Set4 is a chromatin-associated protein, promotes survival during oxidative stress, and regulates stress response genes in yeast

Abstract

The Set4 protein in the yeast contains both a PHD finger and a SET domain, a common signature of chromatin-associated proteins, and shares sequence homology with the yeast protein Set3, the fly protein UpSET, and the human protein mixed-lineage leukemia 5 (MLL5). However, the biological role for Set4 and its potential function in chromatin regulation has not been well defined. Here, we analyzed yeast cell phenotypes associated with loss of Set4 or its overexpression, which revealed that Set4 protects against oxidative stress induced by hydrogen peroxide. Gene expression analysis indicated that Set4 promotes the activation of stress response genes in the presence of oxidative insults. Using ChIP analysis and other biochemical assays, we also found that Set4 interacts with chromatin and directly localizes to stress response genes upon oxidative stress. However, recombinant Set4 did not show detectable methyltransferase activity on histones. Our findings also suggest that Set4 abundance in the cell is balanced under normal and stress conditions to promote survival. Overall, these results suggest a model in which Set4 is a stress-responsive, chromatin-associated protein that activates gene expression programs required for cellular protection against oxidative stress. This work advances our understanding of mechanisms that protect cells during oxidative stress and further defines the role of the Set3-Set4 subfamily of SET domain-containing proteins in controlling gene expression in response to adverse environmental conditions.

Assessing Yeast Cell Survival Following Hydrogen Peroxide Exposure

“…For spot assay tests, plates were imaged using a gel documentation system to qualitatively assess survival following hydrogen peroxide treatment. Examples of these data are shown in Figures 2A and 2E in Tran et al (2018). To quantitatively measure survival using the colony forming unit (cfu) assay, the total number of colonies on each plate was multiplied by the dilution factor (DF).…”

“…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 set4Δ 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).…”

“…We used a set4Δ 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.…”

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5

“…In vitro methylation assays. Methylation assays were performed as described previously (20,42,61). Briefly, different concentrations of GST-purified enzymes were added to 40 pmol of recombinant histone H4 (New England Biolabs) in 50 mM Tris-HCl (pH 8.0), 10% glycerol, 20 mM KCl, 5 mM MgCl 2 , and either 2 Ci of [ 3 H]SAM (Perkin-Elmer Life Sciences) or 0.3 mM unlabeled SAM (Sigma).…”

“…Phosphoproteomic analysis. Wild-type (yEG001) and SET5::SET5-2ϫFLAG::KANMX (yEG139) yeast strains were grown in 2 liters of YPD at 30°C to late log phase, harvested, lysed, and subjected to anti-FLAG immunoprecipitations as described previously (61,63). Phosphatase inhibitors (Roche phos STOP tablet) were included in the lysis buffer to preserve protein phosphorylation.…”