Hypoxia and Antifungal Drug Resistance: Bridging the Gap Between Set Domain-Containing Epigenetic Factors and Phenotypic Plasticity

Nina D Serratore, Purdue University

Abstract

Alteration of gene expression occurs by genetic and epigenetic mechanisms in response to environmental stimuli. SET domain-containing proteins modulate gene expression by methylating histones on chromatin and associating with transcriptional machinery. Here, we define for the first time that Set4, the Set3 paralog, is a chromatin-associating factor that mediates yeast growth under hypoxia, azole antifungal drug treatment and heat stress. Importantly, we define that Set4 is an inducible SET domain-containing protein under sterol-limiting conditions. Set4 expression is controlled by the sterol responsive transcription factors, Upc2 and Ecm22, under hypoxia and azole antifungal drug treatment. To determine the role of Set4 on global gene expression under hypoxia, RNA-sequencing analysis was performed and showed that Set4 is necessary for the global transcriptional adaptations that occur under hypoxia. Specifically, loss of Set4 led to an upregulation of ∼24% of the yeast genome including the majority of ergosterol genes such as ERG11 and ERG3. Therefore, Set4 represses ergosterol genes under hypoxia. Mechanistically, we identified that Set4 interacts with the sterol responsive transcription factors, Hap1, Tup1-Cyc8 and Upc2, to target Set4 to ergosterol gene promoters. Importantly, we define that Set4 is a chromatin-associating factor that is induced under sterol limiting conditions, and demonstrate that Set4 globally alters gene expression under hypoxic conditions. In addition to studying the role of Set4 under hypoxia, we identified that SET domain-proteins govern antifungal drug efficacy. Antifungal drug resistance is a growing threat to human health because there are few targets for antifungal drugs and there has been a steady increase in cases of antifungal drug resistance since the 1980s. Antifungal drug resistance often develops by upregulating genes that encode the drug target or increasing the expression of genes encoding drug efflux pumps. Here, we define that SET domain-containing epigenetic factors alter drug efficacy to the medically relevant class of azole antifungal drugs in Saccharomyces cerevisiae and the opportunistic pathogen, Candida glabrata. Specifically, we demonstrate that Set1, Set2, Set3 and Set6 alter azole drug sensitivity whereas Set4 governs azole drug resistance. Loss of Set4 does not affect genes known to play a role in azole drug resistance; however, a set4Δ strain treated with azole drugs results in increased expression of DAN/TIR and PAU genes which putatively function in cell wall remodeling under conditions that deplete sterol levels. These data propose that Set4 functions in cell wall remodeling and maintenance to alter azole drug efficacy. A double deletion of set4Δ set1Δ suppresses azole drug resistance observed in the set4Δ strain suggesting that a Set1 inhibitor is a potential antifungal drug target. Heat tolerance is another biotic condition that requires large changes in gene expression to survive at non-permissible temperatures. Thermotolerance is a growing area of interest in the biofuel industry because of the economic burden it currently takes to produce ethanol. To make biofuels more affordable thermotolerant facultative anaerobes, including Saccharomyces cerevisiae, are of interest to improve ethanol production using the process of simultaneous saccharification and fermentation. In this final study, we investigated the role of SET domain-containing proteins and other epigenetic factors in heat tolerance. Overall, Set1, Set2, Set3 and Set6 were necessary for yeast growth at 40°C. One interesting finding was that overexpression of Set4 resulted in better yeast growth than WT suggesting that overexpression of SET domain-containing proteins promote thermotolerance. Lastly, we identify that the Set3 and Set4 SET domains are necessary for yeast growth under heat stress, 40°C. These are the first evidence that the SET domains of Set3 and Set4 are necessary for proper growth under the indicated environmental stress conditions. Together, these studies begin to elucidate the biological functions of SET domain proteins, predominantly Set4 and Set3, in phenotypic plasticity. These data suggest that SET domain-containing epigenetic factors mediate yeast growth under conditions like hypoxia, antifungal drug treatment and heat stress because they modulate global gene expression changes in response to environmental variation. Our key findings focused on characterizing the biological and biochemical role of Set4. We characterized that Set4 is the first inducible SET domain protein to our knowledge, and that Set4 functions to repress gene expression under sterol limiting conditions. Overall, these studies provide new insight into the role of epigenetic factors in phenotypic plasticity specifically adaptation to hypoxia, heat stress and azole antifungal drug treatment.

Degree

Ph.D.

Advisors

Briggs, Purdue University.

Subject Area

Molecular biology|Genetics|Biochemistry|Physiology

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