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Specific Interaction between Human Kinetochore Protein CENP-C and a Nucleolar Transcriptional Regulator*

  • Ann F. Pluta
    Correspondence
    To whom correspondence should be addressed: Dept. of Dermatology, Ross Bldg., Rm. 771, Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-550-5032; Fax: 410-955-0520;
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  • William C. Earnshaw
    Footnotes
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  • Author Footnotes
    * This work was supported by an Arthritis Investigator award (to A. F. P.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Present address: Inst. of Cell and Molecular Biology, University of Edinburgh, Michael Swann Bldg., Mayfield Rd., Edinburgh EH9 3JR, Scotland.
Open AccessPublished:August 02, 1996DOI:https://doi.org/10.1074/jbc.271.31.18767
      CENP-C is a human kinetochore protein that was originally identified as a chromosomal autoantigen in patients with scleroderma spectrum disease. To begin to establish a comprehensive protein map of the human centromere, affinity chromatography was used to identify nuclear proteins that specifically interact with CENP-C. Whereas a number of polypeptides appeared to interact with the full-length CENP-C protein, only a pair of similarly sized proteins of ~100 kDa specifically interacted with the isolated carboxyl-terminal third of the CENP-C protein. Neither protein of the doublet bound to control affinity columns. Affinity purification and microsequence analysis of the proteins in the doublet identified them as the two highly related nucleolar transcription factors, UBF1 and UBF2 (also known as the nucleolar autoantigen NOR-90). Immunoblot analysis confirmed that both proteins also interacted with the full-length CENP-C polypeptide with similar affinities. Double indirect immunofluorescence using monospecific antibodies demonstrated that a subset of CENP-C and UBF/NOR-90 is colocalized at nucleoli of interphase HeLa cells, suggesting that the in vitro interaction detected by affinity chromatography may reflect an interaction that occurs in vivo. We discuss the implications of these findings in terms of the properties of interphase centromeres and the role of the nucleolus in scleroderma autoimmunity.

      INTRODUCTION

      The genetic material of eukaryotes is packaged into chromosomes through its interactions with both histones and non-histone chromosomal proteins. Each of the histones is present in vast quantities in the cell and, as a component of nucleosomes, is responsible for the most basic level of chromatin organization. In contrast, individual non-histone chromosomal proteins are considerably less abundant and have more specialized roles, including the structural and functional organization of specific chromosomal elements. One such element is the centromere, the chromosomal region responsible for the precise and accurate segregation of the genetic material during mitosis and meiosis. The centromere directs the segregation of mitotic chromosomes through a differentiated trilaminar structure called the kinetochore, which serves as the binding site for spindle microtubules and for the mechanochemical motors that move chromosomes along those microtubules. The centromere also regulates the separation of sister chromatids at the metaphase-anaphase transition.
      A complete understanding of how the centromere coordinates these important functions requires comprehensive knowledge of the proteins involved. The identification of the protein components of the human centromere initially seemed a daunting task until it was discovered that patients with scleroderma spectrum disease produce circulating autoantibodies that recognize several centromeric polypeptides (
      • Brenner S.
      • Pepper D.
      • Berns M.W.
      • Tan E.
      • Brinkley B.R.
      ,
      • Moroi Y.
      • Hartman A.L.
      • Nakane P.K.
      • Tan E.M.
      ). cDNAs encoding three ntromere roteins, CENP-A, -B, and -C, have now been cloned, and the proteins have been studied in detail. CENP-A is a novel centromere-specific core histone related to histone H3 (
      • Sullivan K.F.
      • Hechenberger M.
      • Masri K.
      ). CENP-B is an α-satellite DNA-binding protein that is localized throughout the centromeric heterochromatin located beneath the kinetochore (
      • Matsumoto H.
      • Masukata H.
      • Muro Y.
      • Nozaki N.
      • Okazaki T.
      ,
      • Earnshaw W.C.
      • Sullivan K.F.
      • Machlin P.S.
      • Cooke C.A.
      • Kaiser D.A.
      • Pollard T.D.
      • Rothfield N.F.
