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1 Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan
2 Bioscience and Biotechnology Center, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
3 Kanagawa Dental College, Inaoka-cho 82, Yokosuka, Kanagawa 238-8580, Japan
4 Division of Biological Science Graduate School of Science Nagoya University Chikusa-ku, Nagoya 464-8601, Japan
5 Fujita Health University Institute for Comprehensive Medical Science, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan
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Abstract |
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Introduction |
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-satellite arrays of centromeres on which CENP-A, CENP-B and CENP-C are assembled (Ando et al. 2002). In this paper, we report the isolation of the CENP-A chromatin complex, the so-called ‘CEN-complex’, and the results of a proteomics approach using tandem mass spectrometry equipped with liquid chromatography (LC/MS/MS) to identify components of the CEN-complex. About 40 different gene products are detected as candidates for components of the CEN-complex. In addition to the previously reported centromere proteins, CENP-A, CENP-B, CENP-C, CENP-H, and CENP-I/hMis 6, we have identified many kinds of proteins of unknown function, and a series of proteins that have been reported not as centromeric proteins but as proteins related to chromatin dynamics (uvDDB-1, XAP8, SNF2H, FACTp140 and FACTp80/SSRP1), polycomb group (PcG) proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), a motor protein (KNL5) and a regulator of G-protein (racGAP). Our results may provide new insights into the structure and function of the centromere complex, which may contain components of various protein networks.
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Results |
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In order to identify the protein components of the centromere complex, CENP-A chromatin was isolated by ChIP using anti-CENP-A monoclonal antibody covalently linked to Sepharose beads (Fig. 1A). We have previously reported that micrococcal nuclease (MNase) digestion of interphase HeLa nuclei in 0.3 M NaCl released CENP-A chromatin into the soluble fraction in the same way as bulk chromatin (Ando et al. 2002). Although extensive DNA cleavage increases the solubility of CENP-A chromatin by up to 70%, it also disrupts the CENP-A/B/C complex, and therefore extensive DNA cleavage reduces recovery of the CENP-A/B/C complex to a lower level than that found after weaker MNase digestion (Ando et al. 2002). To recover the maximum amount of the CENP-A/B/C complex we stopped MNase digestion of HeLa nuclei at the point when approximately 30–50% of the CENP-A chromatin had been released into the soluble fraction. The CENP-A chromatin was isolated by immunoprecipitation using anti-CENP-A beads and eluted with 8 M urea (Fig. 1A). The quantity of DNA fragments in the purified CENP-A chromatin comprised 
0.2% of the bulk chromatin DNA (45 mg), and the sizes of the fragments were 0.15–6.0 kbp (Fig. 1B), which corresponded to 1-30-mer nucleosomes. Approximately 70% of the DNA fragments (52 of 78 cloned fragments) were I-type -satellite sequences, which implies that approximately 70% of the isolated chromatin is derived from the CENP-A chromatin. This is consistent with our argument that CENP-A nucleosomes are selectively formed on I-type satellite arrays on HeLa chromosomes (Ando et al. 2002).
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Next, we analysed the protein components of the purified CENP-A chromatin by Western blotting using various antibodies against known centromere proteins. Figure 2 A shows the quantitative analysis of the recovery of CENP-C. CENP-C was quantitatively co-precipitated with CENP-A and was depleted from the bulk chromatin fraction (Fig. 2A, lane 3) while approximately half of the CENP-B remained in the bulk chromatin fraction, as previously reported (Ando et al. 2002). The amount of the proteins in the purified CENP-A chromatin was 0.4% of the bulk chromatin proteins (see legend to Fig. 2A for details), and CENP-C in the purified CENP-A chromatin fraction was concentrated 120 times compared with bulk chromatin (Fig. 2A, lanes 1 and 2). Furthermore, CENP-H, CENP-I/hMis6 (human homologue of S. pombe Mis 6) and hMis12 were also found in the CENP-A chromatin fraction (Fig. 2B, lanes 3-5, and Fig. 4, lanes 1-4). Thus, the CENP-A chromatin fraction contains all the reported structural proteins of the centromere, suggesting that this fraction represents the whole centromere complex. Therefore we call the isolated CENP-A chromatin the ‘CEN-complex’.
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To analyse extensively the protein components, the CEN-complex isolated by the anti-CENP-A beads and the proteins bound to IgG beads (data not shown) were separated in 12.5% SDS-PAGE and stained with Coomassie Brilliant Blue (Fig. 3). The area of the separation gel corresponding to a molecular mass from about 15–300 kDa was cut at essentially 1 mm intervals (some slices were wider because of the absence of any prominent bands at those positions). All gel slices were subjected to LC/MS/MS analyses to identify proteins, as described under ‘Experimental procedures’. The proteins identified from the CEN-complex fraction but not from the IgG-bound fraction are listed in Table 1, and the proteins identified from both the CEN-complex fraction and from the IgG-bound fraction are listed as contaminant proteins in Table 2. The centromere proteins, CENP-A, CENP-B, CENP-C, CENP-H and CENP-I/hMis 6 were specifically found in the CEN-complex fraction as confirmed by Western blotting (Fig. 2). In addition to the centromere proteins we found 36 proteins specifically in the CEN-complex fraction. There are many kinds of proteins of unknown function, and a series of known proteins that have not previously been reported as centromere proteins, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, PcG proteins (BMI-1, RNF1, RNF2, PHP2 and HPC3), KNL5, and racGAP. These results suggest that many of the proteins shown in Table 1 may be newly recognized components of the CEN-complex, or proteins that interact with the CEN-complex.
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To confirm and examine the apparent molecular weight of the newly identified proteins, Western blot analyses were performed using antibodies against RNF2 (Fig. 4, lanes 5 and 6), BMI-1 (lanes 7 and 8), HPC3 (lanes 9 and 10), XAP8 (lanes 11 and 12), hSNF2H (lanes 13 and 14) and DDB-1 (lanes 15 and 16). As shown in Fig. 4, these factors were specifically found in the CEN-complex fraction as observed for the authentic centromere proteins CENP-B, -C, -H and -I (lanes 1-4). The approximate molecular weight of each protein is as follows: RNF2 appears as doublet bands with molecular weights of 40 and 41 kDa; BMI-1 as three bands of 43, 44 and 45 kDa; HPC3 has a molecular weight of 48 kDa; XAP8 is 240 kDa, SNF2H is 135 kDa, and DDB-1 is 120 kDa. The RNF2 gene codes for 337 amino acids and its expected molecular mass is 37.7 kDa (Table 1), which corresponds well with the observed data. The values for molecular mass of other proteins correspond well with those previously reported (Bardos et al. 2000; Bozhenok et al. 2002; Keeney et al. 1993; Shamay et al. 2002; Voncken et al. 1999). These results support the view that RNF2, BMI-1, HPC3, XAP8, SNF2H and DDB-1 are actually components of the CEN-complex, or are proteins that interact with the CEN-complex.
