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I have pursued a breadth of research that explored the functional genomic study of eukaryotic transcriptional regulation. I have utilized two model organisms, many experimental methodologies, and have developed a suite of computational resources to study the interaction of transcription factors with regulated targets. In Saccharomyces cerevisiae I worked with my collaborator Dr. Zhanzhi (Mike) Hu to characterize the whole-genome transcriptional response of 263 individual transcription factor deletions. We utilized a sophisticated error model and directed-weighted graphs to model a network of high-confidence targets for each transcription factor profiled. We then used regulatory epistasis to elucidate the true set of primary KO-regulated targets and construct a functional transcriptional regulatory network. This network was analyzed for ontological and sequence motif enrichment in order to gain insight into the biological functions represented by transcription factors studied. Functional validation was performed to evaluate the probability of novel functional characterizations. Significant insight was gained from this study with regard to the nature of regulatory cascades and the inability for DNA binding events to predict regulation. This set of analysis was performed with a novel bioinformatic server called ArrayPlex. ArrayPlex is a software package that centrally provides a large number of flexible toolsets useful for functional genomics including microarray data storage, quality assessments, data visualization, gene annotation retrieval, statistical tests, genomic sequence retrieval and motif analysis. It uses a client-server architecture based on open source components, provides graphical, command-line, as well as programmatic access to all needed resources, and is extensible by virtue of a documented API. Using many of the techniques and computational resources developed, I pursued the study of microRNA transcriptional abundance and targeting in H. sapiens cell cultures. Utilizing custom-fabricated microarrays, I measured the whole-genome response of both mRNAs and microRNAs under serum stimulation, c-Myc overexpression, and c-Myc siRNA-mediated knockdown. I then characterized the regulatory interactions between the sets of regulated microRNAs and coordinately regulated transcription factors. Using analytical methods sensitive to regulatory directionality of both populations I was able to determine high-confidence relationships between transcription factors and regulated microRNAs as well as microRNAs and regulated gene targets.

published_or_final_version / abstract / toc / Zoology / Doctoral / Doctor of Philosophy

No description available.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

SUMO-1/2/3 are members of the ubiquitin-like family of protein modifiers. These proteins are covalently attached to numerous proteins in a directed and controlled manner. SUMO conjugation primarily occurs to proteins containing an exposed SUMO conjugation motif, (I, V, L, F)KxE, where x represents any amino acid. SUMO conjugation is controlled by key enzymes, a SUMO activating enzyme, SAE1/2 and a SUMO conjugating enzyme, Ubc9, which is responsible for substrate recognition, and the efficiency of this pathway can be increased by one of many SUMO ligase enzymes. This modification alters the substrate's characteristics and results in a change of state, such as stability, localisation, or activity. p300, a transcriptional co-activator, contains an evolutionary conserved tandem SUMO modification motif, located in a transcriptional repression domain. p300 was efficiently conjugated, both in vitro and in vivo, by SUMO-1/2/3, within this repression domain to both SUMO conjugation motifs. The SUMO conjugation to p300 correlated with p300 ability to repress transcription, requiring both SUMO conjugation motifs for full transcription repression activity. This repression activity was mediated through SUMO recruitment of histone deacetylase 6. Repression could be alleviated through co-expression of a SUMO-specific protease thereby suggesting a potential regulatory mechanism for transcription control of SUMO modified substrates. Despite utilising the same conjugation machinery, there remained the potential for distinct roles for the SUMO isoforms. SUMO -2/3, which form a distinct group from SUMO-1, were shown to preferentially mediate the transcription repression abilities of a number of known SUMO modifiable substrates: p300, Elk-1, and SP3. Further differences were observed in the ability of SUMO-1 and SUMO-2/3 to influence the nuclear organisation of p80 coilin.

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy