characterization of Sim4 shows that it colocalizes with Mis6 and is restricted to the central domain of fission yeast centromeres. The sim4 mutant exhibits altered central core chromatin structure, elevated rates of chromosome loss, and increased sensitivity to MT poisons. Mitosis is frequently aberrant, with lagging chromosomes and nondisjunction of sister chromatids. These phenotypes indicate that Sim4 is required for kinetochore function. Given that neocentromere formation in other organisms requires the association of kinetochore components with noncentromeric DNA, it is of interest that Sim4 is capable of coating a ura4+ gene inserted in the central core.
Sim4 displays many functional interactions with the kinetochore protein Mis6; Sim4 and Mis6 are in the same complex, and overexpression of Sim4 strongly suppresses mis6. Association of Sim4 with the centromere is strongly dependent on Mis6, and the converse is true to a lesser degree. Sim4 is a small coiled-coil protein; many such proteins exist in kinetochore complexes in budding yeast (Cheeseman et al., 2002). Although no definite Sim4 homologues can be identified in metazoa or other organisms, we have noticed a weak similarity in structure and sequence between Sim4 and vertebrate CENP-H (25% similarity; Fig. S2; Sugata et al., 1999, 2000; Fukagawa et al., 2001). In chicken DT40 cells, the Mis6 homologue, CENP-I, is required for kinetochore localization of CENP-H (Nishihashi et al., 2002), as Mis6 is required for Sim4 localization. A two-hybrid interaction between CENP-H and CENP-I has also been reported (Nishihashi et al., 2002), suggesting that they are part of a complex. The observed similarities in behavior between S. pombe Sim4 and chicken CENP-H add weight to the weak homology between the two.
A mis6 mutant is defective in centromere incorporation of newly synthesized GFP-tagged Cnp1 (expressed from a heterologous promoter) at the restrictive temperature, and there is a reduced level of Cnp1CENP-AHA (expressed from its endogenous promoter). These observations have led to the proposal that Mis6 acts as a loading factor for Cnp1CENP-A (Takahashi et al., 2000). We have observed that both mis6 and sim4 mutants display reduced association of Cnp1 with the centromere, and this could be interpreted as evidence for a Mis6/Sim4-containing complex that acts as a specific loading factor for Cnp1. However, the budding yeast Mis6 homologue, Ctf3p, is not required for loading of Cse4p (Measday et al., 2002), the CENP-A counterpart. In addition, chicken CENP-I/Mis6 is not required for the localization of CENP-A, but, like CENP-H, it is required for CENP-C localization (Fukagawa et al., 2001; Nishihashi et al., 2002). These inconsistencies may reflect differences in organization and details between different organisms. However, several central core mutants affect Cnp1CENP-A centromere association, including sim1, sim3, and sim7 (Abbott, J., personal communication; unpublished data) as well as sim4 and mis6. We favor the idea that although specific loading or assembly factors for Cnp1 probably exist, it is the presence of a fully functional kinetochore that directs the incorporation of newly synthesized Cnp1CENP-A into the centromere, cell cycle after cell cycle. One possibility is that Cnp1CENP-A is only correctly incorporated at mitosis when proper kinetochoreMT attachments produce tension at functional kinetochores (Mellone and Allshire, 2003). Such a model is attractive, as it could play a part in the epigenetic inheritance of centromere site and specification.
Similar to other mutants that affect central domain function, sim4 mutants disrupt its unique chromatin structure (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002). Cnp1 is present at high levels in the central core and may take the place of histone H3 in the nucleosomes of this region. mis6 and sim4 mutants, amongst others, have reduced Cnp1CENP-A in the central domain. The smear pattern suggests that central core chromatin is organized in such a way that accessibility to MNase is altered; DNA may be wrapped more loosely around Cnp1-containing nucleosomes. Alternatively, the nucleosomes in this region may not have a regular spacing of 150 bp, but may be nonregularly spaced. Perhaps the fully assembled kinetochore protects the central core chromatin, not only from transcription factors, but also from activities that induce regular nucleosomal spacing or loading of histone H3. Whatever the cause of the unusual chromatin, it correlates with transcriptional silencing, normal levels of Cnp1CENP-A, and kinetochore integrity.