UV-DDB-1 (UV-damaged DNA-binding protein 1) is localized to the centromeric region throughout cell cycle
HeLa cells were immunolabelled with the antibody against DDB-1 (Fig. 5A). Anti-DDB-1C antibody, which recognizes a peptide within the COOH-terminal half of DDB-1, labelled both cytoplasm and nucleus in interphase, and chromosomes in metaphase (Fig. 5A panels b and c). In particular, dot-like labelling was clearly detected in the nucleus (arrowheads) and chromosomes (arrows), which seemed to be characteristic of centromere labelling. To confirm that those dot-like signals in the nucleus are actually originated from DDB-1 protein, we knocked down the protein level in the cells using two kinds of siRNA as described in Experimental procedures. As shown in Fig. 5B, the total DDB-1 protein levels in the cells detected by Western-blot analysis using anti-DDB-1C antibody were reduced at day 3 (lanes 2 and 3) and/or at day 4 (lanes 5 and 6) after transection of the siRNAs. Figure 5C showed that the dot signals in the nuclei (panels b and c for mock experiment) were reduced by transfection with the siRNA site1 (panels e and f) or disappeared in some cells with the siRNA site2 (panels h and i). These results strongly support that anti-DDB-1C actually recognized DDB-1 protein located in the nucleus of HeLa cells. HeLa cells at interphase (Fig. 5D, panels a–d), metaphase (panels e–h) and anaphase (panels i–l) were double-labelled with anti-DDB-1C (panels b, f and j) and anti-CENP-A (panels c, g and k) antibodies. The dot-like signals from anti-DDB-1C completely overlapped those from anti-CENP-A (panels d, h and l). From these results we conclude that DDB-1 is localized to the centromeric region throughout the cell cycle.
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HeLa cells were immunolabelled with antibodies against BMI-1 (Fig. 6A). Dot-like labelling was observed inside the interphase nuclei (Fig. 6A, panels b and c), but these dot-like signals disappeared at prometaphase (panels e and f) and telophase (panels h and i). To see whether these interphase-specific dot-like signals were located to the centromeric regions, HeLa cells (Fig. 6B, panels a–d) and MRC-5 cells (normal fibroblast cells; panels e–h) were double-labelled with anti-BMI-1 (panels b and f) and anti-CENP-C (panels c and g) antibodies. In both cells most of the BMI-1 dots co-localized with the CENP-C dots (panels d and h). All the CENP-C dots co-localized with BMI-1, but a few BMI-1 dots inside the nuclei (arrows in panels d and h) were not co-localized with CENP-C. In order to survey the location of all BMI-1 dots within a single nucleus, HeLa cells were observed with a confocal optical microscope. Figure 6C shows the merged figure of 20 photographs which were taken consecutively at different focal planes from top to bottom of the cells. The CENP-C signals shown in Fig. 6C, panel b, are all co-localized with those of BMI-1 (Fig. 6C, panel c). Observation by confocal microscopy revealed that a few BMI-1 signals devoid of CENP-C which were apparently located inside the nucleus (an arrow in Fig. 6C, panel c) were actually located at the surface or outside the nucleus (data not shown). We therefore conclude that these BMI-1 signals without CENP-C were of cytoplasmic origin. From these results we conclude that BMI-1 is transiently localized at the centromeric region in interphase.
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Discussion |
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70% from the content of I-type -satellite DNA in the isolated chromatin. The purified CEN-complex contained all the reported centromere structural proteins, CENP-A, CENP-B, CENP-H, CENP-I/hMis6 and hMis12. Proteomics analysis of the CEN-complex revealed approximately 40 proteins, which included the known centromere proteins, proteins of unknown function, and a series of known proteins that were not expected to be related to chromosome segregation (Table 1). By Western blotting we confirmed that the purified CEN-complex fraction contained RNF2, BMI-1, HPC3, XAP8, SNF2H and uvDDB-1 (Fig. 4). Among these proteins we focused further studies on uvDDB-1 and BMI-1 and found that uvDDB-1 was localized to the centromeric region throughout the cell cycle (Fig. 5), while BMI-1 was transiently localized to the centromere in interphase (Fig. 6). These results can be expected to give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation. Purity of the isolated CEN-complex
