Candida albicans, a significant human fungal pathogen, switches between two phenotypic states, white and opaque. The white-opaque switch is a unique and tractable system for the study of epigenetic regulation of gene expression. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops on the master regulator WOR1. Translational regulation of the network, as well as chromatin-level regulation of WOR1, have not been extensively explored. This dissertation investigates the means through which WOR1 expression and the white-opaque phenotype are epigenetically regulated, and identifies several novel mechanisms.
The long 5' untranslated region of WOR1 was identified as a translational regulator of the white-opaque transition. Deletion of the WOR1 5'UTR promoted white-opaque switching and stabilized opaque state. Transcripts containing the WOR1 5'UTR show reduced translational efficiency and lower polysome association, and this repressive effect may exist for additional key regulators of cell fate and morphology with long 5'UTR.
The role of the histone variant H2A.Z in chromatin-level epigenetic regulation of switching was investigated. Deletion of SWR1, the major subunit of the SWR1 complex depositing H2A.Z into chromatin, promoted white-opaque switching. Deletion of YNG2, a subunit of NuA4 histone acetyltransferase complex that targets SWR1 activity, produced a similar switching phenotype. Through nucleosome mapping, SWR1 was demonstrated to be required for proper nucleosome positioning and stability on the WOR1 promoter, with effects differing in white and opaque cells. Furthermore, H2A.Z was enriched at the WOR1 promoter in white cells, suggesting stabilization of a repressive chromatin state. An interaction was discovered between swr1 and elevated H3K56ac resulting in a severe synthetic growth defect on nicotinamide, as well as a synergistic effect on white-opaque switching, suggesting altered nucleosome turnover from this interaction.
Finally, the role of the long promoter of WOR1 was investigated. Truncation of the promoter to a 3 kb region, while sufficient to drive opaque-specific gene expression, abrogated switching to opaque, pointing to cis-regulatory elements further upstream. The studies described in this dissertation identify novel translational as well as nucleosome-level regulations of WOR1 expression and white-opaque switching, with further implications for epigenetic regulation.
