|dc.description.abstract||The organisation of DNA varies throughout the cell cycle in a tightly regulated manner. This ranges from the formation of high order structures, during the condensing of chromosomes into sister chromatids in mitosis and meiosis, to smaller scale local interactions between genomic regions, termed ‘topologically associating domains’ (TADs). Cohesin, a highly conserved ring shaped protein complex, facilitates a number of cellular processes via changes in DNA architecture. It is responsible for holding sister chromatids together during cell division until its cleavage at anaphase, extruding DNA loops, and functions in DNA transcription, repair, and regulation of recombination. Mutations in human cohesin and its regulators cause genetic developmental disorders, and are commonly found in cancer genomes.
Cohesin is not randomly distributed across chromosomes, but is rather enriched at specific locations. Previous work in the Marston lab uncovered a mechanism for targeted cohesin loading at the kinetochore in budding yeast, Saccharomyces cerevisiae. The fission yeast Schizosaccharomyces pombe provides a good model organism for cohesin and chromatin dynamics, as it possesses a small, extensively studied and genetically tractable genome, whilst also sharing some of the genetic features of higher eukaryotic chromatin structure which budding yeasts lack. In this study I therefore investigated potential targeted cohesin loading mechanisms in S. pombe. I found that whilst the target of the cohesin loader in S. cerevisiae at the kinetochore is not conserved in S. pombe, the homologous binding patch on the cohesin loader itself is similarly required for cohesin enrichment. The DDK kinase, which performs a multitude of roles in the cell, has also been shown to be involved in targeted cohesin enrichment in budding yeast, and is a candidate for a similar role in fission yeast. Interestingly, the strongest effect on cohesin enrichment found in DDK mutants was at the rDNA.
Through mass spectrometry of the cohesin loader and of the binding patch mutant, cross referenced with the findings of previous studies, I identified candidate targets of the cohesin loader localised on the chromatin. Dnt1, a nucleolar protein, emerged with high significance in pulldowns of both cohesin loader components: an interaction that was lost in the binding patch mutant. Analysing Dnt1 null cells revealed the high levels of cohesin enrichment seen at the nucleolar rDNA in wild type cells to be dependent on Dnt1. The MBF family of proteins regulating the G1/S phase transition were also identified as potential candidate targets of the cohesin loader, with a global increase in cohesin enrichment seen across the genome in G1 arrested cells lacking the MBF protein Res2.
Investigating the effects of cohesin modifications on DNA structure, I used Hi-C experiments to assay the role of the Pef1 kinase and TOR pathway on DNA organisation. This research, which was carried out as part of a collaboration with the Javerzat lab, was based on earlier work highlighting a link between cohesin enrichment and its phosphorylation by Pef1, and identification of Pef1 and TOR components in a screen for suppressors of a cohesin loader mutant. I found that longer range DNA interactions are seen in both G1 arrested Pef1 null and cohesin mutant samples, but not in a TOR mutant sample. Altogether this thesis provides insight into the pathways enabling cohesin loading and chromosomal folding.||en