Background

The unicellular eukaryote Saccharomyces cerevisiae has been an exceptional model for revealing large networks of genes critical to the survival of cells in diverse types of environmental stress (Gasch et al., 2000; Weiner et al., 2012; Ho and Gasch, 2015), largely due to the excellent genetic tools available in this system and the ease with which stress responses can be evaluated. In particular, genetic studies in yeast have uncovered the regulatory mechanisms that control the cells’ response to oxidative stress (Morano et al., 2012). Environmental factors, including radiation and the presence of redox-cycling agents, are a source of oxidative stress which cause increased reactive oxygen species (ROS). These ROS include the superoxide anion, H₂O₂, and hydroxyl radicals, which are highly reactive with proteins, lipids, and nucleic acids, causing oxidative damage to all of these macromolecules (Schieber and Chandel, 2014). Aerobic respiration also generates low levels of endogenous ROS by the leakage of electrons from the electron transport chain, and mitochondrial dysfunction, as occurs with stress or aging, increases ROS levels further, which triggers the induction of cellular defense mechanisms such as activated expression of antioxidant enzymes which detoxify ROS (Schieber and Chandel, 2014). Pathologies including cancer, cardiovascular disease, autoimmune diseases, and aging are associated with deregulated oxidative stress defense systems and altered levels of intracellular ROS. Therefore, identifying and characterizing the network of proteins responsible for protecting cells during oxidative stress is key to defining the pathophysiology underlying these diseases.

To test the role of a given gene in the response to oxidative stress, it is advantageous to analyze yeast strains which are deleted or otherwise mutated for the gene and which overexpress the protein encoded by the gene. If a particular gene is required to protect cells during oxidative stress, it is expected that its deletion would render cells sensitive to the stress, and its overexpression would cause resistance to the stress. The assays described below test the survival of yeast cells following treatment with hydrogen peroxide, which has served as a model compound for inducing oxidative stress in yeast (Morano et al., 2012; Kwolek-Mirek and Zadrag-Tecza, 2014). Specifically, these protocols were used to test the role of the chromatin regulator Set4 during oxidative stress (Tran et al., 2018). We used a set4∆ yeast strain, and a strain carrying a plasmid for the inducible overexpression of SET4 in the presence of β-estradiol, as previously described (McIsaac et al., 2013). The raw data shown here were obtained to generate the results published in Tran et al. (2018) that demonstrate a role for Set4 in protecting cells during oxidative stress.

We present both a semi-quantitative plate spot assay and a quantitative colony forming unit (cfu) survival assay to interrogate the survival of cells following hydrogen peroxide treatment. We found it useful to be able to visualize the growth differences in cells on the plates and validate these differences quantitatively using the cfu assay. Both protocols here appeared to show much more consistent results than plate spot assays in which hydrogen peroxide is added to the medium in the plate prior to spotting the yeast on the plate (Kwolek-Mirek and Zadrag-Tecza, 2014). In addition, we applied methods to optimize the growth conditions and hydrogen peroxide concentration to account for the observations that yeast cells acquire resistance to hydrogen peroxide when grown in nutrient-poor media, such as synthetic complete or dropout media, or following the diauxic shift, which occurs when cells transition from primarily glucose metabolism via glycolysis to aerobic respiration of ethanol (Guan et al., 2012). When these conditions are not accounted for, results are highly variable and may represent confounding factors in the experiment, rather than the consequences of the deletion or overexpression of the gene being investigated. These assays are not resource-intensive and can be used to test multiple mutant or overexpressing strains simultaneously. Overall, these protocols represent a sensitive and highly-reproducible approach to assaying the response of yeast cells to oxidative stress.