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• W1917235046 abstract "•CENP-B binding to alphoid DNA repeats stabilizes CENP-C and kinetochore nucleation•Centromere function is enhanced by mutual dependencies of CENP-A, CENP-B, and CENP-C•The CENP-B free Y and neocentromere chromosomes mis-segregate at elevated frequencies•CENP-B binding to alphoid DNA enhances fidelity of epigenetically defined centromeres Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A’s amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes with CENP-B-containing centromeres. These data demonstrate a DNA sequence-specific enhancement by CENP-B of the fidelity of epigenetically defined human centromere function. Human centromeres are specified by a stably inherited epigenetic mark that maintains centromere position and function through a two-step mechanism relying on self-templating centromeric chromatin assembled with the histone H3 variant CENP-A, followed by CENP-A-dependent nucleation of kinetochore assembly. Nevertheless, natural human centromeres are positioned within specific megabase chromosomal regions containing α-satellite DNA repeats, which contain binding sites for the DNA sequence-specific binding protein CENP-B. We now demonstrate that CENP-B directly binds both CENP-A’s amino-terminal tail and CENP-C, a key nucleator of kinetochore assembly. DNA sequence-dependent binding of CENP-B within α-satellite repeats is required to stabilize optimal centromeric levels of CENP-C. Chromosomes bearing centromeres without bound CENP-B, including the human Y chromosome, are shown to mis-segregate in cells at rates several-fold higher than chromosomes with CENP-B-containing centromeres. These data demonstrate a DNA sequence-specific enhancement by CENP-B of the fidelity of epigenetically defined human centromere function. The centromere is the fundamental unit for ensuring chromosome inheritance. Correct centromere function is required for preventing errors in chromosome delivery that would lead to genomic instability and aneuploidy, both hallmarks of many human cancers. Although not conserved across species, centromere regions from fission yeast to man have arrays of repetitive sequences (Fukagawa and Earnshaw, 2014Fukagawa T. Earnshaw W.C. The centromere: chromatin foundation for the kinetochore machinery.Dev. Cell. 2014; 30: 496-508Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Human centromeres carry extensive (1,500 to >30,000) copies of imperfectly repeated arrays of a 171 bp element, termed α-satellite DNA. In all but the centromere of the Y chromosome (Earnshaw et al., 1987Earnshaw W.C. Sullivan K.F. Machlin P.S. Cooke C.A. Kaiser D.A. Pollard T.D. Rothfield N.F. Cleveland D.W. Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen.J. Cell Biol. 1987; 104: 817-829Crossref PubMed Scopus (331) Google Scholar, Earnshaw et al., 1989Earnshaw W.C. Ratrie 3rd, H. Stetten G. Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads.Chromosoma. 1989; 98: 1-12Crossref PubMed Scopus (257) Google Scholar, Miga et al., 2014Miga K.H. Newton Y. Jain M. Altemose N. Willard H.F. Kent W.J. Centromere reference models for human chromosomes X and Y satellite arrays.Genome Res. 2014; 24: 697-707Crossref PubMed Scopus (137) Google Scholar), this array contains a 17-base pair (bp) consensus motif that is the binding site of CENP-B, the only known mammalian centromeric DNA sequence-specific binding protein (Verdaasdonk and Bloom, 2011Verdaasdonk J.S. Bloom K. Centromeres: unique chromatin structures that drive chromosome segregation.Nat. Rev. Mol. Cell Biol. 2011; 12: 320-332Crossref PubMed Scopus (149) Google Scholar). Nevertheless, human centromere position is epigenetically specified (Ekwall, 2007Ekwall K. Epigenetic control of centromere behavior.Annu. Rev. Genet. 