Ccp1 Homodimer Mediates Chromatin Integrity By Antagonizing Cenp-A Loading

•Ccp1 antagonizes CENP-A loading at both centromeric and non-centromeric regions•Ccp1 is recruited to centromeres at the end of mitosis in a Mis16-dependent manner•Ccp1 cooperates with H2A.Z to evict CENP-A assembled in euchromatin•Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity CENP-A is a centromere-specific histone 3 variant essential for centromere specification. CENP-A partially replaces canonical histone H3 at the centromeres. How the particular CENP-A/H3 ratio at centromeres is precisely maintained is unknown. It also remains unclear how CENP-A is excluded from non-centromeric chromatin. Here, we identify Ccp1, an uncharacterized NAP family protein in fission yeast that antagonizes CENP-A loading at both centromeric and non-centromeric regions. Like the CENP-A loading factor HJURP, Ccp1 interacts with CENP-A and is recruited to centromeres at the end of mitosis in a Mis16-dependent manner. These data indicate that factors with opposing CENP-A loading activities are recruited to centromeres. Furthermore, Ccp1 also cooperates with H2A.Z to evict CENP-A assembled in euchromatin. Structural analyses indicate that Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity. Our study establishes mechanisms for maintenance of CENP-A homeostasis at centromeres and the prevention of ectopic assembly of centromeres. CENP-A is a centromere-specific histone 3 variant essential for centromere specification. CENP-A partially replaces canonical histone H3 at the centromeres. How the particular CENP-A/H3 ratio at centromeres is precisely maintained is unknown. It also remains unclear how CENP-A is excluded from non-centromeric chromatin. Here, we identify Ccp1, an uncharacterized NAP family protein in fission yeast that antagonizes CENP-A loading at both centromeric and non-centromeric regions. Like the CENP-A loading factor HJURP, Ccp1 interacts with CENP-A and is recruited to centromeres at the end of mitosis in a Mis16-dependent manner. These data indicate that factors with opposing CENP-A loading activities are recruited to centromeres. Furthermore, Ccp1 also cooperates with H2A.Z to evict CENP-A assembled in euchromatin. Structural analyses indicate that Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity. Our study establishes mechanisms for maintenance of CENP-A homeostasis at centromeres and the prevention of ectopic assembly of centromeres. The centromere is the part of a chromosome responsible for kinetochore assembly. It is crucial for the proper segregation of chromosomes (Allshire and Karpen, 2008Allshire R.C. Karpen G.H. Epigenetic regulation of centromeric chromatin: old dogs, new tricks?.Nat. Rev. Genet. 2008; 9: 923-937Crossref PubMed Scopus (427) Google Scholar, Malik and Henikoff, 2009Malik H.S. Henikoff S. 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However, the mechanisms involved in preventing the ectopic assembly of CENP-A remain poorly understood. Studies on the centromeres of budding yeast have been instrumental in helping us understand centromere structure and function. Budding yeast contains “point” centromeres, which are genetically defined by a 125 bp DNA sequence (Clarke and Carbon, 1980Clarke L. Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes.Nature. 1980; 287: 504-509Crossref PubMed Scopus (483) Google Scholar, Cottarel et al., 1989Cottarel G. Shero J.H. Hieter P. Hegemann J.H. A 125-base-pair CEN6 DNA fragment is sufficient for complete meiotic and mitotic centromere functions in Saccharomyces cerevisiae.Mol. Cell. Biol. 1989; 9: 3342-3349Crossref PubMed Scopus (91) Google Scholar). In contrast, the fission yeast, Schizosaccharomyces pombe, contains large regional centromeres, which are governed by epigenetic mechanisms (Allshire and Karpen, 2008Allshire R.C. Karpen G.H. Epigenetic regulation of centromeric chromatin: old dogs, new tricks?.Nat. Rev. Genet. 