A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1

Defects in kinetochore proteins often lead to aneuploidy and cancer. Mis12−Mtw1 is a conserved, essential kinetochore protein family. Here, we show that a Mis12 core complex exists in Schizosaccharomyces pombe and human cells. Nine polypeptides bind to human hMis12; two of these, HEC1 and Zwint-1, are authentic kinetochore proteins. Four other human proteins of unknown function (c20orf172, DC8, PMF1 and KIAA1570) correspond to yeast Mis12−Mtw1 complex components and are shown to be required for chromosome segregation in HeLa cells using RNA interference (RNAi). Surprisingly, hMis12 also forms a stable complex with the centromeric heterochromatin components HP1 and HP1. Double HP1 RNAi abolishes kinetochore localization of hMis12 and DC8. Therefore, centromeric HP1 may be the base to anchor the hMis12 core complex that is enriched with coiled coils and extends to outer Zwint-1 during mitosis.

Figure 1. S. pombe proteins that interact with spMis12. (a) Top, extracts of S. pombe cells expressing spMis12−HAhis6 and Mis13−Myc were immunoprecipitated with an anti-Myc antibody, and precipitates were immunoblotted with antibodies against HA and Myc. Bottom, extracts of S. pombe expressing Spc7−Myc were immunoprecipitated with anti-Myc antibodies. Precipitates were immunoblotted with anti-Myc and anti-spMis12 antibodies. IP, immunoprecipitation. (b) A ChIP experiment was performed to determine whether Mis13−Myc and Spc7−Myc associate with the centromeric DNA. The central centromeric cnt1 and imr1 and the outer repetitive centromeric dg DNAs, and the peri-centric arm sequence lys1 were all used as probes. Extracts of S. pombe cells containing the integrated Mis13−Myc and Spc7−Myc were immunoprecipitated with anti-Myc antibodies. PCR amplification was performed for DNAs co-immunoprecipitated with anti-Myc antibodies. Only the central centromeric probes cnt1 and imr1 were amplified. WCE, whole-cell extract. (c) The cellular defective phenotype of mis13-1 at 36 °C was highlighted by DAPI staining and the Cen1 (lys1)−GFP method34. Large and small daughter nuclei (stained with DAPI) revealed 1:1 or 2:0 segregation of CEN1 (lys1) signals. Scale bar represents 10 m. (d) Schematic representation of the genetic interactions observed. The arrow indicates multicopy suppression, whereas the grey broken lines show synthetic lethality. Full Figure and legend (92K)

A critical question that was then addressed was whether higher eukaryotes possess similar kinetochore proteins, as human databases have so far failed to reveal obvious homologues except for hMis12 (ref. 1). Hence, we undertook to identify human proteins bound to hMis12 by immunoprecipitation and subsequent mass spectrometry. A HeLa cell line that stably expresses GFP−hMis12 was created, and nuclear chromatin was extracted with 0.5 M NaCl. The resulting soluble fraction was immunoprecipitated with anti-GFP antibodies, and immunocomplexes were resolved on an SDS−PAGE gel and analysed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after tryptic digestion of gel slices. Nine polypeptides, which included GFP−hMis12, were obtained (Fig. 2a). Two of these, Hec1/Kntc2 (refs 13,14) and Zwint-1 (refs 15, 16), are authentic kinetochore proteins. Zwint-1 contains an extended coiled-coil motif, interacts with ZW10 (ref. 17) and localizes to an outer-plate kinetochore during mitosis. Hec1/Kntc2 is similar to S. cerevisiae Ndc80 and is required for recruiting the checkpoint proteins Mad1 and Mad2. Heterochromatin proteins HP1 and HP1 are components of centrometric heterochromatin^{3, 18, 19, 20}, coexisting with CENP-B and interacting with INCENP, a centromeric heterochromatin protein bound to aurora kinase B^{21, 22, 23}. These results were rather unexpected, as hMis12 was located in the inner plate like CENP-A during mitosis¹.

