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1 Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2 Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1101, Japan
3 Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
4 Kanagawa Dental College, Inaoka-cho, Yokosuka-ku, Kanagawa 238-8580, Japan
5 Department of Gene Mechanism, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
6 Biological Information Research Center, Advanced Industrial Science and Technology, Koto-ku, Tokyo135-0064, Japan
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Introduction |
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-satellite array and a flanking type-II -satellite array (Ikeno et al. 1994). The type-I array is mainly composed of a conserved dimer repeat (171 bp x 2) containing one CENP-B box in one of the dimer units. The CENP-B box in the type-I array is necessary for de novo formation of artificial chromosomes (Ohzeki et al. 2002). The type-II array is composed of a simple monomer repeat with a relatively variable sequence lacking a CENP-B box. Both arrays are of the order of megabases in length. CENP-A chromatin is predominantly formed on 30–50% of the length of the type-I array (Ando et al. 2002), and it is speculated that centromeric heterochromatin is formed outside the CENP-A chromatin region, although the precise structure remains to be elucidated.
We have developed a method to isolate from HeLa interphase nuclei the centromere complexes that contain CENP-A, CENP-B and CENP-C, using monoclonal antibodies against CENP-A (Ando et al. 2002). To isolate the DNA–protein complex and analyze the purified proteins, we avoided sonication or cross-linking reagents such as formaldehyde that are usually used in conventional chromatin immunoprecipitation (ChIP) analysis in yeast (Aparicio 1999). To extract the centromeric chromatin into the soluble fraction in a native state, using the mildest conditions, we only cleaved chromatin DNA using micrococcal nuclease (MNase) and salt (0.3 M NaCl) treatment. To discriminate this method from other conventional ChIP methods we call this method "Native Chromatin Immunoprecipitation" (NChIP). As the CENP-A/B/C chromatin complex purified with NChIP using anti-CENP-A antibodies contained CENP-H, hMis6 and hMis12 as well as CENP-A, CENP-B and CENP-C, which constitute all the reported structural components of the centromere, we call this complex the ICEN (Interphase CENtromere complex). Approximately 70% of the DNA segments in the ICEN are composed of the type-I -satellite sequence, indicating that the minimum purity of the ICEN can be regarded as 70%. By proteomic analysis we have studied these ICEN components extensively and revealed 40 proteins. We have further shown that two of these, DDB1 and BMI1, are actually located in the centromeric regions (Obuse et al. 2004b).
In this paper we have named these proteins ICEN1–ICEN40, as shown in Table 1. To reveal the function of each ICEN component in relation to centromere/kinetochore function, we have examined these proteins in detail using newly prepared antibodies, various EGFP-tagged genes, and siRNA transfection. We have revealed that seven ICEN proteins, ICEN22, 24, 32, 33, 36, 37 and 39, are novel components of the centromeric chromatin complex, and have a role in kinetochore function. ICEN22, 32, 33, 37 and 39 are necessary for loading of both CENP-H and hMis6 proteins onto the centromeric region. On the basis of these results we propose that, although fragmented to < 30 mer chromatin, the ICEN may represent a large centromere chromatin complex, and therefore may contain many other protein components with important roles for structure and function of the centromeric regions.
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Results |
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To observe localization of the ICEN components in a cell, we tagged the EGFP gene or its derivative to the NH2- or COOH-terminus of the genes of ICEN22, 24, 32, 33, 36, 37 and 39, and transfected each of the fused genes into HT1080 cells as described in Experimental procedures. Each of the stable transformant cells were immunolabeled with anti-GFP antibodies and anti-CENP-A antibodies. Figure 1 shows that EGFP-ICEN22, 24, 32, 33, 36, 37 and 39 all co-localize with CENP-A in interphase and metaphase cells. These results suggest that ICEN22, 24, 32, 33, 36, 37 and 39 localize to centromeres in interphase and in metaphase.