2007; 41: 63-81Crossref PubMed Scopus (76) Google Scholar, Karpen and Allshire, 1997Karpen G.H. Allshire R.C. The case for epigenetic effects on centromere identity and function.Trends Genet. 1997; 13: 489-496Abstract Full Text PDF PubMed Scopus (384) Google Scholar). Among the strongest evidence for an epigenetically defined centromere was the discovery of neocentromeres in humans (Amor et al., 2004Amor D.J. Bentley K. Ryan J. Perry J. Wong L. Slater H. Choo K.H. Human centromere repositioning “in progress”.Proc. Natl. Acad. Sci. USA. 2004; 101: 6542-6547Crossref PubMed Scopus (162) Google Scholar, du Sart et al., 1997du Sart D. Cancilla M.R. Earle E. Mao J.I. Saffery R. Tainton K.M. Kalitsis P. Martyn J. Barry A.E. Choo K.H. A functional neo-centromere formed through activation of a latent human centromere and consisting of non-alpha-satellite DNA.Nat. Genet. 1997; 16: 144-153Crossref PubMed Scopus (276) Google Scholar, Ventura et al., 2004Ventura M. Weigl S. Carbone L. Cardone M.F. Misceo D. Teti M. D’Addabbo P. Wandall A. Björck E. de Jong P.J. et al.Recurrent sites for new centromere seeding.Genome Res. 2004; 14: 1696-1703Crossref PubMed Scopus (121) Google Scholar, Warburton, 2004Warburton P.E. Chromosomal dynamics of human neocentromere formation.Chromosome Res. 2004; 12: 617-626Crossref PubMed Scopus (135) Google Scholar) in which the initial functional centromere has moved from its previous location to a new site in formerly euchromatic DNA without α-satellite repeats or binding of CENP-B (Depinet et al., 1997Depinet T.W. Zackowski J.L. Earnshaw W.C. Kaffe S. Sekhon G.S. Stallard R. Sullivan B.A. Vance G.H. Van Dyke D.L. Willard H.F. et al.Characterization of neo-centromeres in marker chromosomes lacking detectable alpha-satellite DNA.Hum. Mol. Genet. 1997; 6: 1195-1204Crossref PubMed Scopus (138) Google Scholar, du Sart et al., 1997du Sart D. Cancilla M.R. Earle E. Mao J.I. Saffery R. Tainton K.M. Kalitsis P. Martyn J. Barry A.E. Choo K.H. A functional neo-centromere formed through activation of a latent human centromere and consisting of non-alpha-satellite DNA.Nat. Genet. 1997; 16: 144-153Crossref PubMed Scopus (276) Google Scholar, Warburton et al., 1997Warburton P.E. Cooke C.A. Bourassa S. Vafa O. Sullivan B.A. Stetten G. Gimelli G. Warburton D. Tyler-Smith C. Sullivan K.F. et al.Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres.Curr. Biol. 1997; 7: 901-904Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Often associated with chromosomal rearrangements and found in some types of cancer, each neocentromere is marked with chromatin stably assembled with CENP-A (Amor et al., 2004Amor D.J. Bentley K. Ryan J. Perry J. Wong L. Slater H. Choo K.H. Human centromere repositioning “in progress”.Proc. Natl. Acad. Sci. USA. 2004; 101: 6542-6547Crossref PubMed Scopus (162) Google Scholar), the centromere-specific histone H3 variant (Earnshaw and Rothfield, 1985Earnshaw W.C. Rothfield N. Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma.Chromosoma. 1985; 91: 313-321Crossref PubMed Scopus (651) Google Scholar, Palmer et al., 1987Palmer D.K. O’Day K. Wener M.H. Andrews B.S. Margolis R.L. A 17-kD centromere protein (CENP-A) copurifies with nucleosome core particles and with histones.J. Cell Biol. 1987; 104: 805-815Crossref PubMed Scopus (318) Google Scholar). CENP-A is essential for centromere identity (Black et al., 2007bBlack B.E. Jansen L.E.T. Maddox P.S. Foltz D.R. Desai A.B. Shah J.V. Cleveland D.W. Centromere identity maintained by nucleosomes assembled with histone H3 containing the CENP-A targeting domain.Mol. Cell. 2007; 25: 309-322Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). It marks and maintains centromere position (Black et al., 2004Black B.E. Foltz D.R. Chakravarthy S. Luger K. Woods Jr., V.L. Cleveland D.W. Structural determinants for generating centromeric chromatin.Nature. 