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Kelly, Joan Alam, Md Ashiqul School of Biological Sciences 2016 http://hdl.handle.net/2440/119465 In A. nidulans, carbon catabolite repression is regulated by the global repressor protein CreA which, in the presence of repressing carbon sources, represses those genes that are required to utilize less preferable carbon sources. Mutational analyses suggested that ubiquitination, mediated by CreD together with ubiquitin ligase HulA, and deubiquitination, mediated by the deubiquitinating enzyme CreB, are involved in the regulatory pathway in A. nidulans. However, the molecular mechanisms are still unknown. Previously, partial loss-of-function alleles of creA and creB indicated genetic interaction, and this was extended to analysis of complete loss-of-function alleles. Both morphological and phenotypic analyses of the double null mutant confirmed genetic interactions between the genes. Moreover, RT-qPCR and enzyme assays also validated genetic interactions as the double null mutant showed synergistic effects for transcript levels and enzyme activity. Co-purifications of CreA and CreB expressed from their native promoters were used to determine whether CreA, or a protein in a complex with it, is a direct target of the CreB deubiquitinating enzyme, and no direct or indirect physical interactions were identified. The Phos-tag system was used to show that CreA is a phosphorylated protein, but no ubiquitination was detected using anti-ubiquitin antibodies and Western analysis. These findings were confirmed using mass spectrometry, which confirmed that CreA was differentially phosphorylated but not ubiquitinated. These results open up new questions regarding the molecular mechanism of CreA action, and how the ubiquitinating pathway involving CreB interacts with this regulatory network. To identify any possible protein(s) that may form a bridge between CreA and CreB, independently purified lysates were analysed by mass spectrometry and, for both CreA and CreB, proteins were identified in repressing and derepressing conditions. Orthologues of the co-purified proteins were identified in S. cerevisiae and humans. Functional annotation analysis revealed that proteins that preferentially interact with CreA in repressing conditions include histones and the histone transcription regulator 3, Hir3. Proteins interacting with CreB were involved in cellular transportation and organization. Similar findings were obtained using yeast and human orthologues, although the yeast background generated a number of other biological processes involving Mig1p which were not present in the A. nidulans or human background analyses. Hir3 was present in repressing conditions for CreA, and in both growth conditions for CreB, suggesting that Hir3, or proteins interacting with Hir3, could be a possible target of CreB. Earlier, genome-wide microarray analysis showed that CreA was involved in the transcriptional regulation of a significant number of genes in A. nidulans, however, this approach cannot show whether the targets are directly or indirectly regulated. To identify the direct targets, and whether CreA binds in derepressing conditions, ChIP-seq analyses were performed. CreA constitutively bound to the promoters of target genes in both growth conditions, indicating that the function of CreA may be controlled on the chromatin by post-translational modifications. A total of 1946 unique targets were identified for both strains in repressing and derepressing conditions including genes that are involved in carbohydrate metabolic/catabolic processes, alcohol biosynthetic processes, secondary metabolism, and sugar and amino acid transporters. Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2016 en An investigation of the role of CreB deubiquitinating enzyme in the regulation of carbon metabolism in Aspergillus nidulans Thesis This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at: http://www.adelaide.edu.au/legalsopen.access