Mutants that alleviate outer repeat silencing have high levels of lagging chromosomes and are supersensitive to MT-disrupting drugs (Ekwall et al., 1996; Ekwall and Partridge, 1999), indicative of defects in MT interaction or in the coordination of MT-binding sites on single kinetochores. It is likely that lagging chromosomes are due to merotelic attachment of kinetochores, in which a single kinetochore interacts with MTs emanating from both spindle poles (Ladrach and LaFountain, 1986; Pidoux et al., 2000; Yu and Dawe, 2000; Stear and Roth, 2002). The Swi6-containing heterochromatin of the outer repeats that is required for centromeric cohesion may also have a role in preventing merotelic attachment (Pidoux et al., 2000; Bernard et al., 2001; Nonaka et al., 2002). Merotelic attachment has been shown to be a major mechanism contributing to aneuploidy in mammalian tissue culture cells (Cimini et al., 2001, 2002). Mutants that alleviate central core silencing or proteins that are located at the central core fall into two classes. Mutants of the first class, e.g., mis6 and cnp1/sim2, display uneven chromosome segregation, with few lagging chromosomes. It has been proposed that Mis6 is required for the biorientation function of the kinetochore, ensuring that the sister kinetochores face in opposite directions (Saitoh et al., 1997); mis6 mutants would not have a defect in kinetochoreMT interaction, per se, which is consistent with their observed wild-type sensitivity to MT drugs. A second class of central core mutants is typified by sim1, sim3, and sim4. These display both uneven segregation and lagging chromosomes and are sensitive to MT drugs. sim1 and 4 also display stronger synthetic interactions with the -tubulin mutant nda3 than does cnp1/sim2. We propose that Sim4, and other members of this class, is required both for the biorientation of sister kinetochores and for assembling a fully functional kinetochore in which the multiple MT-binding sites are locked together so that they interact correctly with the mitotic spindle. Although Mis6 and Sim4 are in the same complex, and may cooperate functionally, genetic evidence suggests that their functions overlap but are not identical; sim4 is synthetically lethal with sim1, sim2/cnp1, and sim3, but mis6 is not. The fact that overexpression of Sim4 suppresses mis6, but not vice versa, suggests that Sim4 acts upstream of Mis6. The lack of interaction with checkpoint components suggests that the spindle checkpoint may be impaired in sim mutants; the fact that these mutants do not exhibit a strong arrest at metaphase is consistent with this. Analyses of Bub1 have suggested that it may play additional roles at kinetochores independent of its role in checkpoint function (Warren et al., 2002).
We have used centromeric silencing in fission yeast as an assay of kinetochore assembly and successfully identified Cnp1CENP-A and a novel kinetochore component, Sim4. This is clearly a very effective approach for the identification of additional kinetochore components. By investigating genetic interactions in conjunction with proteinprotein interactions and dependency relationships for localization, we aim to build up a detailed picture of the architecture and function of this complex kinetochore.
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Materials and methods |
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Yeast strains
S. pombe strains used in this study are shown in Table III. To insert the promoter-crippled arg3+ gene at cnt1, an arg3+ fragment with 181 bp upstream of the ATG, including the TATA box but lacking other promoter elements, was PCR amplified and cloned into NcoI-digested pKS-cnt1, which contains a 5.2-kb EcoRI cnt1 fragment (Fig. 1 A). The cnt1(NcoI):arg3+ fragment was transformed into strain FY1891 (Allshire et al., 1994). An FOA-resistant, arg+ colony was analyzed by Southern blotting. Crosses with strains FY944, 1895, 382, and 1869 created strains FY3027 and 3033.