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In order to study the ICEN 39/PANE1 protein we prepared monoclonal antibodies (3G8) against the synthetic peptides as shown in Experimental procedures. The molecular mass of this protein is expected to be 19.7 kDa from the amino acid sequence (Obuse et al. 2004b) and the antibody recognized an 18 kDa protein in the purified ICEN fraction with Western blotting after SDS-PAGE (Fig. 2A). To examine the distribution of ICEN 39 protein, HeLa cells were fractionated to a cytoplasmic fraction (#1 in Fig. 2B) and two nucleus fractions, 0.3 M NaCl extract (#2) and bulk chromatin (#3), as illustrated in Fig. 2B. As shown in Fig. 2C, ICEN39 distributed both in the cytoplasmic fraction (Fig. 2C, lane 1) and in the two nucleus fractions (Fig. 2C, lanes 2 and 3). ICEN39 in the bulk chromatin fraction (Fig. 2C, lane 3) all localized to the centromere region, since NChIP of the bulk chromatin with anti-CENP-A antibodies depleted ICEN39 from the sup fraction (Fig. 2C, lane 4) and recovered to the ICEN fraction (Fig. 2C, lane 5). Immuno-stain of MRC-5 (human normal fibroblast) cells in Fig. 2D showed that dot-like signals of ICEN39 are detected both in interphase cells (Fig. 2D,b,f) and these signals co-localize with CENP-C (Fig. 2D,d,h), although the intensity of the dots in interphase 1 (Fig. 2D,b) was very weak. The intensity of ICEN39 in cytoplasm and nucleus is variable in interphase 1 and 2 (Fig. 2D,b,f). These results suggest that the endogenous ICEN39/PANE1 distributes both in cytoplasm and in nucleus and also localizes to centromereric regions in interphase.
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To examine comprehensively the physiological role of the ICEN components for chromosome segregation, we prepared siRNA for the ICEN genes and transfected into HeLa cells. In Fig. 3A we examined sequence-specific siRNA activity to deplete target protein, 2 days after transfection of GFP-DNA in the absence (upper panel) or presence (lower panel) of siRNA. The results indicate that each of the transfected siRNAs effectively suppresses production of the target protein, but not the off-target protein (si24 RNA against EGFP-22 DNA; Fig. 3A bottom left panel). As shown in Fig. 3B, abnormal metaphase cells carrying misaligned chromosomes were observed in cells treated with siRNA for each ICEN component. As control experiments we also examined siRNA depletion of hMis6 (ICEN19) and CENP-H (ICEN35). As shown in Fig. 3C, both CENP-H and hMis6 signals disappeared in hMis6 (ICEN19)- and/or CENP-H (ICEN35)-depleted cells, while CENP-A and CENP-C localization were unaffected at 4 days after siRNA transfection. Kinetochore function was severely inhibited and abnormal cells with misaligned chromosomes appeared at an extremely high frequency (Fig. 3C,D). To establish the frequency of abnormal cells, we examined 100–150 metaphase cells for misaligned chromosomes, and the results are summarized in Fig. 3D. In the cells transfected with siRNA for ICEN32, 33 and 37, the frequency of abnormal metaphase cells (79 ± 2%, 59 ± 6% and 67 ± 6%, respectively) were high as was that observed in the cells treated with siRNA for CENP-H and hMis6. While the effect of siRNA for ICEN39 is medium (34 ± 5%) and for ICEN 22, 24 and 36 were low (19 ± 2%, 14 ± 3% and 15 ± 5%) but significantly higher than mock cells (4 ± 2%) (Fig. 3D). These results suggest that ICEN22, 24, 32, 33, 36, 37 and 39 are required for proper kinetochore function.