As the isolated nuclei from HeLa cells were once washed with 0.3 M NaCl (Fig. 1A), the proteins that detached from the chromatin under this salt concentration were removed from nuclei. Therefore MNase digestion of the nuclei will release predominantly the chromatin proteins maintaining its DNA/protein complex form, which we call the ‘bulk chromatin’. The isolated CEN-complex was estimated to occupy 0.2–0.4% of the bulk chromatin and contained DNA fragments of approximately 0.15–6.0 kbp, of which 70% were composed of I-type satellite DNA. These results suggested that 70% of the isolated chromatin was centromeric chromatin with 1–30-mer nucleosomes containing CENP-A. We previously reported that the CENP-A/B/C chromatin complex constituted a repetitive structure in the CEN-complex, reflecting the repetitive sequence of I-type satellite arrays (Ando et al. 2002). Therefore the fragmented CENP-A chromatin complex with 30-mer nucleosomes probably represents the whole of the centromeric chromatin of interphase cells, except that those proteins which detach from the centromere chromatin at 0.3 M NaCl will be lost during purification. Then where does the rest 30% chromatin with unique DNA sequences come from? Two possibilities can be speculated. The first is that the 30% chromatin may be simple contaminants adsorbed to Sepharose beads and/or constant region of mouse IgG. In order to detect these contaminants, we performed the proteomics analysis for the chromatin that adsorbed to non-immune mouse IgG-beads (data not shown). The protein spectrum was very distinctive in the points that the proteins related to RNA, such as RNA polymerase, RNA helicase, transcriptional control factor or hnRNP, were majority and occupied 60% of the adsorbed proteins. And except core histones and other minor components (Table 2), this protein spectrum was totally different from that of the centromeric chromatin (Table 1). These results suggest that the chromatin adsorbed to the IgG-beads might rather represent the chromatin from the area other than the centromere. Therefore we think that contaminant proteins may be minimal in the list presented as Table 1, because we eliminated the proteins that were also found in the IgG-bound fraction and that are listed in Table 2. The second possibility is that it may come from the chromatin that interacts with the CEN-complex. As discussed in later section, the polycomb/chromatin complex might be an example for this possibility. Thus systematic analyses of the CENP-A chromatin by LC/MS/MS offers a large number of candidate proteins which are either components of the CEN-complex or interact with it.
UvDDB-1
DDB-1 was reported to be mostly localized to the cytoplasm, and a small amount was found in the nucleus by biochemical analysis (Otrin et al. 1997). But by cytological analysis DDB-1 has been reported to be localized only to the cytoplasm (Liu et al. 2000). In this report we have found that DDB-1 localized to the centromere regions both by biochemical analysis (Fig. 4 and Table 1) and by cytological analysis (Fig. 5D). Among five different kinds of antibodies against DDB-1 two antibodies recognized the centromere regions. These two antibodies both recognized COOH-terminal region of DDB-1. We further confirmed that the dot-like signals in the nucleus were actually derived from DDB-1 protein, since knock down of the DDB-1 protein level in the cells by transfection of siRNA resulted in reduction of those signals (Fig. 5B,C). DDB-1 was first detected with DDB-2 as one of the proteins classified in xeroderma pigmentosum complementation group E (XP-E) (Keeney et al. 1993), and was extensively studied from the view point of DNA repair (as a review see Tang & Chu 2002). DDB-1/DDB-2 recognizes and binds the DNA damaged by UV radiation, but its function is still unclear (Otrin et al. 1997). After UV radiation, DDB-1 is transferred into the nucleus from the cytoplasm, and therefore it is considered to bind to damaged DNA (Liu et al. 2000; Otrin et al. 1997). But those experiments were performed in a situation of overproduction of DDB-1 protein using an exogenous gene, and it does not necessarily represent the physiological distribution of the endogenous gene product. Takata et al. (2002) argued that D-DDB-1 (Drosophila melanogaster DDB-1) had a role in cell proliferation and development on the basis of the observation that the D-DDB-1 gene was controlled by the DRE/DREF system, and the transcription of