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Centromere protein A (CENP‐A) nucleosomes containing the centromere‐specific histone H3 variant CENP‐A represent an epigenetic mark that specifies centromere position. The Mis18 complex is a licensing factor for new CENP‐A deposition via the CENP‐A chaperone, Holliday junction recognition protein (HJURP), on the centromere chromatin. Chicken KINETOCHORE NULL2 (KNL2) (ggKNL2), a Mis18 complex component, has a CENP‐C‐like motif, and our previous study suggested that ggKNL2 directly binds to the CENP‐A nucleosome to recruit HJURP/CENP‐A to the centromere. However, the molecular basis for CENP‐A nucleosome recognition by ggKNL2 has remained unclear. Here, we present the cryo‐EM structure of the chicken CENP‐A nucleosome in complex with a ggKNL2 fragment containing the CENP‐C‐like motif. Chicken KNL2 distinguishes between CENP‐A and histone H3 in the nucleosome using the CENP‐C‐like motif and its downstream region. Both the C‐terminal tail and the RG‐loop of CENP‐A are simultaneously recognized as CENP‐A characteristics. The CENP‐A nucleosome–ggKNL2 interaction is thus essential for KNL2 functions. Furthermore, our structural, biochemical, and cell biology data indicate that ggKNL2 changes its binding partner at the centromere during chicken cell cycle progression. Synopsis: Cryo‐EM demonstrates the direct interaction of CENP‐A nucleosome of and licensing factor KNL2, and provides an insight into dynamic regulation of centromere localization of KNL2 during cell cycle progression. Mis‐localized KNL2 mutants caused reduction of CENP‐A levels at centromeres in chicken DT40 cells. Chicken KNL2 recognizes the centromeric nucleosome containing CENP‐A via a conserved CENP‐C‐like motif.Chicken KNL2 shares the CENP‐A nucleosome binding mode with kinetochore protein CENP‐C.CENP‐A nucleosome binding of chicken KNL2 is essential for its centromere localization in interphase cells but not mitotic cells.Mitotic centromere localization of chicken KNL2 depends on CENP‐C.Centromere localization of chicken KNL2 is dynamically regulated during the cell cycle. [ABSTRACT FROM AUTHOR]

The CENP‐A nucleosome is a key structure for kinetochore assembly. Once the CENP‐A nucleosome is established in the centromere, additional proteins recognize the CENP‐A nucleosome to form a kinetochore. CENP‐C and CENP‐N are CENP‐A binding proteins. We previously demonstrated that vertebrate CENP‐C binding to the CENP‐A nucleosome is regulated by CDK1‐mediated CENP‐C phosphorylation. However, it is still unknown how the phosphorylation of CENP‐C regulates its binding to CENP‐A. It is also not completely understood how and whether CENP‐C and CENP‐N act together on the CENP‐A nucleosome. Here, using cryo‐electron microscopy (cryo‐EM) in combination with biochemical approaches, we reveal a stable CENP‐A nucleosome‐binding mode of CENP‐C through unique regions. The chicken CENP‐C structure bound to the CENP‐A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP‐C residue. The stable CENP‐A‐CENP‐C complex excludes CENP‐N from the CENP‐A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1‐mediated CENP‐C phosphorylation. Synopsis: Phosphorylation of kinetochore protein CENP‐C regulates its binding to the CENP‐A nucleosome. Cryo‐EM reveals how CENP‐C phosphorylation regulates CENP‐A binding and provides insights into a dynamic kinetochore assembly mechanism during mitosis. The C‐terminal region of CENP‐C adopts a stable fold upon CENP‐A nucleosome binding.CDK1 phosphorylation of CENP‐C stabilizes the complex structure with the CENP‐A nucleosome via an intramolecular interaction.CENP‐C binds the RG loop of the CENP‐A nucleosome, which is recognized by another CENP‐A‐binding protein, CENP‐N.The stable CENP‐A‐ CENP‐C complex excludes CENP‐N from the CENP‐A nucleosome. [ABSTRACT FROM AUTHOR]

The kinetochore forms a dynamic interface with microtubules from the mitotic spindle during mitosis. The Ndc80 complex acts as the key microtubule-binding complex at kinetochores. However, it is unclear how the Ndc80 complex associates with the inner kinetochore proteins that assemble upon centromeric chromatin. Here, based on a high-resolution structural analysis, we demonstrate that the N-terminal region of vertebrate CENP-T interacts with the 'RWD' domain in the Spc24/25 portion of the Ndc80 complex. Phosphorylation of CENP-T strengthens a cryptic hydrophobic interaction between CENP-T and Spc25 resulting in a phospho-regulated interaction that occurs without direct recognition of the phosphorylated residue. The Ndc80 complex interacts with both CENP-T and the Mis12 complex, but we find that these interactions are mutually exclusive, supporting a model in which two distinct pathways target the Ndc80 complex to kinetochores. Our results provide a model for how the multiple protein complexes at kinetochores associate in a phospho-regulated manner. [ABSTRACT FROM AUTHOR]

We previously demonstrated that a fraction of the human Nup107–160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107–160 complex interacts with CENP-F, but that CENP-F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107–160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107–160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1-depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore–microtubule attachment defects. Finally, we show that the presence of the Nup107–160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1–RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107–160 complex at kinetochores. [ABSTRACT FROM AUTHOR]

Dissection of human centromeres is difficult because of the lack of landmarks within highly repeated DNA. We have systematically manipulated a single human X centromere generating a large series of deletion derivatives, which have been examined at four levels: linear DNA structure; the distribution of constitutive centromere proteins; topoisomerase IIα cleavage activity; and mitotic stability. We have determined that the human X major α-satellite locus, DXZ1, is asymmetrically organized with an active subdomain anchored ∼150 kb in from the Xp-edge. We demonstrate a major site of topoisomerase II cleavage within this domain that can shift if juxtaposed with a telomere, suggesting that this enzyme recognizes an epigenetic determinant within the DXZ1 chromatin. The observation that the only part of the DXZ1 locus shared by all deletion derivatives is a highly restricted region of <50 kb, which coincides with the topoisomerase II cleavage site, together with the high levels of cleavage detected, identify topoisomerase II as a major player in centromere biology. [ABSTRACT FROM AUTHOR]

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP- A is an early event that occurs independently of CENP-C and CENP-H. [ABSTRACT FROM AUTHOR]

CENP-C is an evolutionarily conserved centromeric protein. We have used the chicken DT40 cell line to test the idea that CENP-C is sufficient as well as necessary for the formation of a functional centromere. We have compared the effects of disrupting the localization of CENP-C with those of inducibly overexpressing the protein. Removing CENP-C from the centromere causes disassembly of the centromere protein complex and blocks cells at the metaphase—anaphase junction. Overexpressed CENP-C is associated with an increase in errors of chromosome segregation and inhibits the completion of mitosis. However, the excess CENP-C does not disrupt the native centromeres detectably and does not associate with another conserved centromere protein, ZW10. The distribution of the excess CENP-C changes during the cell cycle. In metaphase, the excess CENP-C coats the chromosome arms. At the metaphase—anaphase transition, the excess CENP-C clusters, and during interphase it is present in large bodies which form around pre-existing centromeres which are also clustered. These results indicate that CENP-C is necessary but not sufficient for the formation of a functional centromere and suggest that the structure of CENP-C may be regulated during the cell cycle. [ABSTRACT FROM AUTHOR]