Analysis of the sequence features contributing to centromere organisation and CENP-A positioning and incorporation
Toda, Nicholas Rafael Tetsuo
Centromere identity is integral for proper kinetochore formation and chromosome segregation. In most species chromosomes have a centromere at a defined locus that is propagated across generations. The histone H3 variant CENP -A acts as an epigenetic mark for centromere identity in most species studied. CENP -A is absent from the inactivated centromere on dicentric chromosomes and present at neocentromeres that form on non -centromeric sequences. Thus, the canonical centromere sequence is neither necessary nor sufficient for centromere function. Nevertheless, centromeres are generally associated with particular sequences. Understanding the organisation of centromeric sequence features will provide insight into centromere function and identity.In this study I use the fission yeast Schizosaccharomyces pombe model system to address the relationship between CENP-Aᶜʰᵖ¹ and centromeric sequence features. These analyses reveal that CENP-Aᶜʰᵖ¹ nucleosomes are highly positioned within the central domain by large asymmetric AT-rich gaps. The same sequence features underlying CENP-Aᶜʰᵖ¹ positioning are conserved in the related species S. octosporus, but are not found at neocentromeres, suggesting that they are important but non-essential for centromere function. CENP-Aᶜʰᵖ¹ over -expression leads to ectopic CENP-Aᶜʰᵖ¹ incorporation primarily at sites associated with heterochromatin, including the sites where stable neocentromeres form. Ectopic CENP-Aᶜʰᵖ¹ also occupies additional sites within the central domain that are not occupied in cells with wild -type CENP-Aᶜʰᵖ¹ levels. In wild -type cells CENP-Aᶜʰᵖ¹ occupied sites are likely also occupied by H3 nucleosomes or the CENP- T /W /S /X nucleosome -like complex in a mixed population. Several candidate proteins were investigated to determine a protein residing in the large gaps between CENP-Aᶜʰᵖ¹ nucleosomes could be identified. No proteins could be localised to the AT -rich gaps between CENP-Aᶜʰᵖ¹ nucleosomes, but the origin recognition complex in a promising candidate.The results presented in this thesis demonstrate that nucleosomes within the fission yeast centromere central domain are highly positioned by sequence features in a conserved manner. This positioning also allows for another complex, possibly the origin recognition complex, to bind to DNA. Nucleosome positioning, DNA replication, and transcription could individually and collectively influence CENP-Aᶜʰᵖ¹ assembly and centromere function. Further experiments in fission yeast will continue to provide insight into the general properties of centromere function and identity.