Figure 2. Human hMis12-binding proteins are located in the kinetochore or heterochromatin. (a and e) Extracts of HeLa cells that stably express GFP−hMis12 (a) or c20orf172−GFP (e) were obtained by washing with Triton X-100 and extracting with 0.5 M NaCl, followed by immunoprecipitation using agarose beads coupled to an anti-GFP antibody. As a control, HeLa cells stably expressing GFP−hMis18 were used. Tryptic digests of peptides from gel slices were subjected to mass spectrometric analysis. Proteins specifically co-purified with GFP−hMis12 and c20orf172−GFP are shown in a and e, respectively. Table columns: gene names of proteins; molecular mass of gel slice in which the protein found; molecular mass from the polypeptide sequences; probability scores from the Mascot search33; number of matched peptides; and yeast corresponding homologues. (b−d) Paraformaldehyde-fixed HeLa cell lines that expressed GFP-tagged c20orf172 (b), DC8 (c) and Zwint-1 (d) were stained with anti-hMis12 antibodies and Hoechst 33342. Green, GFP; red, hMis12; and blue, DNA. Scale bar represents 10 m. Full Figure and legend (100K)

Four other proteins (KIAA1570, c20orf172, DC8 and PMF1) have not been studied so far in the context of kinetochore function. We established an additional three HeLa cell lines that stably express GFP-tagged c20orf172, DC8 or Zwint-1 to investigate their localization. Immunofluorescence showed that c20orf172-GFP, GFP-DC8 and GFP-Zwint-1 all localized to the kinetochore (Fig. 2b−d; also see Supplementary Information, Fig. S2a,b). Polyclonal antibodies that detect endogenous c20orf172 were raised and showed kinetochore signals identical to c20orf172−GFP (data not shown). GFP−Zwint-1 was present in kinetochores only during mitosis, whereas localization of KIAA1570 and PMF1 was not determined. To ensure that these identified proteins were not contaminants, reciprocal immunoprecipitation was performed using the cell line that stably expresses c20orf172−GFP. LC/MS/MS analysis of the immunocomplexes produced with the anti-GFP antibodies revealed six proteins, KIAA1570, c20orf172, DC8, hMis12, PMF1 and HP1, consistent with the components obtained from the cell line that stably expresses GFP−hMis12 (Fig. 2e). The discovery of these direct physical interactions suggested that these were most likely to be hMis12 complex components.

To obtain further information about direct interactions among the hMis12 complex proteins, immunoprecipitation was performed with stable cell lines. Anti-GFP antibody immunoprecipitates were examined by western blot analysis with monoclonal antibodies against GFP and HP1, and polyclonal antibodies against hMis12 and c20orf172. Untransfected HeLa cells were used as a control. Stable complex formation was found for the pairs of GFP−hMis12 and c20orf172; c20orf172−GFP and hMis12; GFP−DC8 and hMis12; and GFP-DC8 and c20orf172 (Fig. 3a). However, the amount of hMis12 or c20orf172 bound to GFP−Zwint-1 seemed to be somewhat limited. These interactions should be functionally significant because kinetochore localization of Zwint-1, DC8 and c20orf172 was abolished in hMis12 RNAi knock-down cells (see Supplementary Information, Fig. S2c).

Figure 3. Interactions of the hMis12-containing complex with HP1. (a) Immunoprecipitation of four HeLa cell lines that stably expressed GFP-tagged hMis12, c20orf172, DC8 and Zwint-1. Immunoprecipitation was performed with anti-GFP antibodies. Precipitates were immunoblotted using antibodies against GFP, hMis12, c20orf172 and HP1. I, Input; S, supernatant; P, precipitate. (b) Immunoprecipitation was performed with human 293 cells transiently transfected with pGFP−HP1 or pGFP−HP1 plasmids. Extracts were prepared 24 h after transfection. Precipitates were immunoblotted using anti-GFP, anti-hMis12 and anti-c20orf172 antibodies, and 293 cells transformed with pGFP were used as a control. (c) A HeLa cell line stably expressing hMis12 tagged with 3Flag was constructed and used for immunoprecipitation with anti-Flag antibodies that were subsequently dissociated from the immunocomplex by a 3Flag peptide. The resulting solubilized hMis12−Flag protein complex was analysed on a Superose 6 HR 10/30 size-exclusion chromatography column. Fractions were immunoblotted with anti-Flag, anti-c20orf172 and anti-HP1 antibodies. The standard molecular-weight markers used were bovine serum albumin (BSA; 66K), apoferritin (AF; 443K) and thyroglobulin (TG; 669K). (d) The solubilized hMis12−Flag protein complex was analysed on a 10−30% sucrose gradient. Fractions were immunoblotted with anti-Flag, anti-c20orf172 and anti-HP1 antibodies, and gradient calibration was performed with carbonic anhydrase (2.8 S), bovine serum albumin (4.3 S), alcohol dehydrogenase (7.4 S) and apoferritin (17.6 S). (e) A summary of protein−protein associations detected by LC/MS/MS (black arrows) and immunoprecipitation (red arrows). (f) A schematic representation of functionally related human, S. pombe and S. cerevisiae proteins described in this study. The scale to the right, running from outer to inner, corresponds to the terminology in human kinetochores. Red proteins, those that were identified here as being Mis12-interacting; grey proteins, those that have been suggested to be related to S. cerevisiae proteins, but were not identified here as being Mis12-interacting; black proteins, proteins reported to bind to Mtw1 in S. cerevisiae5, 7, 8, 9, 10, 11. Full Figure and legend (101K)