2004; 430: 578-582Crossref PubMed Scopus (320) Google Scholar, Hori et al., 2013Hori T. Shang W.H. Takeuchi K. Fukagawa T. The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly.J. Cell Biol. 2013; 200: 45-60Crossref PubMed Scopus (148) Google Scholar, Mendiburo et al., 2011Mendiburo M.J. Padeken J. Fülöp S. Schepers A. Heun P. Drosophila CENH3 is sufficient for centromere formation.Science. 2011; 334: 686-690Crossref PubMed Scopus (200) Google Scholar) and recruits additional centromere and kinetochore components (Carroll et al., 2009Carroll C.W. Silva M.C.C. Godek K.M. Jansen L.E.T. Straight A.F. Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N.Nat. Cell Biol. 2009; 11: 896-902Crossref PubMed Scopus (210) Google Scholar, Carroll et al., 2010Carroll C.W. Milks K.J. Straight A.F. Dual recognition of CENP-A nucleosomes is required for centromere assembly.J. Cell Biol. 2010; 189: 1143-1155Crossref PubMed Scopus (230) Google Scholar, Foltz et al., 2006Foltz D.R. Jansen L.E.T. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. The human CENP-A centromeric nucleosome-associated complex.Nat. Cell Biol. 2006; 8: 458-469Crossref PubMed Scopus (520) Google Scholar, Liu et al., 2006Liu S.T. Rattner J.B. Jablonski S.A. Yen T.J. Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells.J. Cell Biol. 2006; 175: 41-53Crossref PubMed Scopus (178) Google Scholar) to both normal and ectopic centromere locations (Barnhart et al., 2011Barnhart M.C. Kuich P.H.J.L. Stellfox M.E. Ward J.A. Bassett E.A. Black B.E. Foltz D.R. HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.J. Cell Biol. 2011; 194: 229-243Crossref PubMed Scopus (232) Google Scholar, Guse et al., 2011Guse A. Carroll C.W. Moree B. Fuller C.J. Straight A.F. In vitro centromere and kinetochore assembly on defined chromatin templates.Nature. 2011; 477: 354-358Crossref PubMed Scopus (175) Google Scholar, Hori et al., 2013Hori T. Shang W.H. Takeuchi K. Fukagawa T. The CCAN recruits CENP-A to the centromere and forms the structural core for kinetochore assembly.J. Cell Biol. 2013; 200: 45-60Crossref PubMed Scopus (148) Google Scholar, Mendiburo et al., 2011Mendiburo M.J. Padeken J. Fülöp S. Schepers A. Heun P. Drosophila CENH3 is sufficient for centromere formation.Science. 2011; 334: 686-690Crossref PubMed Scopus (200) Google Scholar). Indeed, use of gene targeting in both human cells and fission yeast has demonstrated that CENP-A-containing chromatin is the primary epigenetic mark of centromere identity (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar). Centromere identity and function is achieved through a two-step mechanism. First, new CENP-A assembly to centromeric chromatin is mediated by its CENP-A targeting domain (CATD) (Black et al., 2004Black B.E. Foltz D.R. Chakravarthy S. Luger K. Woods Jr., V.L. Cleveland D.W. Structural determinants for generating centromeric chromatin.Nature. 2004; 430: 578-582Crossref PubMed Scopus (320) Google Scholar, Black et al., 2007aBlack B.E. Brock M.A. Bédard S. Woods Jr., V.L. Cleveland D.W. An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes.Proc. Natl. Acad. Sci. USA. 2007; 104: 5008-5013Crossref PubMed Scopus (119) Google Scholar, Shelby et al., 1997Shelby R.D. Vafa O. Sullivan K.F. Assembly of CENP-A into centromeric chromatin requires a cooperative array of nucleosomal DNA contact sites.J. Cell Biol. 1997; 136: 501-513Crossref PubMed Scopus (256) Google Scholar) in conjunction with the CENP-A selective chaperone HJURP (Dunleavy et al., 2009Dunleavy E.M. Roche D. Tagami H. Lacoste N. Ray-Gallet D. Nakamura Y. Daigo Y. Nakatani Y. Almouzni-Pettinotti G. HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.Cell. 2009; 137: 485-497Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar, Foltz et al., 2009Foltz D.R. Jansen L.E.T. Bailey A.O. Yates 3rd, J.R. Bassett E.A. Wood S. Black B.E. Cleveland D.W. Cleveland D.W. Centromere-specific assembly of CENP-a nucleosomes is mediated by HJURP.Cell. 