      • Cleveland D.W.
      ,
      • Cooke C.A.
      • Bernat R.L.
      • Earnshaw W.C.
      ). CENP-C is also a DNA-binding protein and is located at the interface between the centromeric heterochromatin and the innermost region of the kinetochore (
      • Saitoh H.
      • Tomkiel J.
      • Cooke C.A.
      • Ratrie III, H.
      • Maurer M.
      • Rothfield N.F.
      • Earnshaw W.C.
      ).
      Despite these advances, several obstacles have hindered the identification of other proteins that compose the human centromere and kinetochore. First, centromere proteins are very minor constituents in the cell. For example, it has been estimated that CENP-B is present in 20,000-50,000 copies/cell (
      • Muro Y.
      • Matsumoto H.
      • Okazaki T.
      • Ohashi M.
      ,
      • Bernat R.L.
      ). Second, like many structural proteins, centromere proteins are relatively insoluble, a property that complicates their isolation and characterization. Finally, there are no good functional assays available to screen for mammalian centromere proteins, nor are there any widely applicable methods for directly purifying these chromosomal structures.
      Despite these difficulties, two different approaches have identified a handful of new centromere proteins. One of these involves the visual/empirical survey of known proteins suspected of being involved in centromere function using monospecific antibody probes and immunoelectron microscopy of mitotic cells (
      • Mitchison T.J.
      • Kirschner M.W.
      ,
      • Wordeman L.
      • Steuer E.R.
      • Sheetz M.P.
      • Mitchison T.
      ). In the other approach, centromere proteins are identified by generating monoclonal antibody probes to chromosomal protein fractions enriched in known centromeric autoantigens. This type of experiment has now been done using human, chicken, and Xenopus cells as starting material and has resulted in the identification of CENP-E, a kinesin-related microtubule-binding protein (
      • Yen T.J.
      • Compton D.A.
      • Wise D.
      • Zinkowski R.P.
      • Brinkley B.R.
      • Earnshaw W.C.
      • Cleveland D.W.
      ,
      • Yen T.J.
      • Li G.
      • Scharr B.T.
      • Szilak I.
      • Cleveland D.W.
      ); the INCENP proteins (ner tromere roteins), chromosomal passenger proteins (
      • Earnshaw W.C.
      • Cooke C.A.
      ,
      • Cooke C.A.
      • Heck M.M.S.
      • Earnshaw W.C.
      ); and the 3F3/2 kinetochore-localized phosphoepitopes that may be involved in cell cycle signaling (
      • Cyert M.S.
      • Scherson T.
      • Kirschner M.W.
      ,
      • Gorbsky G.J.
      • Ricketts W.A.
      ). In addition, routine screening of patient autoimmune sera continues to occasionally yield a novel centromere-associated autoantigen (e.g. CENP-F) (
      • Casiano C.A.
      • Landberg G.
      • Ochs R.L.
      • Tan E.M.
      ,
      • Rattner J.B.
      • Rao A.
      • Fritzler M.J.
      • Valencia D.W.
      • Yen T.J.
      ). These approaches are unlikely to have identified all (or even a majority) of the protein components of the human centromere and kinetochore. Furthermore, none of these approaches begins to characterize the protein-protein interactions that functionally and structurally define the centromere and kinetochore.
      Mounting evidence suggests that the 140-kDa human centromeric autoantigen, CENP-C, plays a pivotal role in centromere structure and function. First, the demonstration that CENP-C is found only at the active centromere of a stable dicentric chromosome, while CENP-B is found at both the active and inactive centromeres, suggests a direct role for CENP-C in centromere function (
      • Earnshaw W.C.
      • Ratrie H.
      • Stetten G.
      ). Second, CENP-C has been immunolocalized to the inner plate of the kinetochore, a structure intimately involved in the attachment of chromosomes to the mitotic spindle (
      • Saitoh H.
      • Tomkiel J.
      • Cooke C.A.
      • Ratrie III, H.
      • Maurer M.
      • Rothfield N.F.
      • Earnshaw W.C.
