Ccp1 Homodimer Mediates Chromatin Integrity by Antagonizing CENP-A Loading

Abstract

SUMMARY 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.

Ccp1 modulates epigenetic stability at centromeres and affects heterochromatin distribution in Schizosaccharomyces pombe

“…In addition, during interphase (G 1 to G 2 phase) but not in mitosis, Ccp1-GFP forms a distinct bright dot at the edge of the nucleus ( Fig. 2A), a similar cellular localization pattern was also observed by Dong et al (33). In fission yeast cells, all three centromeres cluster adjacent to the spindle pole body (SPB) at the nuclear periphery in interphase.…”

“…Ccp1 shows a high structural similarity to Vps75, suggesting that it may function as an authentic histone chaperone (33). In budding yeast, Vps75 was shown to form a complex with Rtt109 and to promote Rtt109's acetyltransferase activity on Lys-56 of histone H3 (34,35).…”

Cell Cycle-Regulated Transcription of CENP-A by the MBF Complex Ensures Optimal Level of CENP-A for Centromere Formation

“…How CENP-A is prevented from assembling into noncentromeric nucleosomes during replication is unclear. In addition to ubiquitin-dependent proteolysis, the nucleosome assembly protein (NAP) domain-containing Ccp1 and the chromatin remodeling complexes, such as facilitates chromatin transcription (FACT) and human histone cell cycle regulator (HIRA), also play important roles in removing mistargeted CENP-A (Choi et al 2012;Deyter and Biggins 2014;Dong et al 2016;Ciftci-Yilmaz et al 2018). Perhaps these factors maintain the integrity of chromosome arms by excluding ectopic CENP-A during replication.…”

“…Proper centromere assembly is dependent upon the tight regulation of CENP-A levels. Overexpression of CENP-A in many organisms causes misincorporation of CENP-A into noncentromeric regions, leading to chromosome missegregation and growth defects (Heun et al 2006;Olszak et al 2011;Choi et al 2012;Castillo et al 2013;Gonzalez et al 2014;Dong et al 2016;Shrestha et al 2017). CENP-A overexpression has been observed in a number of cancers, which might contribute to chromosome instability (Tomonaga et al 2003;Li et al 2007;Amato et al 2009;Scott and Sullivan 2014;Zhang et al 2016).…”

Antibody Pull-Down Experiments in Fission Yeast

“…In addition, generation of a tagged version of the protein of interest expressing at the endogenous level is straightforward in S. pombe . Tags, such as GFP, HA, Myc, S and TAP, have been successfully used in S. pombe in our lab to study different aspects of cellular biology, such as heterochromatin silencing [1–3] or centromere regulation [4,5]. The protocol can be divided into five steps (Figure 1).…”