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Analysis of genetic interactions
Synthetic lethality.
At least 30 asci were dissected on YES plates and incubated at 25°C. The growth of viable double mutants compared with single mutants was assessed on YES phloxine B at 25°C, 28°C, 30°C, 32°C, 34°C, and 36°C.
Multicopy suppression.
sim mutants were transformed with genomic plasmids (pDB) bearing sim4+, cnp1+/sim2+, and sim1+ ORFs. A genomic fragment bearing the mis6+ ORF was PCR amplified and cloned into pAL-KS. Growth of serial dilutions was assessed on PMG phloxine B and PMG -arg and compared with the relevant empty plasmid.
Micrococcal nuclease digestion of chromatin
MNase digestion of chromatin in permeabilized cells was performed as previously described (Allshire et al., 1994), except that cells were grown in YES and spheroplasted with zymolyase-100T (ICN Biomedicals).
RT-PCR
Total RNA was prepared from strains grown in YES at 25°C or shifted to 36°C for 6 h, and RT-PCR was performed as previously described (Ekwall et al., 1997). The ura4 and ura4-DSE PCR products were separated on 1.5% agarose gels and poststained with ethidium bromide or SYBR green (Molecular Probes) according to the manufacturer's instructions. Quantitation of bands was performed using the Eastman Kodak Co. EDAS 290 system and 1D Image Analysis software. Analysis was performed two to four times at each temperature, and average values from these experiments are presented (Fig. 1 E). The ura4 to ura4-DSE ratio was determined and expressed relative to wild type at 25°C for each site, Rint, cnt1, or otr1.
Production of antibodies, Western blotting, and immunoprecipitations
sim4+ cDNA was PCR amplified and cloned into pGEX-4T1 (Amersham Biosciences). GSTSim4 fusion protein was purified and used to immunize a sheep. -Sim4 antibodies were affinity purified on nitrocellulose. Antibodies for Western blotting were diluted in PBS-Tween as follows:
-Sim4, 1:300;
-HA, 1:300;
-GFP, 1:2,000 (in 1% milk; Molecular Probes). Blots were developed using ECL reagents (Amersham Biosciences). Immunoprecipitations were performed as previously described (Millband and Hardwick, 2002).
ChIP
ChIP was performed as previously described (Ekwall and Partridge, 1999; Jin et al., 2002), except for the following modifications. For growth at restrictive temperature, cells were shifted to 36°C for 6 h. After addition of formaldehyde, incubation was continued at 36°C for 5 min, followed by 3 min in an ice-water bath and 22 min at 18°C (cells grown at 25°C were fixed for 30 min at 18°C). Cells were spheroplasted at 108 cells/ml in PEMS (100 mM Pipes, pH 7, 1 mM EDTA, 1 mM MgCl2, 1.2 sorbitol) + 0.4 mg/ml zymolyase-100T for 25 min at 36°C. Cells were washed twice in PEMS, and cell pellets were frozen at -20°C. Thereafter the standard ChIP procedure was followed (Ekwall and Partridge, 1999). 10 µl -Cnp1 antiserum (Kniola et al., 2001), 30 µl affinity-purified
-Sim4 antibody, or 30 µl
-HA antibody was used in ChIPs. Multiplex PCR analysis was performed as previously described (Jin et al., 2002). PCR products were quantified as described for RT-PCR. For the input PCR, the cnt, imr, and otr values were normalized to the fbp value, giving the input ratio. Enrichment of cnt, imr, and otr bands in the ChIPs was calculated relative to the fbp band and then corrected for the ratio obtained in the input PCR. Steps were taken to check that the multiplex PCR was a good method for quantification of ChIPs. The quantification depends on a quantifiable fbp band being present and if necessary exposure times, or amount of template, were adjusted to ensure that this was the case. PCR performed on different dilutions of input and ChIP'd samples gave very similar results. Although there was variation in the actual values obtained between individual ChIP experiments (producing the error bars in Fig. 7), the fold reductions seen in mutants were consistent between experiments. ChIP performed on strains with ura4+ insertions at Rint or cnt1 was analyzed by PCR as previously described (Ekwall et al., 1997).
