Investigation into the functions of sumoylation in yeast

PhD ThesisSmall ubiquitin modifier (SUMO) is a ubiquitin-like modification that regulates many fundamental processes in eukaryotes such as DNA damage and repair, cell cycle, stress responses and gene expression. Sumoylation is essential for viability in the model organisms Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and is embryonic lethal in mice. Conversely, sumoylation is dispensable for growth in the fission yeast Schizosaccharomyces pombe. High throughput proteomic approaches have detected many sumoylated substrates in a large number of cellular processes, however, the biological function of sumoylation in many of these processes, as well as the essential processes in S. cerevisiae and mammals, have not been elucidated. To gain insight into the biological functions of sumoylation in eukaryotes, we aimed to take advantage of the viability of sumoylation mutants and large scale deletion collections available in S. pombe to study the process via a high throughput genetic screening. Although the sumoylation mutants studied were found unsuitable for genetic screening, characterisation of the cell cycle defects observed in the mutants revealed that cells lacking sumoylation exhibited abnormal expression of cell cycle dependant genes. The gene transcripts affected by lack of sumoylation are normally regulated by a conserved family of forkhead transcription factors, and this study indicates that sumoylation acts specifically to repress forkhead dependant gene expression. A conserved forkhead transcription factor homolog in human cells has been previously identified as a sumoylated substrate, but the effect of sumoylation on the activity of the transcription factor was unclear. Our data therefore suggests that sumoylation may be a conserved negative regulator of cell cycle regulated gene transcription in eukaryotes. To further increase our understanding of sumoylation, a high throughput screening approach was used in S. cerevisiae. The SUMO encoding gene, SMT3, is essential in S. cerevisiae, hence a hypomorphic smt3 allele was screened against a genomic library of mutants. Excitingly, through this approach, we identified that perturbation of highly conserved cytoskeleton proteins, involved in filamentous actin (F actin) dynamics and tubulin, suppressed the slow growth defect associated with misregulated sumoylation. This novel observation suggests that a major role of sumoylation in eukaryotes is in cytoskeleton regulation. Aberrant sumoylation is implicated in many disease states, such as cancer, pathogenic infection, and neurodegenerative disease. Furthermore, forkhead transcription factors have been implicated in human cancers, and F actin dynamics are often misregulated in cancer and viral and bacterial infection. Thus, understanding the mechanism of sumoylation underpinning these processes have potential impacts for human health.Newcastle Biomedical Research Centr