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Dissertation/Thesis Abstract

The Role of Mediator in White-Opaque Switching
by Zhang, Anda, Ph.D., Dartmouth College, 2014, 375; 3634935
Abstract (Summary)

The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. It is also involved in chromatin structure related epigenetic silencing through less well-understood mechanisms. We wanted to develop a novel endogenous epigenetic model to study Mediator’s silencing role in unicellular eukaryotes. White-opaque switching in the human opportunistic fungal pathogen Candida albicans serves as a suitable candidate. We purified Mediator from C. albicans and performed a thorough biochemical analysis of this complex with a focus on the Tail module. This work showed that the Tlo proteins, which are thought to be important to the high virulence of C. albicans, are a stoichiometric component of Mediator Tail module and they associate with Mediator through direct interaction with Med3 subunit. Next we analyzed the white-opaque switching in eight different strains, each lacking a specific Mediator subunit. We investigated the transcriptional levels of key regulators in these mutants, and the impact of overexpressing the master regulator, Wor1, on the switching phenotype in the med3 Δ/Δ and the med12Δ/Δ mutants to begin to uncover the mechanism of how Mediator regulates white-opaque switching. These studies demonstrated that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. These effects, however, were not accompanied by changes in the steady states mRNA level of key white-opaque switch regulators. We found that overexpression of Wor1 could rescue the low white-to-opaque switching frequency phenotype of the med12Δ/Δ mutant but only partially of the med3Δ/Δ mutant. We also studied Wor1 occupancy at its own promoter in the med3Δ/Δ mutant and histone variant H2A.Z occupancy at the WOR1 promoter in the med16Δ/Δ mutant by chromatin immunoprecipitation. Consistent with the lower amount of total Wor1 in the med3Δ/Δ mutant, we detected significant lower Wor1 occupancy at the WOR1 promoter. We also discovered that the H2A.Z/H2A exchange factor, SWR1, is required to maintain a stable white state. Overall, our research built a platform for further mechanistic studies of Mediator’s potential involvement in gene regulatory patterns that underlie C. albicans pathogenesis and epigenetic inheritance.

Indexing (document details)
Advisor: Myers, Lawrence C.
Commitee: Bennett, Richard J., Moseley, James B., Sundstrom, Paula R.
School: Dartmouth College
Department: Biochemistry
School Location: United States -- New Hampshire
Source: DAI-B 76/01(E), Dissertation Abstracts International
Subjects: Microbiology, Biochemistry
Keywords: Candida albicans, Epigenetic switch, Mediator, Transcription regulation, White-opaque switching
Publication Number: 3634935
ISBN: 978-1-321-15707-9
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