Cytology
Immunofluorescence was performed as previously described (Pidoux et al., 2000), except that cells were fixed for 510 min in 3.7% freshly prepared formaldehyde for staining with -Sim4 antibodies. For immunolabeling of MTs, cells were fixed for 1015 min in 3.7% formaldehyde, 0.05% glutaraldehyde. The following antibodies were used: sheep
-Cnp1 antiserum (1:300), mouse 12CA5
-HA (1:30), mouse TAT1
-tubulin (1:15), and affinity-purified sheep
-Sim4 antibody (1:30). FITC (Sigma-Aldrich), Texas red (Jackson ImmunoResearch Laboratories), or Alexa®488 (Molecular Probes)-conjugated secondary antibodies were used at 1:100 or 1:1,000. FISH was performed as previously described (Ekwall et al., 1996). Microscopy was performed as previously described (Pidoux et al., 2000) or using the following setup: 100x Plan Neofluar 1.3 NA objective on a Carl Zeiss MicroImaging, Inc. Axioplan 2 IE fluorescence microscope equipped with Chroma 83000 and 86000 filter sets, Prior ProScan filterwheel (Prior Scientific), and Photometrics CoolSnapHQ CCD camera (Roper Scientific). Image acquisition was controlled using Metamorph software (Universal Imaging Corp.).
For measurement of spindle lengths, cut9 and cut9sim4 strains were grown at 25°C and shifted to 36°C for 4 h. Alternatively, wild-type and sim4 cells containing pREP3X-mad2 (He et al., 1997) were grown at 25°C in the absence of thiamine for 16 h, and then cells were shifted to 36°C for 7 h in the same medium. Spindle length was measured in cells with unseparated chromosomes, where both spindle poles were in focus, using either IPLab or Metamorph software. Spindle length was measured in 100300 cells for each strain.
Online supplemental material
The supplemental figures (Figs. S1 and S2) for this article are available at http://www.jcb.org/cgi/content/full/jcb.200212110/DC1. These figures include minichromosome loss data, sim4+ sequence and alignment with HsCENP-H, as well as Western blotting data and description of the sim4+ knockout.
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Acknowledgments |
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This research was supported by a Caledonian Research Foundation Fellowship (Royal Society of Edinburgh) to A.L. Pidoux and by the Association for International Cancer Research, the Medical Research Council of Great Britain, and The Wellcome Trust. R.C. Allshire is a Principal Research Fellow of The Wellcome Trust.
Submitted: 18 December 2002
Revised: 5 March 2003
Accepted: 7 March 2003
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References |
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Allshire, R.C., J.-P. Javerzat, N.J. Redhead, and G. Cranston. 1994. Position effect variegation at fission yeast centromeres. Cell. 76:157169.[Medline]
Allshire, R.C., E.R. Nimmo, K. Ekwall, J.-P. Javerzat, and G. Cranston. 1995. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 9:218233.[Abstract]
Bahler, J., J.Q. Wu, M.S. Longtine, N.G. Shah, A. McKenzie, A.B. Steever, A. Wach, P. Philippsen, and J.R. Pringle, Jr. 1998. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 14:943951.[CrossRef][Medline]
Bannister, A.J., P. Zegerman, J.F. Partridge, E.A. Miska, J.O. Thomas, R.C. Allshire, and T. Kouzarides. 2001. Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature. 410:120124.[CrossRef][Medline]
Bernard, P., J.F. Maure, J.F. Partridge, S. Genier, J.P. Javerzat, and R.C. Allshire. 2001. Requirement of heterochromatin for cohesion at centromeres. Science. 294:25392542.
Cheeseman, I.M., S. Anderson, M. Jwa, E.M. Green, J. Kang, J.R. Yates, C.S. Chan, D.G. Drubin, and G. Barnes. 2002. Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell. 111:163172.[Medline]
Cimini, D., B. Howell, P. Maddox, A. Khodjakov, F. Degrassi, and E.D. Salmon. 2001. Merotelic kinetochore orientation is a major mechanism of aneuploidy in mitotic mammalian tissue cells. J. Cell Biol. 153:517527.
Cimini, D., D. Fioravanti, E.D. Salmon, and F. Degrassi. 2002. Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells. J. Cell Sci. 115:507515.
Ding, R., K.L. McDonald, and J.R. McIntosh. 1993. Three-dimensional reconstruction and analysis of mitotic spindles from the yeast Schizosaccharomyces pombe. J. Cell Biol. 120:141151.[Abstract]
Ekwall, K., and J.F. Partridge. 1999. Fission yeast chromosome analysis. Fluorescence in situ hybridisation (FISH) and chromatin immunoprecipitation (CHIP). Chromosome Structural Analysis: A Practical Approach. W.A. Bickmore, editor. Oxford University Press, Oxford, UK. 3857.
Ekwall, K., J.-P. Javerzat, K. Lorentz, H. Schmidt, G. Cranston, and R. Allshire. 1995. The chromo domain protein Swi6: A key component at fission yeast centromeres. Science. 269:14291431.[Medline]
Ekwall, K., E.R. Nimmo, J.-P. Javerzat, B. Borgström, R. Egel, G. Cranston, and R. Allshire. 1996. Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. J. Cell Sci. 109:26372648.
Ekwall, K., T. Olsson, B.M. Turner, G. Cranston, and R.C. Allshire. 1997. Transient inhibition of histone acetylation alters the structural and functional imprint at fission yeast centromeres. Cell. 91:10211032.[Medline]
Ekwall, K., G. Cranston, and R.C. Allshire. 1999. Novel fission yeast mutants which alleviate transcriptional silencing in centromeric flanking repeats and disrupt chromosome segregation. Genetics. 153:11531169.
Fukagawa, T., Y. Mikami, A. Nishihashi, V. Regnier, T. Haraguchi, Y. Hiraoka, N. Sugata, K. Todokoro, W. Brown, and T. Ikemura. 2001. CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells. EMBO J. 20:46034617.
Goshima, G., S. Saitoh, and M. Yanagida. 1999. Proper metaphase spindle length is determined by centromere proteins Mis12 and Mis6 required for faithful chromosome segregation. Genes Dev. 13:16641677.
Hahnenberger, K.M., J. Carbon, and L. Clarke. 1991. Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromosome I. Mol. Cell. Biol. 11:22062215.[Medline]
He, X., T.E. Patterson, and S. Sazer. 1997. The Schizosaccharomyces pombe spindle checkpoint protein Mad2p blocks anaphase and genetically interacts with the anaphase-promoting complex. Proc. Natl. Acad. Sci. USA. 94:79657970.
He, X., D.R. Rines, C.W. Espelin, and P.K. Sorger. 2001. Molecular analysis of kinetochore-microtubule attachment in budding yeast. Cell. 106:195206.[Medline]
Garcia, M.A., L. Vardy, N. Koonrugsa, and T. Toda. 2001. Fission yeast ch-TOG/XMAP215 homologue Alp14 connects mitotic spindles with the kinetochore and is a component of the Mad2-dependent spindle checkpoint. EMBO J. 20:33893401.
Jin, Q.W., A.L. Pidoux, C. Decker, R.C. Allshire, and U. Fleig. 2002. The Mal2p protein is an essential component of the fission yeast centromere. Mol. Cell. Biol. 22:71687183.
