Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly.
Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly.
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DOI：
10.1073/pnas.2104459118
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发表时间：
2021-11-30
影响因子：
11.1
通讯作者：
Li F
中科院分区：
文献类型：
--
作者：
Dong Q;Liu XL;Wang XH;Zhao Y;Chen YH;Li F
精确的染色体分离依赖于动粒。着丝粒如何在细胞周期中精确组装在着丝粒上仍然知之甚少。大多数真核生物的着丝粒由含有组蛋白 H3 变体 CENP-A 的核小体进行表观遗传学标记。在这里，我们证明了 Ccp1（一种抗 CENP-A 负载因子）与 CENP-T 的 N 末端相互作用，促进外着丝粒 Ndc80 复合物的组装。这项工作进一步表明，Ccp1 和 Ndc80 在 CENP-T N 末端通过磷酸化进行竞争排斥，确保了有丝分裂过程中着丝粒的精确组装。此外，CENP-T 对于 Ccp1 着丝粒定位至关重要，而 Ccp1 着丝粒定位反过来又调节 CENP-A 的分布。我们的结果揭示了细胞周期中动粒组装的一种以前未被认识的机制。着丝粒是一种组装在着丝粒上的蛋白质复合物，介导染色体分离。在大多数真核生物中，着丝粒由组蛋白 H3 变体 CENP-A 表观遗传指定。 CENP-T 是一种内着丝粒蛋白，在有丝分裂过程中充当外着丝粒 Ndc80 复合体组装的平台。 CENP-T 如何通过细胞周期进行调节仍不清楚。 Ccp1（CENP-A 负载蛋白 1 的反作用物）在间期期间与着丝粒相关，但在有丝分裂期间从着丝粒离域。在这里，我们证明了 Ccp1 直接与 CENP-T 相互作用。 CENP-T 对于 Ccp1 与着丝粒的关联很重要，而 CENP-T 着丝粒定位取决于 Mis16（人 RbAp48/46 的同源物）。我们在 CENP-T 的 N 末端鉴定了一个 Ccp1 相互作用基序 (CIM)，它与 Ndc80 受体基序相邻。 CIM 结构域是 Ccp1 着丝粒定位所必需的，CIM 结构域删除的突变体表型为 ccp1Δ。 CIM 结构域可被 CDK1（细胞周期蛋白依赖性激酶 1）磷酸化。 CIM 的磷酸化削弱了它与 Ccp1 的相互作用。与此一致的是，在 CIM 结构域的磷酸模拟突变体中，Ccp1 在细胞周期的所有阶段都与着丝粒解离，而在该结构域的磷酸无效突变体中，Ccp1 在有丝分裂期间与着丝粒结合。我们进一步表明，磷酸无效突变体在有丝分裂过程中破坏了 Ndc80 复合物的定位，导致染色体错误分离。这项工作表明，CENP-T N 末端的 Ccp1 和 Ndc80 通过磷酸化进行竞争排斥，确保了有丝分裂过程中着丝粒组装的精确性，并揭示了细胞周期中着丝粒组装的先前未被认识的机制。
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.
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影响因子：
16
作者：
Camahort, Raymond;Shivaraju, Manjunatha;Mattingly, Mark;Li, Bing;Nakanishi, Shima;Zhu, Dongxiao;Shilatifard, Ali;Workman, Jerry L.;Gerton, Jennifer L.
通讯作者：
Gerton, Jennifer L.
影响因子：
16
作者：
Dong, Qianhua;Yin, Feng-Xiang;Gao, Feng;Shen, Yuan;Zhang, Faben;Li, Yang;He, Haijin;Gonzalez, Marlyn;Yang, Jinpu;Zhang, Shu;Su, Min;Chen, Yu-Hang;Li, Fei
通讯作者：
Li, Fei
影响因子：
21.1
作者：
He, Haijin;Gonzalez, Marlyn;Zhang, Fan;Li, Fei
通讯作者：
Li, Fei
影响因子：
64.5
作者：
Black BE;Cleveland DW
通讯作者：
Cleveland DW
影响因子：
3.3
作者：
Aristizabal-Corrales, David;Yang, Jinpu;Li, Fei
通讯作者：
Li, Fei