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To check if depletion of ICEN proteins causes aneuploidy as the result of a mitotic defect, FISH analysis using several centromere probes was performed after transfection with siRNA for each of the seven ICEN genes to a diploid colorectal cancer cell line, RKO. Figure 4A shows examples of aneuploidy observed by FISH with the DNA sequence from pericentromeric region of chromosome 7 (CEP7) probe on RKO cells, 3 days after transfection with siRNA. Compared to control cells, after transfection with siRNA for ICEN 33, 37 or 39, the cells frequently contain more than two centromere signals, suggesting that the cells have failed to segregate chromosome 7 in the previous mitosis (Fig. 4A). To calculate the frequency of aneuploidy, the cells carrying more than two centromeres of CEP7, CEP12 and CEP15 were counted. As shown in Fig. 4B, the frequency of aneuploidy for each chromosome after transfection with siRNA for ICEN22, 24, 32, 33, 36, 37 or 39 was increased by 1.2–2.5 times compared with that of control cells. These results suggest that the damage to kinetochore function due to the depletion of each of the seven ICEN proteins caused defects in chromosome segregation and resulted in aneuploidy. It is noteworthy that the frequency of aneuploidy in si36 cells, in spite of the low frequency of abnormal cells with misaligned chromosomes as shown in Fig. 3D, is as high as that in si32 and si37 cells that show high frequency of misaligned chromosomes.
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As shown in Fig. 5, we next examined the effect of siRNA depletion of each of the seven ICEN proteins on loading of the known centromeric proteins onto centromeres. While loading of CENP-A and CENP-C onto centromeres was unaffected by depletion of any of these proteins (Fig. 5A and Fig. 3B, respectively), we observed that in interphase and/or metaphase cells depleted of ICEN22, 32, 33, 37 or 39, both CENP-H and hMis6 signals were often lost from the centromeric regions. The frequencies of loss of the CENP-H and hMis6 signals in interphase and/or metaphase cells were 40–76% for CENP-H and 70–78% for hMis6 (data not shown). Interestingly, the abnormal cells carrying lagging chromosomes produced by siRNA for ICEN 22, 32, 33, 37 or 39-transfection had often lost all the CENP-H and hMis6 signals, as shown in Fig. 5. CENP-H and hMis6 signals in cells depleted of each ICEN were measured for at least 50 normal and abnormal metaphase cells in each case, and the results are summarized in Fig. 6, demonstrating that the appearance of misaligned chromosomes for si22, 32, 33, 37 and 39 clearly correlates with the loss of CENP-H and hMis6 signals in metaphase cells. These results suggest that ICEN22, 32, 33, 37 and 39 proteins are necessary for CENP-H and hMis6 loading onto centromeres, and that abnormal cells with lagging chromosomes may be produced as a consequence of the loss of CENP-H and hMis6 at the centromeric regions. On the other hand, depletion of ICEN24 or 36 proteins does not affect the loading of CENP-H or hMis6.
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Discussion |
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ICEN39 is the same as PANE1 (Proliferation Associated Nuclear Element 1) that is induced upon transformation of mouse mammary epithelium by an activated ß-catenin (Renou et al. 2003). This protein was reported to localize in both the cytoplasm and the nucleus, and its nuclear localization is predominant in actively proliferating cells (Renou et al. 2003). We also observed that the intensity of the ICEN39 signals in nuclei and centromeres are variable (Fig. 2D), which may be dependent on the state of the cell. These results suggest that this protein might have a role at the centromeric regions in the regulation of cell proliferation.
ICEN22, 32, 33, 37 and 39
CENP-H and hMis6 associate with each other to form a complex and are dependent on each other for centromere localization (Fig. 3C) (Nishihashi et al. 2002). Centromere localization of the CENP-H/hMis6 complex also requires ICEN22, 32, 33, 37 and 39 (Figs 5 and 6). These results suggest that the CENP-H/hMis6 complex functionally associates with each of the ICEN22, 32, 33, 37 or 39 proteins, implying that some or all of these proteins might physically associate with each other to form a complex, and that the stoichiometry of these proteins might be important for centromere targeting and kinetochore function. Tomonaga et al. (2005) reported that CENP-H over-expression in mouse 3T3 cells inhibited the loading of endogenous CENP-H onto the centromeric regions, which could be explained if over-expression of CENP-H disturbed the stoichiometry of the multiprotein complex.