2008; 9: 923-937Crossref PubMed Scopus (427) Google Scholar, Carroll and Straight, 2006Carroll C.W. Straight A.F. Centromere formation: from epigenetics to self-assembly.Trends Cell Biol. 2006; 16: 70-78Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). In fission yeast, the CENP-A homolog, Cnp1 (CENP-Acnp1), is enriched within 10–12 kb of the central domain region of centromeres, which are flanked with pericentromeric heterochromatin. The loading of CENP-Acnp1 also depends on the CENP-A loading factor, HJURPscm3. Recruitment of HJURPscm3 is cell cycle-regulated and requires the conserved Mis16-Mis18 complex (Pidoux et al., 2009Pidoux A.L. Choi E.S. Abbott J.K. Liu X. Kagansky A. Castillo A.G. Hamilton G.L. Richardson W. Rappsilber J. He X. Allshire R.C. Fission yeast Scm3: A CENP-A receptor required for integrity of subkinetochore chromatin.Mol. Cell. 2009; 33: 299-311Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Williams et al., 2009Williams J.S. Hayashi T. Yanagida M. Russell P. Fission yeast Scm3 mediates stable assembly of Cnp1/CENP-A into centromeric chromatin.Mol. Cell. 2009; 33: 287-298Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Like in other eukaryotes that harbor regional centromeres, the mislocalization of CENP-Acnp1 at non-centromeric regions can lead to assembly of ectopic CENP-Acnp1 chromatin and consequent chromosome mis-segregation (Castillo et al., 2013Castillo A.G. Pidoux A.L. Catania S. Durand-Dubief M. Choi E.S. Hamilton G. Ekwall K. Allshire R.C. Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.PLoS ONE. 2013; 8: e69673Crossref PubMed Scopus (23) Google Scholar, Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar, Ishii et al., 2008Ishii K. Ogiyama Y. Chikashige Y. Soejima S. Masuda F. Kakuma T. Hiraoka Y. Takahashi K. Heterochromatin integrity affects chromosome reorganization after centromere dysfunction.Science. 2008; 321: 1088-1091Crossref PubMed Scopus (150) Google Scholar). In addition to ubiquitin-mediated proteolysis and the FACT complex (Choi et al., 2012Choi E.S. Strålfors A. Catania S. Castillo A.G. Svensson J.P. Pidoux A.L. Ekwall K. Allshire R.C. Factors that promote H3 chromatin integrity during transcription prevent promiscuous deposition of CENP-A(Cnp1) in fission yeast.PLoS Genet. 2012; 8: e1002985Crossref PubMed Scopus (72) Google Scholar, Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar), Pht1, the homolog of histone H2A.Z in S. pombe, has also been implicated in preventing ectopic assembly of CENP-Acnp1 (Ogiyama et al., 2013Ogiyama Y. Ohno Y. Kubota Y. Ishii K. Epigenetically induced paucity of histone H2A.Z stabilizes fission-yeast ectopic centromeres.Nat. Struct. Mol. Biol. 2013; 20: 1397-1406Crossref PubMed Scopus (18) Google Scholar). In this study, we identify an uncharacterized protein in fission yeast, Ccp1, antagonizing the loading of CENP-Acnp1 at both centromeric and non-centromeric regions. Our data show that Ccp1 physically associates with CENP-Acnp1. Like HJURPscm3, Ccp1 is recruited to centromeres by Mis16 at the end of mitosis. The simultaneous recruitment of CENP-A loading and anti-loading factors provides an explanation for how the proper ratio of CENP-A to histone H3 is maintained at centromeres. Ccp1 also functions together with H2A.Z to displace ectopic CENP-A at non-centromeric regions. We report the crystal structure of Ccp1. Our data suggest that Ccp1 functions as a novel CENP-A chaperone specifically for preventing CENP-A loading. Together, these findings provide insights into how the right balance of CENP-A and histone H3 levels is achieved at centromeres and uncover a mechanism for how cells protect themselves from excessive assembly of CENP-A at non-centromeric regions. To identify genes required for proper CENP-A distribution, we performed a candidate visual genetic screen using fission yeast cells carrying CENP-Acnp1-GFP under the native promoter at the ade6+ locus. In wild-type interphase cells, all centromeres are clustered at the nuclear envelope; thus, a single fluorescent focus was observed in cells expressing CENP-Acnp1-GFP at endogenous level (Figure 1A) (Takahashi et al., 2000Takahashi K. Chen E.S. Yanagida M. Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast.Science. 2000; 288: 2215-2219Crossref PubMed Scopus (316) Google Scholar). Through the screen, we found that deletion of the gene SPBC36B7.08C results in more than 12% of cells exhibiting multiple foci of CENP-Acnp1-GFP (Figure 1A). We also found that the centromere clustering and the level of CENP-Acnp1-GFP are not affected in the mutant (Figures S1A and S1B). These results indicate that the gene is important for preventing mis-targeting of CENP-Acnp1-GFP to non-centromeric regions. To confirm this, we also examined the distribution pattern of N- or C-terminal GFP-tagged CENP-Acnp1 under the control of its native promoter at endogenous locus in the mutant background. Both fusion proteins display a single fluorescent focus in wild-type (Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar, Takayama