      ). Third, evidence from antibody microinjection experiments indicates that CENP-C is involved in the assembly of a structurally sound, morphologically normal kinetochore and is required for the cell's timely transition into anaphase (
      • Tomkiel J.
      • Cooke C.A.
      • Saitoh H.
      • Bernat R.L.
      • Earnshaw W.C.
      ).
      We have developed a biochemical approach to identify chromosomal proteins involved in centromere function. By exploiting CENP-C as an affinity handle to dissect protein interactions at the centromere, we have identified a specific interaction with two protein components of the nucleolus. Our results are consistent with known properties of interphase centromeres and the observed involvement of the nucleolus in the autoimmune response in scleroderma spectrum disease.

      DISCUSSION

      We developed a biochemical approach to identify proteins that interact with the human kinetochore protein CENP-C and have found that two related nucleolar proteins specifically bind affinity columns coupled with CENP-C via residues at its carboxyl terminus. Several facts suggest that the interaction we have detected between CENP-C and UBF/NOR-90 by protein affinity chromatography is significant. First, the interaction of UBF/NOR-90 with the CENP-C carboxyl terminus was highly reproducible and allowed the affinity purification of sufficient quantities of the proteins from nuclear extracts for direct microsequence analysis. Second, we have demonstrated that CENP-C and UBF show limited colocalization in interphase nuclei. Finally, it has recently been shown that transient expression of CENP-C amino acid residues 638-829 (which do not contain the centromere targeting signal) in HeLa cells results in a protein that specifically accumulates in nucleoli (
      • Yang C.H.
      • Tomkiel J.
      • Saitoh H.
      • Johnson D.H.
      • Earnshaw W.C.
      ). Thus, the potential for the carboxyl-terminal region of CENP-C to interact with nucleoli in vivo appears to be regulated in the context of the whole protein.
      Both CENP-C and UBF have previously been functionally characterized. CENP-C is a basic protein capable of binding DNA in vitro (
      • Saitoh H.
      • Tomkiel J.
      • Cooke C.A.
      • Ratrie III, H.
      • Maurer M.
      • Rothfield N.F.
      • Earnshaw W.C.
      ). The DNA-binding domain of CENP-C has been localized to a short region (~116 amino acid) located in the center of the protein (residues 422-537) (
      • Yang C.H.
      • Tomkiel J.
      • Saitoh H.
      • Johnson D.H.
      • Earnshaw W.C.
      ). It is not yet known whether CENP-C binds a specific DNA sequence. The DNA-binding region overlaps with a domain (residues 478-537) that is necessary and sufficient to target CENP-C to centromeres in transfected HeLa cells (
      • Yang C.H.
      • Tomkiel J.
      • Saitoh H.
      • Johnson D.H.
      • Earnshaw W.C.
      ). No function has yet been identified for the carboxyl-terminal third of CENP-C (residues 635-943), which we have shown here to interact with UBF/NOR-90. However, several observations suggest that the carboxyl terminus of CENP-C is functionally important. First, the carboxyl terminus of CENP-C is the most highly conserved region of the protein between humans and mice (
      • Meluh P.B.
      • Koshland D.
      ). Second, this part of CENP-C shares two regions of amino acid similarity (40% identity over 27 amino acids and 28% identity over 52 amino acids) with an essential budding yeast protein, Mif2p (
      • Meluh P.B.
      • Koshland D.
      ,
      • Brown M.T.
      ). Properties of MIF2 mutants are consistent with Mif2p being a centromere protein (
      • Meluh P.B.
      • Koshland D.
      ). Interestingly, two mutant alleles of MIF2 that display defects in chromosome segregation and genetically interact with mutants in other yeast centromere proteins have base pair changes that lead to amino acid substitutions in the region of high CENP-C homology. Thus, if this region of CENP-C/MIF2 is functionally conserved, one of those functions may involve interactions with other proteins, including UBF/NOR-90. In this regard, it is noteworthy that one of the centromere proteins with which mutant Mif2p interacts genetically is Cpf1p (
      • Meluh P.B.
      • Koshland D.