ICEN24/KLIP1/MLF1IP/CENP-50
The frequency of abnormal cells carrying misaligned chromosomes in ICEN24 siRNA-transfected cells is rather low (14 ± 3%) compared to that found in the case of ICEN32, 33 or 37 (60–80%) (Fig. 3D), implying that the production of misaligned chromosomes in this case might be attributable to a different cause. Centromere localization of ICEN24 was confirmed using GFP-tagged genes (Fig. 1). It is noteworthy that ICEN24 protein does not participate in CENP-H and hMis6 loading onto centromeres (Figs 5 and 6). It could therefore be considered that inhibition of kinetochore function by depletion of ICEN 24 may involve a pathway different from the CENP-H/hMis6 complex pathway. A series of protein interaction pathways originating from hMis12 were reported to take part in kinetochore functions (Obuse et al. 2004a). The hMis12 interacting proteins, HEC1 and Zwint1, are components of the outer kinetochore and HP1 and HP1
are centromeric chromatin components. Another five proteins are unknown, and none corresponds to ICEN components except HP1 (ICEN38). We detected hMis12 signals on the centromeres of the lagging chromosomes produced by ICEN24 siRNA (data not shown). ICEN24 was also identified as KLIP1/MLF1IP/CENP-50 (Pan et al. 2003; Hanissian et al. 2004; Minoshima et al. 2005). Studies of CENP-50-deficient DT40 cells indicate that the CENP-50 gene is nonessential, except that recovery from mitotic arrest induced by nocodazole treatment is delayed substantially (Minoshima et al. 2005). In human cells, ICEN24 depletion produces misaligned chromosomes (Fig. 3) and causes aneuploidy (Fig. 4B), which is different from the situation with CENP-50 in the chicken. Like human ICEN24, chicken CENP-50 does not participate in CENP-H/hMis6 loading onto centromeres (Fig. 5). ICEN24/CENP-50 was isolated by two-hybrid selection using MgcRacGAP as a fishing bait, suggesting that ICEN24/CENP-50 directly interacts with MgcRacGAP (Minoshima et al. 2005). The ICEN includes MgcRacGAP (ICEN17) as one of its components (Table 1) (Obuse et al. 2004b). These results suggest that ICEN24 may physically, and therefore functionally, interact with MgcRacGAP. We showed that the MgcRacGAP/hMKLP1 complex localized to centromeres from interphase to early metaphase, and had a role in kinetochore function (our unpublished observation). Recently, it was also reported that the Ect2/Cdc42/mDia3 and MgcRacGAP signaling pathway positively regulated the dynamics of spindle microtubule/kinetochore interaction (Yasuda et al. 2004; Oceguera-Yanez et al. 2005).
Aneuploidy and cancer
The frequency of misaligned chromosomes (Fig. 3D) and aneuploidy (Fig. 4B), both of which may be produced by the defect of kinetochore function owing to siRNA transfection, were not necessarily correlated with each other. In fact, the frequency of misaligned chromosomes in si22, 24 or 36 cells were only a little higher than that of control cells (Fig. 3D), but that of aneuploidy is high (Fig. 4B). This tendency is especially conspicuous in si36 cells. On the other hand in si33 cells, the frequency of aneuploidy is low, in spite of that of misaligned chromosomes being relatively high. This discrepancy may be because the cells become lethal if they suffer substantial damage to their chromosome. Conversely, if the damage is minimal, the cells may be able to stay alive with the accumulation of aneuploid chromosomes, which leads to the development of cancer. It is interesting to investigate whether the expression and/or the function of ICEN components are deregulated in human cancers.