There was also a physical association of HP1 with hMis12 and DC8, and also with c20orf172, but not with Zwint-1 (Fig. 3a; bottom). To confirm the interaction of HP1 with these kinetochore proteins, extracts from human 293 cells transformed with the expression plasmids pGFP−HP1 or pGFP−HP1 were immunoprecipitated with anti-GFP antibodies. Both HP1 and HP1 co-immunoprecipitated with hMis12 and c20orf172 (Fig. 3b). HP1 was not found in the hMis12 complex, the reason of which was unclear. Therefore, HP1 and HP1 might be distinct in function from HP1²⁴.

To determine the size of the hMis12 complex, exclusion chromatography with Superose 6 was employed (Fig. 3c). A HeLa cell line stably expressing hMis12 tagged with a 3Flag epitope was constructed and used for immunoprecipitation with anti-Flag antibodies that were subsequently dissociated from the immunocomplex by a 3Flag peptide. The resulting solublized hMis12−Flag protein complex was eluted, and three peaks containing both hMis12−Flag and c20orf172 were found. However, HP1 was detected only in the middle peak (similar to the elution of thyroglobulin; relative molecular mass (M_r) 669,000; Fig. 3c). Sucrose gradient centrifugation of the complex showed that hMis12−Flag, c20orf172 and HP1 co-sedimented at an unexpectedly small S value (5S; Fig. 3d). As the presumed complex consisting of hMis12(3Flag)−c20orf172−DC8−PMF1−HP1 had an M_r of 142K, this suggested that the shape of the complex might be asymmetric. A summary of the interactions detected by LC/MS/MS (grey arrows) and immunoprecipitations (yellow arrows) are illustrated in Fig. 3e. These protein interactions were not mediated by interactions with DNA or RNA, as the treatment of cell lysates with excess units of benzonase — an active endonuclease towards all forms of DNA and RNA — followed by protein purification, did not affect results of the protein composition of the hMis12 immunoprecipitates (data not shown).

Figure 4. Single RNAi of c20orf172 and double RNAi of HP1 and HP1. (a) Immunoblotting and immunostaining of HeLa cells with anti-c20orf172 after c20orf172 RNAi for 24 h. Luciferase siRNA was used as a control. The level of c20orf172 greatly decreased. PSTAIR, anti-Cdc2 antibody. (b) Living cells expressing histone H2B−GFP26 were observed under a Delta-Vision microscope after c20orf172 RNAi for 30 h. Cells were greatly delayed in the metaphase. (c) After c20orf172 RNAi for 24 h, cells were fixed and stained with anti-CENP-C and anti-tubulin antibodies. Misaligned kinetochore signals were numerous in the arrested metaphase spindle. (d) Double RNAi for HP1 and HP1 was performed in cells expressing GFP−hMis12. Immunoblotting of whole-cell HeLa extracts 72 h after RNAi with anti-GFP, HP1 and PSTAIR antibodies. (e) DNA (Hoechst), CENP-A and GFP−hMis12 were observed in HeLa cells 72 h after HP1 & HP1 RNAi. Control HeLa cells 72 h after luciferase RNAi. (f) Quantification of the number of dot signals per cell for CENP-A and GFP−Mis12 in cells 72 h after RNAi. All scale bars represent 10 m. Full Figure and legend (91K)

To further examine the functional implication of interactions between HP1 and the hMis12-containing complex, HP1 RNAi of HeLa cells stably expressing GFP-hMis12 was performed. As functional redundancy was found for HP1 and HP1 by single RNAi, double RNAi of HP1 and HP1 was performed. The concentration of HP1 decreased below 10%, whereas the level of hMis12 detected by immunoblot remained constant 72 h after RNAi (Fig. 4d). The kinetochore signal of GFP−hMis12 was greatly diminished, whereas the signal of CENP-A was not affected at all (Fig. 4e, f). In Drosophila melanogaster, the loss of HP1 does not affect the concentration of CENP-A²⁷. HP1 should not affect kinetochore formation through CENP-A. As observed for hMis12, the kinetochore signals of DC8 were also abolished in double HP1 RNAi cells (see Supplementary Information, Fig. S5). Micronuclei, indicative of aberrant segregation in mitotic cells, were frequently seen in double RNAi cells (HP1 RNAi, 34.2%, n = 226; luciferase RNAi, 4.4%, n = 228; Fig. 4e; also see Supplementary Information, Fig. S5). These results strongly suggest that HP1 and HP1 are implicated in the formation of a functional kinetochore through interaction with hMis12 complex proteins.

