Determining the role of the epigenetic factor SET4 in antifungal drug resistance in budding yeast

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Fungal pathogens and their role in the increasing prevalence of systemic infections are of critical concern for human health. Systemic infections are regularly caused by drug resistant pathogens, a phenomenon that results from genetic mutation or overexpression. These changes in gene expression can be a result of mutations in epigenetic factors. Epigenetic factors remodel DNA and histones within the nucleosome by adding or removing chemical groups from the aminoterminal tail of the histone protein. These modifications cause changes in gene expression or recruitment of additional factors to the epigenetic site. Classes of epigenetic factors include ATP-dependent chromatin remodelers and histone modifiers such as histone acetyltransferases (HATs) and methyltransferases (HMTs). The SET domain superfamily is a class of epigenetic factors in yeast that include HATs and HMTs. This family includes SET4, a possible HMT that we hypothesize is regulating antifungal drug resistance genes in budding yeast. Set4 is induced under stress conditions, such as hypoxia and drug treatment. Additionally, a SET4 deletion is resistant to antifungal drug treatment. While the interactions of Set4 in the hypoxia stress condition have been studied, it is currently unclear what Set4 targets during antifungal drug treatment. Interestingly, overexpression of SET4 results in hypersensitivity to antifungal drugs. This study utilizes suppressor screens, overexpressed SET4 colonies plated on media containing antifungal drugs, to identify suppressors of the hypersensitive phenotype. The suppressor colonies overcome the hypersensitive phenotype to grow under the antifungal treatment. Replating the suppressors showed drug resistance, suggesting that a genetic mutation(s) occurred. The suppressor colonies were finally analyzed for SET4 expression and found to have little to no visible change in Set4 levels from the original overexpressed SET4 strain. This suggests that the suppressor mutations are novel and not due to mutations in SET4 or the constitutive promoter. In the future, genome-wide sequencing will be conducted in order to identify the suppressor mutation(s) and further analysis will determine whether the suppressor proteins have roles in antifungal resistance or a function in the drug resistant mechanism of Set4. Support or Funding Information Purdue Department of Biochemistry Purdue Summer Undergraduate Research Fellowship (SURF) S. cerevisiae wildtype and PYK1p-SET4 strains grown for 6 hours in SC media from OD600 0.1. Samples diluted to OD600 0.0001 and plated on SC agar plates with 8 μg/mL fluconazole. Pictures taken over a period of five days. The screen produced six colonies from plates with a concentration of 8 μg/mL. They are named SET4 (OE) 8-1, 8-2, 8-3, 8-4, 8-5, and 8-6. S. cerevisiae wildtype, PYK1p-SET4, and suppressor strains 8-1, 8-2, 8-3, 8-4, 8-5, and 8-6. Cells grown for 6 hours in SC media from OD600 0.1. Dilutions performed from OD600 0.01 to 0.00001 and spotted on SC agar plates with 8 μg/mL. Pictures taken over a period of three days. S. cerevisiae wildtype, 3xFLAG-SET4 wildtype, and suppressor strains 8-1, 8-2, 8-3, 8-4, 8-5 and 8-6. Cells grown for 7 hours in SC media from OD600 0.1 under aerobic conditions. Sample dilutions performed to reach similar concentrations. 8 μL sample used for the α-Rabbit FLAG blot and 8 μL sample used for the α-G6PDH blot. Blot exposed 10 seconds. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.