      ), which, in addition to binding the centromere DNA element CDEI, acts (like UBF/NOR-90) as a transcription factor at other loci in the yeast genome (
      • Cai M.
      • Davis R.W.
      ,
      • Bram R.J.
      • Kornberg R.D.
      ,
      • Mellor J.
      • Jiang W.
      • Funk M.
      • Rathjan J.
      • Barnes C.A.
      • Hinz T.
      • Hegemann J.H.
      • Philippsen P.
      ).
      UBF1 is a highly charged protein that binds DNA sequences in the ribosomal RNA promoter via four high mobility group box motifs and activates transcription by interacting with other factors, including SL1 and RNA polymerase I (
      • Jantzen H.-M.
      • Admon A.
      • Bell S.P.
      • Tjian R.
      ,
      • Bell S.P.
      • Learned R.M.
      • Jantzen H.-M.
      • Tjian R.
      ). UBF1 contains a dimerization domain at its amino terminus and an acidic carboxyl-terminal tail composed of two uninterrupted tracts of glutamic and aspartic acid residues that are 21 and 18 amino acids long (
      • Jantzen H.-M.
      • Chow A.M.
      • King D.S.
      • Tjian R.
      ). At present, we do not know whether UBF/NOR-90 interacts with CENP-C directly or indirectly. Our inability to demonstrate a direct interaction using partially purified GST-C635-943 and UBF may reflect, in part, our lack of success in accurately replicating the conditions that permitted binding in the affinity chromatography experiments. For example, unknown post-translational modifications of UBF, not present on in vitro translated UBF, may be required for interactions with CENP-B. Additionally, while the functional significance of the naturally occurring deletion variant NOR-90 is not clear, it, as well as UBF, may be important for CENP-C interactions. Alternatively, these results may suggest that CENP-C and UBF/NOR-90 do not interact directly, but rather require an additional protein(s) not detectable on our silver-stained gels. If the interaction of UBF/NOR-90 with CENP-C is direct, we suspect that binding is unlikely to involve nonspecific interactions with the UBF1 acidic tail. Nuclei contain many polypeptides with acidic regions that rival or exceed that of UBF/NOR-90 (
      • Earnshaw W.C.
      ). However, only the UBF/NOR-90 doublet was observed to bind to the affinity column baited with the carboxyl-terminal third of CENP-C. Furthermore, we have failed to detect any interaction between CENP-C and the acidic centromere protein CENP-B (
      • Earnshaw W.C.
      • Sullivan K.F.
      • Machlin P.S.
      • Cooke C.A.
      • Kaiser D.A.
      • Pollard T.D.
      • Rothfield N.F.
      • Cleveland D.W.
      ) either by affinity chromatography or in the yeast two-hybrid interaction assay.3
      CENP-C is not the first centromere protein that has been found to interact with a non-centromere protein. The centromere-associated human autoantigen CENP-F was also cloned under another name (mitosin) based on its ability to bind a portion of the retinoblastoma protein in vitro (
      • Zhu X.
      • Mancini M.A.
      • Chang K.-H.
      • Liu C.-Y.
      • Chen C.-F.
      • Shan B.
      • Jones D.
      • Yang-Feng T.L.
      • Lee W.-H.
      ). The biological significance of this interaction remains unclear, and the function of CENP-F is unknown. In addition to CENP-F, the retinoblastoma protein has also been found to interact with UBF (
      • Shan B.
      • Zhu X.
      • Chen P.-L.
      • Durfee T.
      • Yang Y.
      • Sharp D.
      • Lee W.-H.
      ,
      • Cavanaugh A.H.
      • Hempel W.H.
      • Taylor L.J.
      • Rogalsky V.
      • Todorov G.
      • Rothblum L.I.
      ), providing an indirect link between a centromere protein and a nucleolar transcription factor. Here we demonstrate a direct biochemical link between CENP-C and UBF.
      Another intriguing recent result also strengthens the link between centromeres and nucleoli. A novel nucleolar protein from rat liver called NAP57 was found to co-immunoprecipitate with Nopp140, a shuttling nucleolar phosphoprotein (
      • Meier U.T.
      • Blobel G.