2009; 137: 472-484Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, Shuaib et al., 2010Shuaib M. Ouararhni K. Dimitrov S. Hamiche A. HJURP binds CENP-A via a highly conserved N-terminal domain and mediates its deposition at centromeres.Proc. Natl. Acad. Sci. USA. 2010; 107: 1349-1354Crossref PubMed Scopus (147) Google Scholar) whose activity is tightly controlled across the cell cycle (Müller and Almouzni, 2014Müller S. Almouzni G. A network of players in H3 histone variant deposition and maintenance at centromeres.Biochim. Biophys. Acta. 2014; 1839: 241-250Crossref PubMed Scopus (44) Google Scholar). In the second step, either the amino- or carboxy-terminal tail of CENP-A is required for nucleation of assembly of a kinetochore that mediates high fidelity chromosome segregation indefinitely (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar). Curiously, amino-terminal tail-dependent kinetochore nucleation requires the presence of CENP-B (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar). Starting from this suggestion of a CENP-B-dependent role in kinetochore function, we now use a combination of gene targeting and replacement in human and mouse cells, coupled with in vitro approaches, to identify a DNA sequence-dependent contribution to fidelity of human centromeric function that is mediated by CENP-B binding to centromeric α-satellite repeats. To test the effect that complete loss of the CENP-A amino-terminal tail has on centromere-bound CENP-B and on overall cell viability, we stably expressed (by retroviral integration) a full-length CENP-A or a CENP-A variant lacking its amino-terminal tail (ΔNH2CENP-A) in human cells containing one disrupted endogenous CENP-A allele and one floxed allele (CENP-A−/F) (Figure 1A). After Cre-recombinase mediated inactivation of the floxed allele and subsequent loss of endogenous CENP-A protein (Figures S1A and S1B), long-term cell viability was rescued by ΔNH2CENP-A (Figure 1B), albeit with a 4-fold increase in chromosome mis-segregation and micronuclei formation (scored by live cell imaging in cells stably expressing H2B-mRFP to label chromosomes) (Figures 1A and 1C). Furthermore, loss of the CENP-A amino-terminal tail was accompanied by reduced CENP-B binding at centromeres (Figures 1A and 1D), as measured by quantifying centromeric CENP-B intensity by immunofluorescence. To determine if this CENP-A-dependent binding of CENP-B at centromeres could result from a direct interaction, recombinant CENP-B was incubated with GST or GST-tagged CENP-A fragments and GST-containing proteins were affinity purified on glutathione-immobilized beads (Figures 1E and 1F). CENP-B bound directly to the amino-terminal tail of CENP-A (GST-CENP-A1–44), but not to GST alone or to a CENP-A mutant lacking its amino-terminal tail (GST-CENP-AΔ1–44) (Figure 1F). The first 29 amino acids of the CENP-A tail were sufficient for this interaction (Figure S1C), in agreement with the observation that the first 29 amino acids of CENP-A’s amino terminal tail stabilize CENP-B binding at centromeres (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar). Deletion of the CENP-A amino-terminal tail not only affected CENP-B binding, but also reduced by half centromere-bound CENP-C (Figure 1D), a major centromere component required for kinetochore assembly (Fukagawa et al., 1999Fukagawa T. Pendon C. Morris J. Brown W. CENP-C is necessary but not sufficient to induce formation of a functional centromere.EMBO J. 1999; 18: 4196-4209Crossref PubMed Scopus (89) Google Scholar). In vitro (Carroll et al., 2010Carroll C.W. Milks K.J. Straight A.F. Dual recognition of CENP-A nucleosomes is required for centromere assembly.J. Cell Biol. 2010; 189: 1143-1155Crossref PubMed Scopus (230) Google Scholar, Guse et al., 2011Guse A. Carroll C.W. Moree B. Fuller C.J. Straight A.F. In vitro centromere and kinetochore assembly on defined chromatin templates.Nature. 