Future scope for the ICEN
We have previously reported that DDB1 (ICEN9) is a component of the centromere complex and suggested that DDB1 might take part in regulation of heterochromatin dynamics around the centromeric region (Obuse et al. 2004b). Jia et al. (2005) have recently reported that Rik1, Cul4, Swi-6, HDAC and Clr-4 are necessary for heterochromatin formation at the mating type locus and the pericentromeric region of Schizosaccaromyces pombe, and that Rik1 is functionally and structurally related to DDB1 and interacts with Cul4. As shown in Table 1, the ICEN contains DDB1 (ICEN 9), cullin-4 A (ICEN15), HP1 (ICEN38), which is one of the Swi6 homologs, and HDAC1 (ICEN28). These results strongly suggest that the ICEN contains a set of proteins necessary for centromeric heterochromatin formation. We have shown in this study that seven ICEN components localize to the centromeres and have a role in kinetochore function. These results strongly support the idea that, although fragmented, the ICEN represents a large CENP-A chromatin complex and therefore may contain many other protein components with important roles for structure and function of the centromeric regions. We still have many ICEN components with unknown centromere functions, and we believe that studies of these proteins will open up a new horizon in the structure and function of centromeres.
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Experimental procedures |
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HeLa S3 cells and HT1080 cells were grown at 37 °C in DMEM (Sigma) for monolayer culture or RPMI 1640 (Nissui, Japan) for suspension culture, supplemented with 5% or 10% calf serum and antibiotics. MRC-5 and RKO cells were grown in Iscove's modified Dulbecco's medium (IMDM < Invitrogen) supplemented with 10% fetal calf serum and antibiotics.
Bulk chromatin and NChIP
Methods for preparation of bulk chromatin from HeLa cells and NChIP were fundamentally the same as previously reported (Obuse et al. 2004b; Yoda & Ando 2004).
Antibodies
Monoclonal antibodies against ICEN39 (clone 3G8) were prepared as described previously (Kimura et al. 1994). A mixture of amino- and carboxyl-terminal peptides (MSVLRPLDKLPGLNTAC, and CSLLRSSEGPSLEDL, respectively) was used as antigens. Antibodies against CENP-A, CENP-C, CENP-H, hMis6 and hMis12 were described previously (Obuse et al. 2004b). Anti-CENP-A monoclonal antibodies clone 3–19, anti-CENP-C from guinea pig, anti-CENP-H and anti-hMis12 from rabbit, and anti-hMis6 from rat were used. Anti-GFP antibodies (rabbit) were obtained from MBL and anti-ß-tubulin (mouse monoclonal) from Sigma.
Western blotting
The methods for Western blotting were as described previously (Ando et al. 2002). Five to ten microliters of the sample was separated using a 12.5% SDS-PAGE. The separated proteins were transferred to a PVDF membrane (Millipore), and immunoreaction was for 10–12 h at 4 °C.
Construction of the EGFP-ICEN fusion gene
Coding regions of ICEN24, 33, 37 and 39 were amplified by RT-PCR from HT1080 total RNA, using the following primers:
24: forward: 5'-AAGCTTCGATGGCCCCGCGGGGGCGGCGGCGGCCG-3'
reverse: 5'-GTCGACTCATCCCTGGTCAAGGAGCTTCTC-3'
33: forward: 5'-GAATTCTATGGATTCTTACAGTGCACCAGA-3'
reverse: 5'-GTCGACTCAATTTGAAAAATTGCCAGTTCT-3'
37: forward: 5'-GAATTCTATGAATCAGGAGGATCTAGATCC-3'
reverse: 5'-GTCGACTTACTGATGGAAAGCTTCTAATCT-3'
39: forward: 5'-CACCATGTCGGTGTTGAGGCCCCTG-3'
reverse: 5'-TCACAGGTCCTCCAAGGGAGGG-3'
PCR products of ICEN24, 33 and 37 were cloned into pDrive vector (QIAGEN) and re-cloned into pEGFP-C1 vector (Clontech) after digestion with EcoRI and SalI for ICEN33 and 37, and HindIII and SalI for ICEN24. The PCR product of ICEN39 was cloned into pCR2.1-TOPO vector (Invitrogen) and re-cloned into pEGFP-C1 after digestion with EcoRI. Coding sequences for ICEN22, 32 and 36 were amplified by PCR from human FLJ cDNA clones (Ota et al. 2004) and cloned into pDONR201 vector (Invitrogen). These clones were recloned into pDESTMN-VenusA206K vector or pDESTMC-VenusA206K vector, as described in the company's instruction manual. pDESTMN-VenusA206K-ICEN22 and ICEN32 and pDESTMC-VenusA206K-ICEN36 were used in this experiment. Venus protein is a derivative of EGFP protein (Nagai et al. 2002).