Karpen, G.H., and R.C. Allshire. 1997. The case for epigenetic effects on centromere identity and function. Trends Genet. 13:489496.[CrossRef][Medline]
Kniola, B., E. O'Toole, J.R. McIntosh, B. Mellone, R. Allshire, S. Mengarelli, K. Hultenby, and K. Ekwall. 2001. The domain structure of centromeres is conserved from fission yeast to humans. Mol. Biol. Cell. 12:27672775.
Ladrach, K.S., and J.R. LaFountain. 1986. Malorientation and abnormal segregation of chromosomes during recovery from colcemid and nocodazole. Cell Motil. Cytoskeleton. 6:419427.[Medline]
McIntosh, J.R., E.L. Grishchuk, and R.R. West. 2002. Chromosome-microtubule interactions during mitosis. Annu. Rev. Cell Dev. Biol. 18:193219.[CrossRef][Medline]
Measday, V., D.W. Hailey, I. Pot, S.A. Givan, K.M. Hyland, G. Cagney, S. Fields, T.N. Davis, and P. Hieter. 2002. Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore. Genes Dev. 16:101113.
Mellone, B.G., and R.C. Allshire. 2003. Stretching it: putting the CEN(P-A) in centromere. Curr. Opin. Genet. Dev. 13:191198.[CrossRef][Medline]
Millband, D.N., and K.G. Hardwick. 2002. Fission yeast Mad3p is required for Mad2p to inhibit the anaphase-promoting complex and localizes to kinetochores in a Bub1p-, Bub3p-, and Mph1p-dependent manner. Mol. Cell. Biol. 22:27282742.
Moreno, S., A.J.S. Klar, and P. Nurse. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194:795823.[Medline]
Musacchio, A., and K.G. Hardwick. 2002. The spindle checkpoint: structural insights into dynamic signalling. Nat. Rev. Mol. Cell Biol. 3:731741.[CrossRef][Medline]
Nakaseko, Y., G. Goshima, J. Morishita, and M. Yanagida. 2001. M phase-specific kinetochore proteins in fission yeast: microtubule-associating Dis1 and Mtc1 display rapid separation and segregation during anaphase. Curr. Biol. 11:537549.[CrossRef][Medline]
Nakayama, J., J.C. Rice, B.D. Strahl, C.D. Allis, and S.I. Grewal. 2001. Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science. 292:110113.
Nonaka, N., T. Kitajima, S. Yokobayashi, G. Xiao, M. Yamamoto, S.I. Grewal, and Y. Watanabe. 2002. Recruitment of cohesin to heterochromatic regions by Swi6/HP1 in fission yeast. Nat. Cell Biol. 4:8993.[CrossRef][Medline]
Nasmyth, K. 2002. Segregating sister genomes: the molecular biology of chromosome separation. Science. 297:559565.
Nimmo, E.R., A.L. Pidoux, P.E. Perry, and R.C. Allshire. 1998. Defective meiosis in telomere-silencing mutants of Schizosaccharomyces pombe. Nature. 392:825828.[CrossRef][Medline]
Nishihashi, A., T. Haraguchi, Y. Hiraoka, T. Ikemura, V. Regnier, H. Dodson, W.C. Earnshaw, and T. Fukagawa. 2002. CENP-I is essential for centromere function in vertebrate cells. Dev. Cell. 2:463476.[Medline]
Partridge, J.F., B. Borgstrom, and R.C. Allshire. 2000. Distinct protein interaction domains and protein spreading in a complex centromere. Genes Dev. 14:783791.
Partridge, J.F., K.S. Scott, A.J. Bannister, T. Kouzarides, and R.C. Allshire. 2002. Cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site. Curr. Biol. 12:16521660.[CrossRef][Medline]
Pidoux, A.L., and R.C. Allshire. 2000a. Centromeres: getting a grip of chromosomes. Curr. Opin. Cell Biol. 12:308319.[CrossRef][Medline]
Pidoux, A.L., and R.C. Allshire. 2000b. The structure of yeast centromeres and telomeres and the role of silent heterochromatin. The Yeast Nucleus. J. Beggs and P. Fantes, editors. Oxford University Press, Oxford, UK. 212245.