      ). NAP57 shows striking similarity (71% identity and 85% homology) over ~82% of its length to the product of the essential budding yeast gene CBF5. Cbf5p is a low affinity centromere-binding protein that interacts genetically with CBF3, a multiprotein, high affinity centromere-binding complex in Saccharomyces cerevisiae (
      • Jiang W.
      • Middleton K.
      • Yoon H.-J.
      • Fouquet C.
      • Carbon J.
      ). Interestingly, Cbf5p is reported to localize to the nucleolus in yeast (
      • Meier U.T.
      • Blobel G.
      ). It has been proposed that NAP57 and its highly conserved homologs may serve as chaperones for newly made ribosomal components (
      • Meier U.T.
      • Blobel G.
      ).
      Compelling evidence for an intimate relationship between interphase centromeres and nucleoli comes from studies using isolated human nucleoli (
      • Ochs R.L.
      • Press R.I.
      ). Immunofluorescence and immunoelectron microscopy using anti-centromere antibodies from scleroderma patients unambiguously placed centromeric autoantigens within the chromatin surrounding and, at times, embedded in isolated nucleoli. In addition, specific centromere proteins could be identified in immunoblots of protein extracts from isolated nucleoli using human autoimmune sera. A 140-kDa protein recognized by such sera in a nucleolar extract was enriched relative to an equivalent protein load of nuclear extract (see Fig. 7 in Ref.
      • Ochs R.L.
      • Press R.I.
      ). The speculation that this protein is CENP-C appears reasonable considering that CENP-C is the only autoantigen of this size known to be recognized by human anti-centromere antibodies. The results from this study suggest that the presence of CENP-C in isolated nucleoli may be mediated by specific interactions with the nucleolar transcription factor UBF/NOR-90.
      The finding that centromere proteins can be localized ultrastructurally and biochemically to nucleoli is relevant for considering mechanisms of autoimmunity. The presence of circulating autoantibodies that are diverse, yet highly specific and unique for a particular disease is the hallmark of many human autoimmune disorders. This has led to the hypothesis that the immune response in systemic autoimmunity is antigen-driven, a mechanism most easily understood if the antigens in question reside together at some point in a common subcellular particle that serves as the dominant autoantigen in the disease (
      • Tan E.M.
      • Chan E.K.L.
      • Sullivan K.F.
      • Rubin R.L.
      ). The antigens recognized by the immune system in scleroderma spectrum disease all reside, at least transiently, in the nucleolus (
      • Ochs R.L.
      • Press R.I.
      ,
      • Pollard K.M.
      • Reimer G.
      • Tan E.M.
      ). Our demonstration that CENP-C physically interacts either directly or indirectly with UBF/NOR-90 provides a possible biochemical link between the anti-centromere and anti-nucleolar antibodies that characterize scleroderma spectrum disease.
      The biological significance of a biochemical interaction between a kinetochore protein and a nucleolar protein is unclear. It is possible that in vivo such an interaction could serve to regulate the function of CENP-C during interphase by sequestering the protein and its bound centromeric DNA, UBF/NOR-90, or both. Alternatively, the association of CENP-C with nucleoli could play an architectural role in the organization of the interphase nucleus. In any case, the observation that only a subset of interphase centromeres is juxtaposed with nucleoli at any point during interphase suggests that such an interaction must be subject to control mechanisms that are not currently understood. The theme of biochemical associations between centromeres and nucleoli promises to lead to interesting insights into novel aspects of centromere function during interphase.

      Acknowledgments

      We thank D. Thrower for the generous supply of HeLa nuclei, T. Formosa for advice about affinity chromatography, H. Saitoh for the construction of pTCATG, D. Hernandez-Verdun for the gift of S14 serum, and H. Beckmann and R. Tjian for affinity-purified anti-UBF antibodies and UBF protein. We are grateful to I. Goldberg and Drs. A. Takahashi, C. Yang, and M. Monteiro for critical comments on the manuscript. A. F. P. thanks Drs. A. Rosen, L. Casciola-Rosen, and G. Anhalt for generosity and support during the completion of this work.

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