2011; 477: 354-358Crossref PubMed Scopus (175) Google Scholar) and in vivo (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar) findings have reported that the small (six amino acid) carboxy-terminal tail of CENP-A is one element for CENP-C recruitment to centromeres. Complete loss of the CENP-A carboxy-terminal tail did not, however, abolish centromeric CENP-C binding (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar), indicating the existence of another pathway for its recruitment. Because the CENP-A amino-terminal tail binds to CENP-B and its deletion reduced both CENP-B and CENP-C bound to centromeres (Figure 1), we tested if CENP-B was required for the maintenance of a fraction of centromeric CENP-C. Long-term dependency of centromere recruitment of CENP-C on CENP-B was tested by disrupting both CENP-B alleles in human diploid RPE-1 cells using a CRISPR/Cas9 nuclease (Figure 2A and Figure S2A). Complete loss of CENP-B (Figures 2B–2D) resulted in a 50% reduction of CENP-C at centromeres but not of its total cellular levels (Figures 2B and 2D), with only a slight decrease of centromeric CENP-A levels (Figure 2D), a reduction insufficient to explain the observed CENP-C reduction (CENP-A must be depleted >75% to produce 2-fold decrease of centromere-bound CENP-C [Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar]). To determine if short-term reduction of CENP-B also had consequences on CENP-C maintenance at centromeres, we integrated (at a unique genomic locus using the Flp-In system in DLD-1 cells) an siRNA-resistant, doxycycline-inducible gene encoding CENP-B that was dually tagged with EYFP and AID (auxin-inducible degron), the latter to enable rapid degradation upon addition of the synthetic auxin indole-3-acetic acid (IAA) (Holland et al., 2012Holland A.J. Fachinetti D. Han J.S. Cleveland D.W. Inducible, reversible system for the rapid and complete degradation of proteins in mammalian cells.Proc. Natl. Acad. Sci. USA. 2012; 109: E3350-E3357Crossref PubMed Scopus (179) Google Scholar). In agreement with our initial gene inactivation approach (Figure 2D), siRNA-mediated CENP-B depletion led to decrease in centromeric CENP-C by half (Figures S2B–S2D). In contrast, doxycycline-induced expression of CENP-BAID-EYFP maintained the initial level of CENP-C at each centromere (Figures S2B–S2D), while IAA-induced rapid degradation of CENP-BAID-EYFP was accompanied by reduction (again by half) of CENP-C bound at centromeres (Figure S2D). To directly measure the level of CENP-C after siRNA-mediated CENP-B depletion, we EYFP-tagged one or both alleles of the endogenous human CENP-C gene using TALEN-mediated gene targeting (Figures 2E–2G). As expected, quantification of centromere fluorescence intensity of single centromeres in live cells revealed that CENP-C in CENP-C+/EYFP cells was half that of centromeres in which both alleles were targeted (CENP-CEYFP/EYFP) (Figure 2H). Importantly, in agreement with our indirect immunofluorescence analysis, siRNA-mediated depletion of CENP-B led to loss of half of centromeric EYFP intensity (Figure 2H). To determine whether the CENP-B influence on the level of centromere-bound CENP-C in cells could be mediated by physical interaction between CENP-B and CENP-C, we tested direct binding of the proteins to each other in vitro (Figure 3A). After co-incubation of GST-CENP-C and His-CENP-B, either protein was affinity purified (Figure S2E), followed by immunoblot. GST pull-down for CENP-C co-purified a portion of CENP-B and vice versa (Figure 3B), indicative of direct binding and in agreement with a previous two-hybrid study (Suzuki et al., 2004Suzuki N. Nakano M. Nozaki N. Egashira S. Okazaki T. Masumoto H. CENP-B interacts with CENP-C domains containing Mif2 regions responsible for centromere localization.J. Biol. Chem. 2004; 