Determining the role of epigenetic factors in antifungal drug resistance

Epigenetic factors are proteins that regulate gene expression by altering transcriptional machinery access to nucleosomes, DNA wrapped around histone proteins. Two classes of epigenetic factors are ATP-dependent chromatin remodelers and histone modifiers such as histone methyltransferases (HMTs), proteins that add methyl groups to histone tails. This study focuses on AIF4 (Antifungal-Induced Factor 4), a possible HMT induced upon neutral lipid depletion that we hypothesize is regulating antifungal drug resistance genes. Overexpression of AIF4 results in hypersensitivity to antifungal drugs. Studying epigenetic factors in the yeast Saccharomyces cerevisiae, including AIF4, can lead to better understanding of cell adaptation to their environments and insight into antifungal drug resistance of pathogenic yeast. This project will focus on identifying suppressors of AIF4’s hypersensitive phenotype and exploring whether genes in the neutral lipid pathway are necessary for AIF4 expression. To support our hypothesis, I will grow yeast colonies with overexpressed AIF4 on media containing antifungal drugs. Overexpressing AIF4 strains exposed to antifungal drugs over time suppressed the grow defect. Re-plating the suppressor colonies showed drug resistance, suggesting that a genetic mutation(s) occurred. Suppressor colonies will be analyzed for AIF4 expression and genome-wide sequencing to identify the suppressor mutation(s). In addition, I have generated deletions for genes that encode neutral lipid production enzymes, and I will determine if AIF4 expression is affected. Single and double deletions will determine if a particular neutral lipid is required for the expression of AIF4. Overall, my work will help to characterize a pathway required for AIF4 expression and drug resistance

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity

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

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

Characterizing the Role of AIF4 in Saccharomyces Cerevisiae

Chromatin remodelers are important regulatory mechanisms that eukaryotic cells use to modify the structure of chromatin, which is made up of DNA and proteins. DNA wraps around histone proteins to make up chromatin. When these proteins are modified, the shape of the chromatin is altered. “Loosening” the chromatin structure by chromatin modifications allows for active gene expression whereas “tightening” or compaction of chromatin results in gene repression. Therefore the modifications on chromatin modulate gene expression in all eukaryotes. It has been shown that mis-regulation of chromatin remodelers contribute to various cancers. Understanding the biochemistry behind how chromatin associating proteins modify chromatin, and ultimately gene expression, can help provide insight into developing anti-cancer drugs. This study focused on characterizing AIF4, a chromatin associating protein. Since other chromatin associating proteins are known to be histone modifiers, we hypothesized that AIF4 is another chromatin remodeler. Many chromatin remodelers are found in protein complexes. These protein complexes have been shown to be important for the functions of chromatin modifying proteins. Therefore this study tested whether AIF4 interacts with other proteins. Purifying AIF4 and testing if it binds to the nucleosome will support the hypothesis that AIF4 is chromatin associating protein. Co-immunoprecipitation will be used to determine if AIF4 interacts with other proteins, indicating that AIF4 functions in a protein complex. However, we were unable to detect an interaction between AIF4 and known chromatin associating proteins. Future work will aim to determine whether AIF4 acts alone or is involved in a unique protein complex

H3K4 methyltransferase Set1 is involved in maintenance of ergosterol homeostasis and resistance to Brefeldin A

Set1 is a conserved histone H3 lysine 4 (H3K4) methyltransferase that exists as a multisubunit complex. Although H3K4 methylation is located on many actively transcribed genes, few studies have established a direct connection showing that loss of Set1 and H3K4 methylation results in a phenotype caused by disruption of gene expression. In this study, we determined that cells lacking Set1 or Set1 complex members that disrupt H3K4 methylation have a growth defect when grown in the presence of the antifungal drug Brefeldin A (BFA), indicating that H3K4 methylation is needed for BFA resistance. To determine the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate-limiting enzyme HMG-CoA reductase. Consequently, deletion of SET1 leads to a reduction in HMG-CoA reductase protein and total cellular ergosterol. In addition, the lack of Set1 results in an increase in the expression of DAN1 and PDR11, two genes involved in ergosterol uptake. The increase in expression of uptake genes in set1Δ cells allows sterols such as cholesterol and ergosterol to be actively taken up under aerobic conditions. Interestingly, when grown in the presence of ergosterol set1Δ cells become resistant to BFA, indicating that proper ergosterol levels are needed for antifungal drug resistance. These data show that H3K4 methylation impacts gene expression and output of a biologically and medically relevant pathway and determines why cells lacking H3K4 methylation have antifungal drug sensitivity