Regulation and assembly of the associated network in human cells
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Centromeres are the chromosomal loci which are responsible for the equal segregation of sister chromatids to daughter cells during mitosis, by directing the assembly of kinetochores. Despite being involved in the faithful inheritance of the genome, centromeres are themselves epigenetically determined. Centromeres of higher organisms are determined by their centromeric chromatin which consists of unique nucleosomes, in which canonical histone H3 is replaced by the histone H3 variant CENP-A. A large group of additional proteins associate with the centromere throughout the cell cycle, collectively known as the constitutive centromere associated network (CCAN), and these are required for kinetochore function. In this thesis I have investigated the regulation and assembly of these CCAN proteins in human cells. Chapter 3 focuses on the regulation of the abundance of CCAN components during the cell cycle. Our analysis of the relative transcript abundance of CCAN members using real-time PCR did not reveal strong regulation of gene expression for the CCAN as a whole. However our results were suggestive of the centromere and kinetochore being regulated in a "just-in-time" manner, with the robust periodic expression of CENP-C and CENP-E and F during G2/M, making them ideal candidates to act as the 'pacesetters' for assembly of the inner and outer kinetochore respectively. Chapter 4 examines the assembly and inheritance of the histone fold domain proteins CENP-S and CENP-X at centromeres. Both proteins were found to assemble late in the cell cycle during late S/G2 phase similar to the CENP-T/-W, and to exhibit multi-generational persistence. FCCS and FRET analysis confirmed that CENP-S and CENP-X form a complex with one another. In addition we found CENP-S and CENP-T to be in close proximity to each other using FRET. These results with CENP-S/-X are consistent with the complex playing a role in kinetochore assembly before cells enter mitosis, coordinate with CENP-T/-W.