The S. pombe mutants used in this study were described previously^{4, 12, 28, 29}. To tag the carboxyl terminus with 8Myc or GFP, the termination codon of the mis13⁺ and spc7⁺ genes were changed to the NotI restriction site to incorporate 8Myc or GFP, or CFP and the polyA terminal sequences of the nmt1⁺ gene. Complete YPD and minimal EMM2 media were used for the cultures of S. pombe. HeLa cells were grown at 37 °C in DME supplemented with 10% FCS, 1% penicillin−streptomycin and 1% antibiotic−antimycotic. All reagents were purchased from Invitrogen (Carlsbad, CA).

The genes for KIAA1570, c20orf172, Zwint-1, DC8, HP1 and HP1 were cloned by PCR using a HeLa cDNA library as template. The plasmids, pGFP−Zwint-1, pGFP−DC8, pGFP−HP1, and pGFP−HP1, which expressed proteins tagged with GFP at their amino termini were constructed as described¹. The c20orf172−GFP plasmid contains the coding region of c20orf172 with 20 bp of the upstream non-coding region and the C-terminal fusion to GFP (derived from pEGFP-N1) placed under the control of the CMV promoter of pcDNA3-11 (Invitrogen). The coding region of hMis12 was inserted into p3Flag−CMV-10 (Sigma, St Louis, MO) and the resulting p3Flag−hMis12 plasmid expresses hMis12 tagged with Flag at its amino terminus. To construct HeLa cell lines that stably express GFP-tagged hMis12, Zwint-1, DC8 or c20orf172, plasmids and the Flag-tagged hMis12 plasmid described above were used for transfection of HeLa cells with FuGENE 6 (Roche, Basel, Switzerland). HeLa cells that could grow in the presence of Geneticin were isolated. Zwint-1, clone 9; hMis12, clone 20; DC8, clone 23; and c20orf172, clone 7 (which are all GFP-tagged proteins) and hMis12, clone 3 (which is Flag-tagged) were all selected. Their expression levels were similar or several-fold higher than that of the endogenous protein.

Immunoblots were performed with the following antibodies: anti-GFP (7.1 and 13.1; Roche), anti-Flag (M2; Sigma), anti-tubulin (DM1A; Sigma), anti-HP1 (Mab3446; Chemicon, Temecula, CA) and anti-CENP-C (a gift from K. Yoda). The polyclonal antibodies against c20orf172 used in this study were made using New Zealand White rabbits immunized with a recombinant c20orf172, the N-terminus of which was fused with GST.

All the fractionation and extraction procedures using cytoskeleton (CSK) buffer (100 mM NaCl, 10 mM PIPES at pH 7.0, 300 mM sucrose, 0.1% Triton X-100, 1 mM MgCl₂, 1 mM EGTA, 0.1 mM phenylmethylsulphonyl fluoride and 2 g ml^-1 leupeptin) were performed as described³¹. The 0.5 M NaCl extracts were obtained using CSK containing 0.5 M NaCl and incubated with beads coupled to anti-GFP (clone RQ2; MBL, Nagaya, Japan) or anti-Flag (M2; Sigma) antibodies for 2 h. The beads were then washed twice with CSK containing 0.5 M NaCl. Eluates were obtained by incubating with 0.1 M glycine at pH 2.0 (for the anti-GFP antibody) or 0.2 mg ml^-1 3Flag peptide (Sigma; for anti-Flag antibody). Human 293 cell extracts were directly extracted with CSK containing 0.5 M NaCl after washing with PBS.

Annealed siRNAs specific for hMis12 (ref. 1), c20orf172 (5'-GGCGUUUCAGAGGAAAGAATT-3'), HP1 (5'-CCUGAGAAAAACUUGGAUUTT-3'), HP1 (5'-UAUUUCCUGAAGUGGAAGGTT-3') and luciferase³¹ were transfected into the indicated cell line with Oligofectamine (Invitrogen) as described¹. Cells were fixed and observed by microscopy at the indicated times after transfection.

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Competing interests statement:  The authors declare that they have no competing financial interests.