Evaluation of genomic binding locations of the Candida albicans transcription factor Brg1 during early hyphal induction




Stassen, Nicholas A.

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Candia albicans is a common commensal organism and an opportunistic fungal pathogen of humans. Its ability to cause disease is dependent on a morphological change from yeast to true hyphae. It is widely known that the transcriptional repressor Nrg1 is a negative regulator of hypha formation and when this protein is overexpressed, the organism stays in the yeast form under hyphae inducing conditions. Recent studies have demonstrated that the transcription factor Brg1 is a positive regulator of hypha formation and its role is critical in driving the change from yeast to true hyphae. Previous work in our laboratory uncovered that Brg1 and Nrg1 function in a genetic feedback loop and, specifically that BRG1 expression leads to rapid down-regulation of NRG1 transcript levels. It was subsequently shown that this down-regulation was independent of the promoter and 3’ UTR regions of NRG1 and entirely dependent on the coding sequence of the gene. Finally, we were able to detect the presence of an NRG1 antisense transcript that is induced in wild-type strains but not in brg1Δ mutants, which fail to filament under hyphal growth conditions. Currently, it is not known whether Brg1 acts directly or indirectly on the NRG1 coding sequence to up-regulate the antisense NRG1 transcript production. The purpose of this study was to determine whether Brg1 binds to the coding sequence of NRG1 during the early stages of hyphal induction. In order to do this, a novel Candida albicans strain was constructed in which both BRG1 alleles are tagged with a 13XMyc sequence. The tagged Brg1 transcription factor remains functional in its ability to drive filamentation and can be purified through immunoprecipitation. Quantitative PCR analysis on DNA immunoprecipitated from this strain suggests that Brg1 does not bind directly to a Brg1 consensus motif that was identified within the NRG1 coding sequence. However, now that we have the novel Myc-tagged Candida albicans strain, it will be possible to conduct a more comprehensive analysis of Brg1 binding sites during the early stages of hyphal induction using ChIP-Seq. That information will be very useful in helping us to unravel the cellular machinery through which Brg1 controls the pivotal virulence attribute of hyphal formation and may identify new targets for antifungal drug development.


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Integrative Biology