D-DDB-1 changed greatly during development of Drosophila embryos without UV radiation. Gene targeting of S. pombe DDB-1 showed that the pombe DDB-1 gene is not essential, but the phenotype of the mutant cells; aberrant nuclear structures, appearance of lagging chromosomes or enhanced sensitivity to TBZ, suggested a role for pombe DDB-1p in chromosome segregation (Zolezzi et al. 2002). The DDB-1/DDB-2 complex is a target of Cullin 4A (Chen et al. 2001), a ubiquitin ligase, and activates a transcription regulation factor, E2F, under cell-cycle regulation (Shiyanov et al. 1999). DDB-1 is associated with the STAGA complex (SPT3-TAFII31-GCM5L acetylase) (Martinez et al. 2001) and physically interacts with histone acetyltransferase subunits (Rapic-Otrin et al. 2002). Recently, it was found that DDB-1 separately interacts with DDB-2 and/or CSA forming two different complexes that commonly contain cullin 4A, Roc 1 and COP 9 signalosome (CSN) and the ubiquitin ligase activity in these two complexes is differntially regulated by the COP9 signalosome in response to DNA damage (Groisman et al. 2003). Therefore DDB-1 might play multiple roles, as a factor for DNA repair and a cofactor for transcription regulation in the context of regulation in altering chromatin structure. The amino acid sequence analysis of DDB-1 suggested that the whole DDB-1 sequence forms a tertiary repetitive structure of the ß-propeller type (Neuwald & Poleksic 2000) and the fission yeast Rik1p protein, which plays a role in gene silencing in centromeric regions, and in chromosome segregation, forms the same structure (Allshire et al. 1995). Therefore DDB-1 might take part in regulation of heterochromatin dynamics around the centromeric region.
BMI-1 and other PcG proteins, HPC3, RING1, RNF2 and PHP2
PcG proteins appear to act as an epigenetic mark on the chromatin template that maintains the silent state at specific target loci (Cavalli & Paro 1998). Although direct DNA binding of individual PcG proteins has not been demonstrated, PcG complexes can bind to DNA and establish repressive chromatin structures (Francis et al. 2001). The interactions of various sets of the PcG proteins shown in Table 1 have been reported (Bardos et al. 2000; Gunster et al. 1997; Satjin et al. 1997; Saurin et al. 1998; Voncken et al. 2003). These proteins, BMI-1, HPC3, RING1, RNF2 and PHP2, probably interact with each other to form a distinct PcG complex. The distribution of BMI-1, Ring1 (Ring1a) and Rnf2 (Ring1b) within the cells has been reported (Suzuki et al. 2002; Saurin et al. 1998; Voncken et al. 1999). Saurin et al. (1998) reported that the human polycomb complexes (Ring 1, BMI-1 and hPc2) form foci within the nucleus that are partly associated with pericentromeric heterochromatin. Voncken et al. (1999) confirmed that BMI-1 was localized to the pericentromeric region of chromosome 1, dependent on the cell cycle. We observed that BMI-1 was transiently localized at the centromeres of all chromosomes only at interphase (Fig. 6). These results suggest two possibilities. The first is that BMI-1 may constitute part of the CEN-complex, but in this case it is difficult to explain its transient localization to the centromeres. The second is that the PcG complexes, including BMI-1, may be formed at specific loci other than centromeres and only associate with the CEN-complexes at specific stages of the cell cycle. In the latter case the PcG complexes would bind specifically to chromatin, and therefore H3 nucleosomes and DNA with unique sequences may be systematically co-precipitated with the CENP-A chromatin. Brown et al. (1997) have shown using human B cells that Ikaros, together with transcriptionally inactivated target genes, was localized to centromeric heterochromatin in interphase nuclei, and proposed a model of organization of the nucleus in which repressed genes are selectively recruited into centromeric domains. In this context, localization of BMI-1 to the CEN-complex or association of the BMI-1 complex with the CEN-complex in interphase nuclei might suggest a role for the CEN-complex in the establishment of heterochromatin structure to repress gene expression and to ensure epigenetic transmission during cell differentiation.
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Experimental procedures |
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HeLa S3 cells were grown in spinner flasks and the nuclei were isolated as previously described (Ando et al. 2002).