Transfection of EGFP-ICEN genes and/or siRNA and isolation of stable transformants
Transfection of DNA and/or RNA was performed using Lipofectamine 2000 (Invitrogen) according to the company's instruction manual. To isolate stable transformant cells, 1 µg DNA was transfected into HT1080 cells (1 x 106) and cultured for a few weeks with DMEM medium containing 400 µg/mL of G418.
Depletion of ICEN proteins by short RNA interference
The following short double-stranded RNAs were obtained from Invitrogen as stealth siRNA:
ICEN22: 5'-AGUUGAGUGGCCAAACAAGGACGAU-3'
ICEN24: 5'-GCAAGCCUAUUGACGUGUUCGACUU-3'
ICEN32: 5'-GCUGCCCTGUUAGACAUCAUUUAUA -3'
ICEN33: 5'-GCCACAAGAUUAGUUCGUGUUUCAA-3'
ICEN36: 5'-CCUUGCAGAGAAACCCACUGUGUAA-3'
ICEN37: 5'-GACUGAAGACGUUCUCAUAACAUUA-3'
ICEN39:5'-UUGACCUGAUCGUGUUUGUGGUUAA-3'
ICEN19 (HMIS6): 5'-AUCAUCAGCAUGUUCAUAAUCUCCC-3'
ICEN35 (CENP-H): 5'-AACAAUUUCCUUAAGGGCAGGAUCC-3'
We usually used four-well slide glasses (Nalge Nunc) coated with 0.01% poly L-lysine (mol. wt. 150 000–300 000, Sigma). Twenty picomoles of each siRNA and 1 µL of Lipofectamine 2000 were used in 0.5 mL DMEM medium.
Optical-microscopy observations
The cells were fixed with 2% paraformaldehyde for 30 min at room temperature or with cold acetone (98%, –20 °C) for 30 min, and stained with antibodies as indicated in each figure. Second antibodies were conjugated with FITC (green) or TRITC (red), and DNA was stained with DAPI (blue). The cells were observed with a BX51 microscope (Olympus) and images were taken with a CoolSNAP monochrome camera (Roper Scientific Ltd) under Openlab version 2 (Improvision Ltd).
FISH analysis
After culture, slides were washed twice with PBS, incubated in 75 mM KCl for 10 min, fixed in 3 : 1 methanol: acetic acid for 10 min at room temperature, and treated with 0.1 mg/mL of RNase A in 2 x SSC for 30 min at 37 °C. After washing in PBS, slides were dehydrated by passage through an ethanol series (70%, 85% and 100%), then incubated in 2 x SSC/0.1% Nonidet P-40 solution for 30 min at 37 °C, and dehydrated again. Target DNA was denatured for 5 min at 73 °C in 70% formamide/2 x SSC (pH 7.3). Probes (10 µL) to the pericentromeric regions of chromosome 7 (CEP7 Spectrum GreenTM), chromosome 12 (CEP12 Spectrum OrangeTM), and 15 (CEP15 Spectrum GreenTM) (Vysis, Downers Grove, IL, USA) were also denatured for 5 min at 73 °C, then hybridized to the target DNA by incubation overnight at 37 °C. Post-hybridization washes were performed 3 times in 50% formamide/2 x SSC (pH 7.0) for 10 min at 45 °C, once in 2 x SSC and in 2 x SSC/0.1% Nonidet P-40 solution for 5 min at 45 °C. Hybridization signals were observed and analyzed with Leica QFISH (Leica QFISH; Leica Microsystems, Tokyo, Japan). At least 200 nuclei of each sample were evaluated for chromosome counts.