279: 5934-5946Crossref PubMed Scopus (54) Google Scholar). We then tested if CENP-B was also responsible for CENP-C deposition at centromeres besides its role in CENP-C stabilization. To test the initial loading of CENP-C at centromeres and its dependency on CENP-B, a tetracycline-inducible CENP-C tagged with EYFP and AID was stably integrated at a specific chromosomal locus. After culturing in IAA to induce degradation of the accumulated tagged CENP-C assembled at centromeres, IAA was withdrawn and immunofluorescence was used to monitor CENP-CAID-EYFP re-accumulation and its assembly at centromeres (Figures S3A and S3B). Interestingly, siRNA-mediated reduction in CENP-B by more than 90% (Figure S3C) did not significantly change the percentage of cells that efficiently loaded new CENP-CAID-EYFP (Figure S3D), but did reduce the intensity of centromeric CENP-CAID-EYFP (Figure S3E). This result indicates that CENP-B is not required for CENP-C deposition at centromeres, but only for its retention. To determine the consequence of CENP-B loss on the fidelity of centromere function, we measured chromosome mis-segregation rates using live cell imaging (Figure 3C) in CENP-A−/− cells deleted in both endogenous CENP-A alleles and with centromere function rescued by stable expression of full-length CENP-A or a CENP-A variant (CENP-AH3-C) in which its CENP-C binding site (CENP-A’s carboxy terminus) was replaced with the corresponding region of histone H3 (Figures 3C and 3E). In CENP-A−/− cells rescued with full-length CENP-A, reduction in CENP-B lowered centromeric CENP-C by 50% and produced a 2-fold increase in mitotic errors relative to the siRNA control (Figures 3C–3F). In CENP-A−/− cells rescued by CENP-AH3-C, almost all centromere-bound CENP-C was lost following siRNA-mediated depletion of CENP-B (Figures 3C–3E). This CENP-B-dependent loss of CENP-C at centromeres was accompanied by more than half of mitoses with misaligned chromosomes and micronuclei found within 60% of interphase cells (Figures 3C and 3F). The KMN network (comprised of the Mis12, Knl1, and Ndc80 complexes) is essential for connection between the centromere and spindle microtubules (Hori and Fukagawa, 2012Hori T. Fukagawa T. Establishment of the vertebrate kinetochores.Chromosome Res. 2012; 20: 547-561Crossref PubMed Scopus (29) Google Scholar). Recognizing that CENP-C has been proposed to be required for kinetochore function through recruitment of the Mis12 complex (Przewloka et al., 2011Przewloka M.R. Venkei Z. Bolanos-Garcia V.M. Debski J. Dadlez M. Glover D.M. CENP-C is a structural platform for kinetochore assembly.Curr. Biol. 2011; 21: 399-405Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, Screpanti et al., 2011Screpanti E. De Antoni A. Alushin G.M. Petrovic A. Melis T. Nogales E. Musacchio A. Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore.Curr. Biol. 2011; 21: 391-398Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), we measured the centromeric levels of Dns1 and Hec1, subunits of the Mis12 and Ndc80 complexes, respectively, on metaphase centromeres in cells with normal or reduced centromere-bound CENP-C (Figures S3F and S3G). Reduction of centromeric CENP-C in CENP-B−/− cells or in CENP-A−/− cells rescued by ΔNH2CENP-A lead to correspondingly reduced levels of Dsn1 and slightly reduced Hec1, similar to what was observed in CENP-A−/− cells rescued by CENP-AH3-C (Figures S3H and S3I) (Fachinetti et al., 2013Fachinetti D. Folco H.D. Nechemia-Arbely Y. Valente L.P. Nguyen K. Wong A.J. Zhu Q. Holland A.J. Desai A. Jansen L.E.T. Cleveland D.W. A two-step mechanism for epigenetic specification of centromere identity and function.Nat. Cell Biol. 2013; 15: 1056-1066Crossref PubMed Scopus (182) Google Scholar). Thus, diminution of centromeric CENP-C drives chromosome mis-segregation at least in part from reduced recruitment of the Mis12 complex. We next tested if a role of CENP-B in supporting centromere function via maintenance of centromeric CENP-C in human cells was conserved in an additional mammalian species. We tested if chromosome mis-segregation rates and centromeric CENP-C loading were affected in immortalized mouse embryonic fibroblasts (MEFs) in which both CENP-B alleles had been inactivated (Kapoor et al., 1998Kapoor M. Montes de Oca Luna R. Liu G. Lozano G. Cummings C. Mancini M. Ouspenski I. Brinkley B.R. May G.S. The cenpB gene is not essential in mice.Chromosoma. 