Solubilization of bulk chromatin
The procedures were as previously described (Ando et al. 2002) with slight modifications. The isolated nuclei from 5 x 109 cells were washed once with ice cold washing buffer (WB: 20 mM HEPES pH 8.0, 20 mM KCl, 0.5 mM EDTA, 0.5 mM DTT and 0.05 mM PMSF), and then with WB containing 0.3 M NaCl to remove the proteins eluted from the chromatin at this salt concentration. The nuclei were resuspended with WB-0.3 M NaCl to a concentration of approximately 1–2 x 108 nuclei equivalent/mL, CaCl2 was added to a final concentration of 3 mM, and the nuclei were digested with 6 U/mL of MNase for 30 min at 37 °C. The reaction was stopped by the addition of EGTA to a final concentration of 5 mM and quick chilling. The digest was centrifuged with a swinging rotor at 1000 g for 10 min at 4 °C and the supernatant was centrifuged again with SS34 rotor (Beckman) at 16 000 g for 20 min at 4 °C to remove the residual pellet.
Antibodies
ACA (MI), mouse anti-CENP-A3-19 monoclonal antibody, and guinea pig anti-CENP-C antibody were previously described (Ando et al. 2002). Anti-CENP-H antibody was raised by immunizing rabbits with recombinant CENP-H expressed using the baculovirus system. Anti-CENP-I was raised by immunizing rats with the recombinant CENP-I NH2-half protein (amino acid number from NH2-terminus, #1-527). These antibodies were affinity purified using the same recombinant proteins. Anti-human-Mis12 was a rabbit antibody kindly provided by Drs Goshima and Yanagida (Goshima et al. 2003). Anti-DDB-1C (Zymed Laboratory) was a rabbit antibody raised against a peptide located within the COOH-terminal half of the human DDB-1 protein. Anti-BMI-1 was a mouse polyclonal antibody raised against a peptide from the COOH-terminal region of human BMI-1 (#310–326). The antibodies against DDB-1, RNF2, HPC3, XAP8, SNF2F, KNL5 and racGAP were raised by immunizing mice with the following peptides: RNF2, #20–37 and #320–336; HPC3, #1–19 and #376–380; XAP8, #1–13 and #1413–1431; SNF2H, #1034–1052; KNL5, #1–19 and #838–856; racGAP, #13–27 and #606–624.
Chromatin immunoprecipitation (ChIP)
The ChIP procedures were essentially as described in Ando et al. (2002), with slight modifications. The purified anti-CENP-A monoclonal IgG (5 mg) and non-immune IgG (CHEMICON; 5 mg) were chemically linked to CNBr-activated Sepharose 4B (Amersham-Pharmacia, 2 mL). The bulk chromatin solution (25-50 ml) containing 0.1% NP-40 was suspended in 2 mL of IgG-beads prewashed with WB-0.3M NaCl-0.1% NP-40, and the mixture was incubated with inversion rotation for 1 h at room temperature. The IgG-beads were removed by centrifugation at 30 g for 5 min at 4 °C, and the supernatant solution was then added to 2 mL of anti-CENP-A-beads and rotated for 12 h at 4 °C. After the reaction, the supernatant solution was removed by centrifugation at 30 g for 5 min at 4 °C. The anti-CENP-A beads and the IgG beads were applied to disposable columns (0.8 cm diameter x 14 cm) after being washed three times with WB-0.3M NaCl-0.1% NP-40 at 4 °C. Proteins adsorbed on to the IgG beads and anti-CENP-A beads were eluted with 8 M/urea2% CHAPS-18 mM/DTT00 µg/mL CENP-A3-19 peptide (a peptide of amino acid numbers #3–19 from the NH2-terminus). The eluate was fractionated into 10 fractions of 1.0 mL each. Elution of CENP-A, -B and -C was detected by Western blotting using ACA serum. Peak fractions, no. 2–4, of each column were combined and dialysed against CEN buffer (50 mM Tris-HCl pH 8, 0.1% SDS, 0.5 mM DTT, 0.1 mM PMSF, 0.5 µg/mL pepstatin A and 2 µg/mL leupeptin). The proteins were recovered by acetone precipitation and resuspended in 80–300 µL of the CEN buffer and stored at –80 °C until use.