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work 
* Correspondence: E-mail: i45156a{at}cc.nagoya-u.ac.jp |
References |
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|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Aparicio, O.M. (1999) Characterization of proteins bound to chromatin by immunoprecipitation from whole-cell extracts. In: Current Protocols in Molecular Biology, Vol. 4 (eds F.M. Ausubel, R. Brent, R.E. Kingston et al.), pp. 21.3.1–21.3.12. New York: John Wiley and Sons, Inc.
Belotserkovskaya, R., Oh, S., Bonarenko, V.A., Orphanides, B., Studitsky, V.M. & Reinberg, D. (2003) FACT facilitates transcription-dependent nucleosome alteration. Science
301, 1090–1093.
Black, B.E., Foltz, D.R., Chakravarthy, S., Luger, K., Woods, V.L. Jr & Cleveland, D.W. (2004) Structure determinants for generating centromeric chromatin. Nature 430, 578–582.[CrossRef][Medline]
Buchwitz, B.J., Ahmad, K., Moore, L.L., Roth, M.B. & Henikoff, S. (1999) A histone-H3-like protein in C. elegans. Nature 401, 547–548.[CrossRef][Medline]
Cleveland, D.W., Mao, Y. & Sullivan, K.F. (2003) Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407–421.[CrossRef][Medline]
Dobie, K.W., Hari, K.L., Maggert, K.A. & Karpen, G.H. (1999) Centromere proteins and chromosome inheritance: a complex affair. Curr. Opin. Genet. Dev. 9, 206–217.[CrossRef][Medline]
Goshima, G., Kiyomitsu, Y., Yoda, K. & Yanagida, M. (2003) Human centromere chromatin protein hMis12, essential for equal segregation, is independent of CENP-A loading pathway. J. Cell Biol.
160, 25–39.
Hanissian, S.H., Akbar, U., Teng, B., et al. (2004) cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1 IP. Oncogene 23, 3700–3707.[CrossRef][Medline]
Henikoff, S., Ahmad, K., Platero, J.S. & Steensel, B.V. (2000) Heterochromatic deposition of centromeric histone H3-like proteins. Proc. Natl. Acad. Sci. USA
97, 716–721.
Ikeno, M., Masumoto, H. & Okazaki, T. (1994) Distribution of CENP-B boxes reflected in CREST centromere antigenic sites on long-range -satellite DNA arrays of human chromosome 21. Hum. Mol. Genet.
3, 1245–1257.
Jia, S., Kobayashi, R. & Grewal, S.I.S. (2005) Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin. Nat. Cell Biol. 7, 1007–1013.[CrossRef][Medline]
Kimura, K., Nozaki, N., Saijo, M., Kikuchi, A., Ui, M. & Enomoto, T. (1994) Identification of the nature of modification that causes the shift of DNA topoisomerase II beta to apparent higher molecular weight forms in the M phase. J. Biol. Chem.
269, 24523–24526.
Kops, G.J.P.L., Foltz, D.R. & Cleveland, D.W. (2004) Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. Proc. Natl. Acad. Sci. USA
101, 8699–8704.
Lengauer, C., Kinzler, K.W. & Vogelstein, B. (1998) Genetic instabilities in human cancers. Nature 396, 643–649.[CrossRef][Medline]
Liu, S.T., Hittle, J.C., Jablonski, S.A., Campbell, M.S., Yoda, K. & Yen, T.J. (2003) Human CENP-I specifies localization of CENP-F, MAD1 and MAD2 to kinetochores and is essential for mitosis. Nat. Cell Biol. 5, 341–345.[CrossRef][Medline]
Loyola, A., Huang, J.Y., LeRoy, G., et al. (2003) Functional analysis of the subunits of the chromatin assembly factor RSF. Mol. Cell. Biol.