et al., 2008Takayama Y. Sato H. Saitoh S. Ogiyama Y. Masuda F. Takahashi K. Biphasic incorporation of centromeric histone CENP-A in fission yeast.Mol. Biol. Cell. 2008; 19: 682-690Crossref PubMed Scopus (82) Google Scholar), but they also mislocalize in the mutant (Figures S1C–S1E), similar to CENP-Acnp1-GFP at ade6+ locus. We have thus named the gene counteracter of CENP-A loading protein 1 (ccp1+). ccp1+ encodes an uncharacterized nucleosome assembly protein (NAP) domain-containing protein of 244 amino acids, which is 26% and 27% identical to human SET protein and budding yeast Vps75, respectively. NAP family proteins often act as histone chaperones and play crucial roles in both assembly and disassembly of nucleosomes (Lorch et al., 2006Lorch Y. Maier-Davis B. Kornberg R.D. Chromatin remodeling by nucleosome disassembly in vitro.Proc. Natl. Acad. Sci. USA. 2006; 103: 3090-3093Crossref PubMed Scopus (173) Google Scholar, Park and Luger, 2008Park Y.J. Luger K. Histone chaperones in nucleosome eviction and histone exchange.Curr. Opin. Struct. Biol. 2008; 18: 282-289Crossref PubMed Scopus (152) Google Scholar). To confirm that CENP-Acnp1-GFP is misincorporated into non-centromeric region in the absence of Ccp1, we analyzed ccp1Δ mutant cells using chromatin immunoprecipitation (ChIP) along with primers specific to an rDNA region. Consistent with our cytological observation, we found that CENP-Acnp1-GFP is significantly enriched at the rDNA region, indicating that CENP-Acnp1-GFP associates with ectopic regions (Figure 1B), consistent with our cytological observation. Intriguingly, our ChIP assay showed that the CENP-Acnp1-GFP level at the centromere in the mutant is largely unchanged (Figure 1C). However, we found that a significant number of ccp1Δ mutant cells displaying a single fluorescence focus has disproportionately brighter CENP-Acnp1-GFP signal (Figure 1D). These results suggest that in ccp1Δ cells exhibiting a single GFP focus, the level of CENP-Acnp1-GFP at this region can substantially increase. To further verify the mislocalization of CENP-Acnp1 in the ccp1Δ mutant, we analyzed ccp1Δ cells carrying CENP-Acnp1-GFP by ChIP followed by high-throughput sequencing (ChIP-seq). Our ChIP-seq data showed that CENP-Acnp1-GFP is significantly enriched in euchromatin in ccp1Δ mutant relative to wild-type (Figure S1F). Together, our results indicate that Ccp1 is required for maintaining proper amounts of CENP-Acnp1 at both centromeric and non-centromeric regions. Consistent with this, we found that the mutant cells exhibited sensitivity to the microtubule-depolymerizing drug, thiabendazole (TBZ) (Figure 1E), which indicates that chromosome segregation in the ccp1 mutant is abnormal. Chromosome mis-segragation defects were further confirmed by DAPI staining (Figure S1G). Previous studies have shown that CENP-Acnp1-GFP still largely associates with centromeres when lowly overexpressed in S. pombe (Castillo et al., 2013Castillo A.G. Pidoux A.L. Catania S. Durand-Dubief M. Choi E.S. Hamilton G. Ekwall K. Allshire R.C. Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.PLoS ONE. 2013; 8: e69673Crossref PubMed Scopus (23) Google Scholar). To further examine the role of Ccp1 in counteracting the CENP-Acnp1 loading at non-centromeric regions, we induced a low level of overexpression of CENP-Acnp1-GFP using the intermediate-strength, thiamine-repressible nmt42 promoter in the ccp1Δ mutant. After a 32-hr induction, 94% of wild-type cells display one or two fluorescence foci. In contrast, multiple CENP-Acnp1-GFP foci were observed in 46% of the ccp1Δ cells analyzed under the same induction conditions (Figures S2A and S2B). This indicates that low level overexpression of CENP-Acnp1-GFP in ccp1Δ cells results in overall euchromatic localization. Overexpression of CENP-Acnp1-GFP in wild-type cells causes slow cell growth (Castillo et al., 2013Castillo A.G. Pidoux A.L. Catania S. Durand-Dubief M. Choi E.S. Hamilton G. Ekwall K. Allshire R.C. Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.PLoS ONE. 2013; 8: e69673Crossref PubMed Scopus (23) Google Scholar, Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar). We found that the overexpression of CENP-Acnp1-GFP under the strong nmt1 promoter in the ccp1Δ mutant results in even stronger inhibition of cell growth (Figures 1F and S2B). This is in agreement with the idea that overexpression of CENP-Acnp1 enhances the ectopic distribution of CENP-Acnp1-GFP in ccp1Δ cells. This is reminiscent of the overexpression of N-terminal deleted CENP-Acnp1, which leads to increased level of misincorporation of CENP-Acnp1 and slower growth (Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar). To further investigate the role of Ccp1 in CENP-Acnp1 positioning at centromeres, we overexpressed Ccp1 from the strong nmt1 promoter in wild-type cells expressing CENP-Acnp1-GFP at endogenous level. We found that the single CENP-Acnp1 focus is lost from centromeres in 76% of the cells analyzed (Figure 2A), even though the level of CENP-Acnp1-GFP in these cells remained similar to that of wild-type (Figure S3A). These results demonstrate that overexpression of Ccp1 causes significant eviction of CENP-Acnp1-GFP from native centromeres. Consistent with this, cells overexpressing Ccp1 exhibit severe growth defects and are highly sensitive to TBZ (Figures 2B and 2C). The overexpression of CENP-Acnp1 can induce the assembly of stable ectopic CENP-A chromatin at non-centromeric regions (Castillo et al., 2013Castillo A.G. Pidoux A.L. Catania S. Durand-Dubief M. Choi E.S. Hamilton G. Ekwall K. Allshire R.C. Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins.PLoS ONE. 2013; 8: e69673Crossref PubMed Scopus (23) Google Scholar, Gonzalez et al., 2014Gonzalez M. He H. Dong Q. Sun S. Li F. Ectopic centromere nucleation by CENP--a in fission yeast.Genetics. 2014; 198: 1433-1446Crossref PubMed Scopus (39) Google Scholar). To investigate whether Ccp1 can promote the exclusion of CENP-Acnp1 from ectopic loci, we overexpressed Ccp1 in wild-type cells carrying ectopic CENP-Acnp1 chromatin induced by massive overexpression of CENP-Acnp1-GFP. Consistent with previous studies, multiple distinct, stable fluorescent foci were observed in control cells overexpressing CENP-Acnp1-GFP alone, after 24 hr induction (Figure 2D). However, when we overexpressed Ccp1 in cells also overexpressing CENP-Acnp1-GFP, we found that almost all cells exhibited diffusion of the CENP-Acnp1-GFP signal, indicating that the ectopically assembled CENP-Acnp1-GFP was evicted from non-centromeric chromatin (Figures 2D and S3B). Together, our results show that overexpression of Ccp1 results in the eviction of CENP-Acnp1 from both centromeric and ectopic loci, consistent with its role as an anti-CENP-A loading factor. To further investigate the relationship between Ccp1 and CENP-Acnp1, ccp1Δ was crossed into a CENP-Acnp1 mutant, cnp1-1, to create a ccp1Δ cnp1-1 double mutant. The double mutant displays severe growth impairment at 32°C. The synthetic growth defects are even more apparent at 36°C (Figure 3A). Our data predict that Ccp1 physically associates with CENP-Acnp1. To test the idea, we created a TAP-tag version of Ccp1 under the control of its native promoter at its endogenous locus. This strain shows no sensitivity to TBZ (Figure S4A), indicating that Ccp1-TAP is functional. In the Ccp1-TAP strain massively overexpressing CENP-Acnp1-GFP, we found that Ccp1-TAP co-immunoprecipitated (coIP) with CENP-Acnp1-GFP (Figure 3B), supporting the idea that Ccp1 physically associates with CENP-Acnp1. We also conducted coIP experiments using cells carrying Ccp1-S tag and GFP-tagged histone H3. Ccp1-S tag was not able to co-immunoprecipitate H3-GFP from cell lysates (Figure S4B). To further confirm the interaction between Ccp1 and CENP-Acnp1, we purified chromatin-bound CENP-Acnp1 using the TAP-tag purification method and followed with mass spectrometry (MS) analysis to identify its interacting proteins. As expected, in addition to CENP-Acnp1, histones were identified by our MS analysis of purified chromatin-bound CENP-Acnp1 (Figure 3C). We also found that the chromatin-remodeling complex FACT, including Pof3 and Spt16, interacts with CENP-Acnp1 (Figure 3C). Consistent with these findings, CENP-A has been shown to be associated with the FACT complex in Drosophila and human cells (Boltengagen et al., 2016Boltengagen M. Huang A. Boltengagen A. Trixl L. Lindner H. Kremser L. Offterdinger M. Lusser A. 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Cell. 2009; 20: 3986-3995Crossref PubMed Scopus (104) Google Scholar). In addition, a conserved centromere protein, CENP-T (Cnp20 in fission yeast) copurified with CENP-Acnp1. CENP-T also copurified with CENP-A nucleosome in human cells (Foltz et al., 2006Foltz D.R. Jansen L.E. Black B.E. Bailey A.O. Yates 3rd, J.R. Cleveland D.W. The human CENP-A centromeric nucleosome-associa