| Summary: | <p>The centromere is a vital locus for mediating chromosome segregation. It defines the location where sister chromatids are joined, and constitutes the foundation of the kinetochore, facilitating microtubule binding in mitosis. The position of the centromere is defined by nucleosomes containing CENP-A, a centromere-specific histone which is a necessary and sufficient locus-specifying seed to facilitate deposition of further CENP-A nucleosomes via an epigenetic self-templating feedback loop. Once incorporated into chromatin, CENP-A is stably maintained across multiple cell divisions with a high degree of retention. No turnover of CENP-A has ever been observed, aside from its replicative halving during passage of the DNA replication fork, prior to deposition of newly synthesised CENP-A in G1 following mitosis.</p>
<p>This thesis explores the roles of recently identified proteins which may actively regulate CENP-A. These factors include SENP6, a SUMO protease found to be vital for stable CENP-A maintenance, and p97, a SUMO/ubiquitin segregase whose depletion was found to increase levels of centromeric CENP-A. The latter finding is unexpected, given that CENP-A turnover has not previously been detected. The experiments described herein confirm these initial observations and investigate mechanistic explanations. The increase in centromeric CENP-A levels in p97 depleted cells was found not to be a consequence of increased CENP-A loading, nor of reduction in cell cycle progression. Therefore, this represents the first observation of a factor capable of physiologically destabilising CENP-A nucleosomes. Simultaneous perturbation of SENP6 and p97 was found to fully rescue defects in CENP-A maintenance otherwise caused by SENP6 depletion. These findings lead to the hypothesis that SUMOylation of centromeric proteins destabilises CENP-A with the aid of the p97 segregase, which is kept in check by SENP6 as it cleaves SUMO chains.</p>
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