23, 6759–6768.
Minoshima, Y., Hori, T., Okada, M., et al. (2005) The constitutive centromere component CENP-50 is required for recovery from spindle damage. Mol. Cell. Biol.
25, 10315–10328.
Nagai, T., Ibata, K., Park, E.S., Kubota, M., Mikoshiba, K. & Miyawaki, A. (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90.[CrossRef][Medline]
Nishihashi, A., Haraguchi, T., Hiraoka, Y., et al. (2002) CENP-I is essential for centromere function in vertebrate cells. Dev. Cell 2, 463–476.[CrossRef][Medline]
Obuse, C., Iwasaki, O., Kiyomitsu, T., Goshima, G., Toyoda, Y. & Yanagida, M. (2004a) A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1. Nat. Cell Biol. 6, 1135–1141.[CrossRef][Medline]
Obuse, C., Yang, H., Nozaki, N., Goto, S., Okazaki, T. & Yoda, K. (2004b) Proteomics analysis of the centromere complex from HeLa interphase cells: uv-Damaged DNA Binding Protein-1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently colocalized with the centromeric region in interphase. Genes Cells
9, 105–120.
Oceguera-Yanez, F., Kimura, K., Yasuda, S., et al. (2005) Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis. J. Cell Biol.
168, 221–232.
Ohzeki, J., Nakano, M., Okada, T. & Masumoto, H. (2002) CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA. J. Cell Biol.
159, 765–775.
Ota, T., Suzuki, Y., Nishikawa, T., et al. (2004) Complete sequencing and characterization of 21,243 full-length human cDNAs. Nat. Genet. 36, 40–45.[CrossRef][Medline]
Pan, H.Y., Zhang, Y.J., Wang, X.P., Deng, J.H., Zhou, F.C. & Gao, S.J. (2003) Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus. J. Virol.
77, 9758–9768.
Pidoux, A.L. & Allshire, R.C. (2004) Kinetochore and heterochromatin domains of the fission yeast centromere. Chromosome Res. 12, 521–534.[CrossRef][Medline]
Renou, J.P., Bierie, B., Miyoshi, K., et al. (2003) Identification of genes differentially expressed in mouse mammary epithelium transformed by an activated ß-catenin. Oncogene 22, 4594–4610.[CrossRef][Medline]
Shelby, R.D., Vafa, O. & Sullivan, K.F. (1997) Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites. J. Cell Biol.
136, 501–513.
Stoler, S., Keith, K.C., Curnick, K.E. & Fitzgerald-Hayes, M. (1995) A mutation in CSE4, an essential gene encoding a novel chromatin-associated protein in yeast, causes chromosome nondisjunction and cell cycle arrest at mitosis. Genes Dev.
9, 573–586.
Sugata, N., Li, S., Earnshaw, W.C., et al. (2000) Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes. Hum. Mol. Genet.
9, 2919–2926.
Sullivan, K.F., Hechenberger, M. & Masri, K. (1994) Human CENP-A contains a histone H3 related histone fold domain that is required for targeting to the centromere. J. Cell Biol.
127, 581–592.
Takahashi, K., Chen, E.S. & Yanagida, M. (2000) Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast. Science
288, 2215–2219.
Tomonaga, T., Matsushita, K., Ishibashi, M, et al. (2005) Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy. Cancer Res.
65, 4683–4689.
Yasuda, S., Oceguera-Yanez, F., Kato, T., et al. (2004) Cdc43 and mDia3 regulate microtubule attachment to kinetochores. Nature 428, 767–771.[CrossRef][Medline]
Yoda, K. & Ando, S. (2004) Immunological analysis and purification of centromere complex. Methods Enzymol. 375, 270–277.[Medline]
Yoda, K., Ando, S., Morishita, S., et al. (2000) Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc. Natl. Acad. Sci. USA
97, 7266–7271.
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