32P-labelling and cloning of the chromatin DNA after ChIP
The DNA in 6 µL of the CEN-complex was extracted with phenol and resuspended with 20 µL of TE buffer. One microlitre of the DNA was labelled with 32P using T4 polynucleotide kinase and 
32P-ATP. For cloning of the DNA fragments, 5' protruding DNA was trimmed by incubation with T4 DNA polymerase in the presence of 4 x dXTPs at 37 °C for 10 min after removal of 5'-terminal phosphates by calf intestine phosphatase (CIP) treatment. The trimmed DNA was then cloned into the pCR 2.1-TOPO vector (Invitrogen) after addition of a 3'-dA overhang by incubation with Taq DNA polymerase at 72 °C for 10 min
Western blotting
The method for Western blotting was as described by Ando et al. (2002). 0.5–5 µL of the sample was separated by 12.5% SDS-PAGE. The separated proteins were transferred to a PVDF membrane (Millipore), and the immunoreaction was for 10–12 h at 4 °C.
Mass spectrometry
All procedures were done essentially as previously described (Ohta et al. 2002) The CEN-complex fraction and the IgG-bound fraction were separated on a 12.5% SDS-PAGE gel, and the gel area from about 15 kDa to 300 kDa (Fig. 2) was cut at about 1 mm intervals. Proteins in the gel slices were subjected to reduction, alkylation and tryptic digestion with modified trypsin. The product peptides were extracted with 5% formic acid and acetonitrile, dried in a vacuum and dissolved in 5% formic acid. Multiply digested peptides from each gel slice were separated by microcapillary C18-reverse phase chromatography (200 µm x 5 cm capillary, Michrom Bio-Resource, USA) and applied directly to an LCQ Advantage quadrupole ion-trap mass spectrometer (Finnigan, USA). The primary ion spectrum data generated by LC/MS/MS were screened against the NCBI non-redundant protein database by the MASCOT program (Matrix Science, UK) to pick up highly fitting scored proteins.
Indirect immunofluorescence microscopy
HeLa cells were cultured in poly D-lysine cell ware two-well or four-well culture slides (Beckton Dickinson) for 2–3 days at 37 °C. The cells were washed with PBS (phosphate-buffered saline; 10 mM sodium phosphate pH 7.6, 0.14 M NaCl) at room temperature, and fixed with 95% acetone (pre-cooled to –20 °C) for 30 min at –20 °C. The cells were dried by cool air, rinsed with PBS, and incubated with 0.1% skimmed milk for 30 min at 37 °C. Incubations with first and second antibodies were for 60 min at 37 °C. The cells were washed three times after each incubation with PBS. DNA was stained with DAPI. The cells were observed with a BX51 microscope (Olympus) and images were taken with a Cool SNAP monochrome (Roper Scientific Ltd) under the operating system of Openlab version 2 (Improvision Ltd).
RNAi method
siRNA(Elbashir et al. 2001) was synthesized for RNAi of DDB-1 site1 (5'-GAUUGCGGUCAUGGAGCUUTT-3'), DDB-1 site 2 (5'-CAGAGUGGCGAGAGCAUUGTT-3'), Lamin A/C (5'-CUGGACUUCCAGAAGAACATT-3'), and Lucierase (5'-CGUACGCGGAAUACUUCGATT-3') by Jbios. The procedures of cell culture and transfection were according to (Goshima et al. 2003) using Oligofectamine (Invitrogen).
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Acknowledgements |
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Footnotes |
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aPresent address: Department of Gene Mechanisms Graduate School of Biostudies Kyoto University, Kitashirakawa— Oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan
* Correspondence: Email: i45156a{at}nucc.cc.nagoya-u.ac.jp |
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Received: 23 July 2003
Accepted: 10 November 2003
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