A genome-wide analysis of carbon catabolite repression in Schizosaccharomyces pombe
AffiliationSchool of BioSciences
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr. Dane Aaron McKay Vassiliadis
The process by which eukaryotic microorganisms preferentially utilise glucose as a carbon source is coordinated by a network of sensory and signalling pathways, which converge at the transcriptional level to control the function of a conserved regulatory mechanism known as carbon catabolite repression (CCR). In the fission yeast, Schizosaccharomyces pombe, CCR is mediated by Scr1, a C2H2 zinc finger transcriptional repressor orthologous to Saccharomyces cerevisiae Mig1, and Aspergillus nidulans CreA. In addition, a conserved co-regulatory complex, comprised of Tup11, Tup12 and Ssn6 proteins (hereafter Tup/Ssn6), is required for maximal transcriptional repression. Also implicated in this process is the transcriptional activator, Rst2, which in the absence of glucose, induces the expression of gluconeogenesis and sexual differentiation genes. To date, the molecular mechanism of CCR in S.ipombe has not been characterised in depth, and so there is limited knowledge of the range of genes that are subject to transcriptional repression, or of the functional relationship between Scr1, Tup/Ssn6, Rst2, or other factors that influence the establishment and/or maintenance of CCR. This study combined genetic techniques with a suite of high-throughput sequencing approaches to investigate the process of CCR in S. pombe. RNA-seq and ChIP-seq approaches showed that Scr1 represses approximately 2% of the S. pombe genome in the presence of glucose including hexose uptake, glycolysis, TCA cycle, pentose phosphate pathway, and gluconeogenesis genes. In addition, unexpected roles for Scr1 in the regulation of iron homeostasis and stress-induced meiosis were discovered, integrating Scr1 and CCR more broadly into the regulation of general metabolism and stress responses in S.ipombe. Biochemical pulldown approaches showed that Scr1 physically interacts with the Tup/Ssn6 complex in vitro and further ChIP-seq showed co-localisation of Tup11 with Scr1 at gene promoters in glucose-sufficient conditions. Interestingly, Tup11 was shown to remain at the promoter of certain target genes that were activated in the absence of glucose, suggesting roles for the Tup/Ssn6 complex in gene activation. Additional ChIP-seq analysis of Rst2 in the absence of glucose revealed localisation to gene promoters formerly repressed by Scr1 in glucose-sufficient conditions. Surprisingly, Rst2 was also found to co-localise with Scr1 and Tup11 at certain genes in the presence of glucose suggesting unforeseen regulatory roles for this factor in glucose-sufficient conditions and hinting at a potential competitive or co-operative relationship between Scr1 and Rst2 at these genes. In addition to increasing knowledge of CCR, these findings also have important biotechnological implications. S. pombe is industrially utilised to produce bioethanol, a renewable biofuel of significant environmental and economic importance. In a concurrent approach, an S. pombe isolate used for industrial scale bioethanol production from sugarcane molasses was analysed for modifications to carbon metabolism, CCR or other processes. Whole genome sequencing identified structural variation, including a 100kb duplication of a subtelomeric region in chromosome III, and potential evidence for horizontal gene transfer of coding sequences from Schizosaccharomyces octosporus. Further transcriptomic analysis identified a distinct transcriptional signature of this industrial isolate in both laboratory media and the molasses feedstock. Importantly, regulatory rewiring of central carbon metabolism and stress response pathways was evident. Finally, direct examination of CCR within this strain via Scr1 ChIP-seq, revealed significant plasticity with respect to the number of Scr1 targets, particularly in the molasses feedstock where Scr1 was associated with multiple actively transcribed genes, suggesting alteration of the CCR pathway within the industrial strain genetic background. Overall, this study has shown that CCR in S. pombe forms a core regulatory network that responds to glucose primarily at the transcriptional level to facilitate the regulation of a range of metabolic processes in both laboratory and industrial contexts. Thus, this work significantly improves our understanding of the CCR process in S. pombe and forms an important resource for the study of carbon regulation in eukaryotes. These findings will also be useful for the development of fission yeast strains that possess improved bioethanol production characteristics.
KeywordsSchizosaccharomyces pombe; transcriptional regulation; carbon metabolism; carbon catabolite repression; Scr1; Tup11; Rst2; RNA-seq; ChIP-seq; whole genome sequencing; bioethanol production; horizontal gene transfer
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