Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.
The centromere is a strange locus that derives its identity from the proteins that shape it rather than the DNA sequences it contains. It also functions in a remarkably singular way, providing a motor and command control center for the chromosome in conjunction with the kinetochore. Key to centromere identity is the chromatin that comprises it, which has a unique nucleosomal “bead on a string” including a special centromeric histone H3, called CENP-A. Found in alternating clusters of nucleosomes with “regular” histone H3, CENP-A is crucial for propagating centromere identity as well as for regulating kinetochore function. In this study, we have analysed the cell cycle dynamics of CENP-T and CENP-W, another two components of the constitutive centromere associated network. We show that, unlike CENP-A, CENP-T/W are not inherited stringently by daughter cells. Instead, these complexes - which are bound to the interstitial “regular” H3 nucleosome domains - assemble after DNA replication and are required for kinetochore formation. Thus, we propose that a stable CENP-A nucleosome population plays a role in centromere locus inheritance to daughter cells, while dynamic CENP-T/W and H3 nucleosomes provide a cycling function that triggers kinetochore assembly as cells enter mitosis in each new cell cycle.