1998; 107: 570-576Crossref PubMed Scopus (112) Google Scholar, Okada et al., 2007Okada T. Ohzeki J. Nakano M. Yoda K. Brinkley W.R. Larionov V. Masumoto H. CENP-B controls centromere formation depending on the chromatin context.Cell. 2007; 131: 1287-1300Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). As in the human RPE-1 or DLD-1 cells following CENP-B reduction, CENP-B null MEFs displayed a 50% reduction in centromeric CENP-C, relative to wild-type MEFs, despite unchanged levels of CENP-A (Figure 4A). This was accompanied by an increased mitotic error frequency (measured by imaging of fixed cells or by live imaging of cells expressing H2B-mRFP), with accumulation of micronuclei at more than twice the rate seen in CENP-B wild-type cells (Figures 4B–4E). Using release from nocodazole arrest to enrich for mitotic errors (Thompson and Compton, 2011Thompson S.L. Compton D.A. Chromosome missegregation in human cells arises through specific types of kinetochore-microtubule attachment errors.Proc. Natl. Acad. Sci. USA. 2011; 108: 17974-17978Crossref PubMed Scopus (182) Google Scholar), higher error frequencies were again observed in CENP-B deleted MEFs relative to wild-type MEFs (Figures 4B–4D). We then tested whether it was the presence of CENP-B per se or the sequence-dependent CENP-B binding at centromeres that was required to reinforce both CENP-C binding at centromeres and fidelity of chromosome segregation. To address this point, we examined a patient-derived cell line (PD-NC4) containing an alphoid DNA-free neocentromere on chromosome 4 (hereafter Neo4) (Amor et al., 2004Amor D.J. Bentley K. Ryan J. Perry J. Wong L. Slater H. Choo K.H. Human centromere repositioning “in progress”.Proc. Natl. Acad. Sci. USA. 2004; 101: 6542-6547Crossref PubMed Scopus (162) Google Scholar) (Figure 5A). The intensities of CENP-A/B/C were measured by immunofluorescence on chromosome spreads. The neocentromere was identified by scoring for a chromosome in which CENP-A (at the Neo4 centromere) and CENP-B (at the inactive, original chromosome 4 centromere DNA locus containing CENP-B boxes in its alphoid DNA repeats) did not colocalize (Figure 5A). CENP-A level at the Neo4 centromere was nearly normal (83% of the average level at the other centromeres; Figure 5B, top), in agreement with previous reports (Bassett et al., 2010Bassett E.A. Wood S. Salimian K.J. Ajith S. Foltz D.R. Black B.E. Epigenetic centromere specification directs aurora B accumulation but is insufficient to efficiently correct mitotic errors.J. Cell Biol. 2010; 190: 177-185Crossref PubMed Scopus (58) Google Scholar, Bodor et al., 2014Bodor D.L. Mata J.F. Sergeev M. David A.F. Salimian K.J. Panchenko T. Cleveland D.W. Black B.E. Shah J.V. Jansen L.E. The quantitative architecture of centromeric chromatin.eLife. 2014; 3: e02137Crossref Scopus (140) Google Scholar). In contrast" @default.
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• W1917235046 date "2015-05-01" @default.
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• W1917235046 title "DNA Sequence-Specific Binding of CENP-B Enhances the Fidelity of Human Centromere Function" @default.
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• W1917235046 doi "https://doi.org/10.1016/j.devcel.2015.03.020" @default.
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