Friday, April 19, 2024
4/19/2024
PROJECT SUMMARY In eukaryotes, cell states transitions often require major transcriptional shifts that are subsequently reinforced through feedback loops that maintain stable expression of the newly acquired cellular phenotype. Candida albicans, a diploid commensal yeast and significant opportunistic pathogen, has been found to adopt a number of morphologically-distinct cell states that modulate its interaction with the human host. The phenotypic switch between the white and opaque cell states governs entry into the parasexual mating system by opaque cells and regulates the balance between virulence and commensalism in different host niches. Previous work has shown that switching between these states is regulated, in part, by the Mediator complex, a major transcriptional regulatory complex in eukaryotes. However, mutants within the Tail module subunits of Mediator that are necessary for formation of the opaque state affect incorporation of other subunits into the complex. More specifically, deletion of MED3 from the Tail module also causes the loss of Med2 from Mediator. In C. albicans, MED2 is encoded by the telomere-associated (TLO) expanded gene family, which can function as interchangeable Med2 subunits of Mediator. Thus, it is unclear if C. albicans phenotypic switching is affected by inactivation of MED3 or TLO genes in Mediator. Individual TLO genes have diverse functions based on overexpression studies with multiple genes often regulating a single trait. Therefore, we hypothesize that the TLO gene family as a whole is necessary for the white-opaque switch and progression through the parasexual cycle, with individual TLO paralogs possessing unique regulatory impacts on parasexual processes. Aim 1 will test necessity of the TLO gene family on interconversion between the white and opaque cell states by utilizing a recently built TLO-null strains in comparison to the med3¿/¿ and wild-type strains. Furthermore, we will evaluate the phenotypic impact of complete TLO loss on the pheromone response and mating efficiency of opaque cells, as MED3 mutants ectopically induced into the opaque state also suffered from reduced mating efficiency. To determine if specific TLO paralogs are responsible for alterations in cell state transition and mating, Aim 2 will produce a panel of strains containing a single epitope-tagged TLO paralog with their native promoters reintroduced into the null background. These strains will be tested for restoration of wild-type phenotypes for traits identified in Aim 1. These TLO reintroduction strains will also be used to identify paralog-specific bound DNA targets by chromatin immunoprecipitation sequencing (ChIP-Seq) and affected regulons via RNA sequencing (RNA-Seq). This work will determine the role of the TLO gene family and individual paralogs in the parasexual cycle of C. albicans. Furthermore, it will begin to determine the extent of functional diversification among members of a large paralogous gene family, which is largely untested beyond gene duplicates, and provide greater understanding into how genes may be retained in tightly packed evolutionary spaces.
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