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标题：Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly
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作者：Qianhua Dong ; Xue-lei Liu ; Xiao-hui Wang 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：48
DOI：10.1073/pnas.2104459118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance Precise chromosome segregation relies on kinetochores. How kinetochores are precisely assembled on centromeres through the cell cycle remains poorly understood. Centromeres in most eukaryotes are epigenetically marked by nucleosomes containing the histone H3 variant, CENP-A. Here, we demonstrated that Ccp1, an anti–CENP-A loading factor, interacts with the N terminus of CENP-T to promote the assembly of the outer kinetochore Ndc80 complex. This work further suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis. In addition, CENP-T is critical for Ccp1 centromeric localization, which in turn regulates CENP-A distribution. Our results reveal a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle. Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.
关键词：kinetochore; centromere; CENP-A; phosphorylation; CENP-T