Pidoux, A.L., S. Uzawa, P.E. Perry, W.Z. Cande, and R.C. Allshire. 2000. Live analysis of lagging chromosomes during anaphase and their effect on spindle elongation rate in fission yeast. J. Cell Sci. 113:41774191.
Pluta, A.F., A.M. Mackay, A.M. Ainsztein, I.G. Goldberg, and W.C. Earnshaw. 1995. The centromere: hub of chromosomal activities. Science. 270:15911594.[Abstract]
Polizzi, C., and L. Clarke. 1991. The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function. J. Cell Biol. 112:191201.[Abstract]
Rea, S., F. Eisenhaber, D. O'Carroll, B.D. Strahl, Z.W. Sun, M. Schmid, S. Opravil, K. Mechtler, C.P. Ponting, C.D. Allis, and T. Jenuwein. 2000. Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature. 406:593599.[CrossRef][Medline]
Saitoh, S., K. Takahashi, and M. Yanagida. 1997. Mis6, a fission yeast inner centromere protein, acts during G1/S and forms specialized chromatin required for equal segregation. Cell. 90:131143.[Medline]
Stear, J.H., and M.B. Roth. 2002. Characterization of HCP-6, a C. elegans protein required to prevent chromosome twisting and merotelic attachment. Genes Dev. 16:14981508.
Sugata, N., E. Munekata, and K. Todokoro. 1999. Characterization of a novel kinetochore protein, CENP-H. J. Biol. Chem. 274:2734327346.
Sugata, N., S. Li, W.C. Earnshaw, T.J. Yen, K. Yoda, H. Masumoto, E. Munekata, P.E. Warburton, and K. Todokoro. 2000. Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromerekinetochore complexes. Hum. Mol. Genet. 9:29192926.
Sullivan, B.A., M.D. Blower, and G.H. Karpen. 2001. Determining centromere identity: cyclical stories and forking paths. Nat. Rev. Genet. 2:584596.[CrossRef][Medline]
Takahashi, K., S. Murakami, Y. Chikashige, H. Funabiki, O. Niwa, and M. Yanagida. 1992. A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere. Mol. Biol. Cell. 3:819835.[Abstract]
Takahashi, K., E.S. Chen, and M. Yanagida. 2000. Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. Science. 288:22152219.
Volpe, T., V. Schramke, G.L. Hamilton, S.A. White, G. Teng, R.A. Martienssen, and R.C. Allshire. 2003. RNA interference is required for normal centromere function. Chromosome Res. 11:137146.[CrossRef][Medline]
Volpe, T.A., C. Kidner, I.M. Hall, G. Teng, S.I. Grewal, and R.A. Martienssen. 2002. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science. 297:18331837.
Warren, C.D., D.M. Brady, R.C. Johnston, J.S. Hanna, K.G. Hardwick, and F.A. Spencer. 2002. Distinct chromosome segregation roles for spindle checkpoint proteins. Mol. Biol. Cell. 13:30293041.
Wigge, P.A., and J.V. Kilmartin. 2001. The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J. Cell Biol. 152:349360.
Winey, M., C.L. Mamay, E.T. O'Toole, D.N. Mastronarde, T.H. Giddings, K.L. McDonald, and J.R. McIntosh. 1995. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J. Cell Biol. 129:16011615.[Abstract]
Wood, V., R. Gwilliam, M.A. Rajandream, M. Lyne, R. Lyne, A. Stewart, J. Sgouros, N. Peat, J. Hayles, S. Baker, et al. 2002. The genome sequence of Schizosaccharomyces pombe. Nature. 415:871880.[CrossRef][Medline]
Yu, H.G., and R.K. Dawe. 2000. Functional redundancy in the maize meiotic kinetochore. J. Cell Biol. 151:131142.