Fine-tuning of the Msn2/4-mediated yeast stress responses as revealed by systematic deletion of Msn2/4 partners

Abstract

The Msn2 and Msn4 transcription factors play major roles in the yeast general stress response by mediating the transcription of hundreds of genes. Despite extensive information on Msn2/4-mediated gene expression profiles, much less is known regarding the network of proteins that regulate its activity. Here we describe a systematic approach designed to examine the roles of 35 Msn2/4 partners in regulating Msn2/4 transcriptional activity in the face of four different environmental conditions. Our analysis indicates that single deletions of 26 Msn2/4 partners significantly affect Msn2/4 transcription activity under four different conditions. The low functional redundancy of the Msn2 regulatory network indicates that Msn2/4 activity is finely tuned by many of Msn2/4 partners to provide an optimized stress response through differential activation, nuclear localization, degradation, and chromatin remodeling. Our specific analysis of Msn2 activity showed that a relatively large number of partners act to suppress Msn2 activity under nonstress conditions through independent mechanisms, including cytoplasmic retention, proteosome-mediated Msn2 degradation, and chromatin remodeling. Such negative regulation is crucial to minimize the cost of uncontrolled stress response gene expression and ensures a high growth rate in the absence of stress.

FIGURE 1:

A systematic approach for the identification of Msn2 partners that regulate Msn2 gene expression activity under different stress conditions. (A) Yeast strains deleted of Msn2 partners were generated on the background of a strain expressing the HSP12-GFP, the TPS2-GFP, or the PNC1-GFP reporter gene. Following exposure to diverse environmental conditions, reporter gene expression levels were analyzed by flow cytometry. Selected deletion strains with increased or decreased Hsp12-GFP expression relative to the nondeleted strain were further analyzed for survival, Msn2 nuclear localization, and Msn2 protein degradation. (B) Deletion of Msn2 partners can result in no change (left), in decrease (middle), or in increase (right) in reporter gene expression level. Deletion of Msn2 partners that resulted in decrease or increase in the reporter gene expression level allowed for identification of the absent protein as an Msn2 activator (green) or suppressor (red), respectively.

FIGURE 2:

Msn2 activity in 35 Msn2 partner–deleted strains generated on the S288c background analyzed by flow cytometry measurements of Hsp12-GFP reporter gene expression. The Hsp12-GFP expression level in the different strains was measured following yeast exposure to four different stress conditions and normalized to the wild-type strain (see Supplemental Figure S2 for examples and Supplemental Table S2 for raw data). The yeast cells were exposed to 40 min of stress, including oxidative stress (0.6 mM H₂O₂), osmotic stress (0.5 M NaCl), and heat stress (temperature shift from 30 to 37°C). Msn2 partner deletions that result in at least a 30% decrease or increase in Hsp12-GFP expression levels relative to the nondeleted strain led to the identification of the absent protein as a Msn2 activator or suppressor, highlighted in green and red, respectively. The glc7, cyr1, tor2, and cdc25 mutant strains contained a DaMP fusion (see Materials and Methods and Supplemental Table S5).

FIGURE 3:

Msn2 activity in 18 Msn2 partner–deleted strains analyzed by flow cytometry measurements of HSP12-GFP, TPS2-GFP, or PNC1-GFP reporter gene expression. The expression levels of the PFK2-GFP housekeeping gene is used as a negative control for the experiments. The 18 partner-deleted strains were generated on the background of W303 msn4-deleted strain to specifically analyze the effects of partner deletion on Msn2 activity. Gene expression levels in the different strains were measured following yeast exposure to four different stress conditions, as described in the legend to Figure 2, and normalized to the msn4-deleted strain (WT*) strain (see Supplemental Table S3 for raw data). Msn2 partner deletions that result in at least a 30% decrease or increase in Hsp12-GFP expression levels relative to the reference strain led to the identification of the absent protein as an Msn2 activator or suppressor, highlighted in green and red, respectively.

FIGURE 4:

Correlations between Hsp12-GFP expression following exposure of the different deletion strains to osmotic stress (NaCl) and heat (37°C) (A), oxidative stress (H₂O₂) and osmotic stress (NaCl) (B), and oxidative (H₂O₂) stress and heat (37°C) (C). The r values of the linear fit for the data in A–C are 0.83, 0.81, and 0.9, respectively. Each point represents the Hsp12-GFP expression level of a specific strain in the face of two different environmental conditions. The high correlation between the Hsp12-GFP expression levels of the different strains indicates that different partners regulate Msn2 similarly following exposure to different conditions. Data were derived from analysis of deletion strains generated on the S288c background (Supplemental Table S2).

FIGURE 5:

Yeast stress survival and generation time are significantly affected by deletion of several Msn2 partners. Linear correlations between yeast stress survival and Hsp12-GFP expression levels in the different deletion strains relative to the msn4-deleted strain following exposure to heat stress (A) or oxidative stress (B). The r values for the linear fit in A and B are 0.89 and 0.84, respectively. (C) Correlation between the increase in yeast generation time and Hsp12-GFP fluorescence of the different deletion strains (r = 0.8). The increase in generation time was calculated by comparing the strain growth rate at 30°C to that observed at 37°C (see Materials and Methods for a detailed description). Hsp12-GFP fluorescence values were obtained by flow cytometry analysis of the different strains in the absence of stress. All partner deletions were generated on the background of the W303 msn4-deleted strain.

FIGURE 6:

Different effects of deletion of TPK isoforms on Msn2 activity and yeast stress survival. (A) Hsp12-GFP, Tps2-GFP, and Pnc1-GFP expression levels in the different single and double TPK deletion strains following exposure to multiple stress conditions. Reporter gene expression levels were normalized to that of the msn4-deleted strain. (B) Survival and Hsp12-GFP expression levels of the different TPK isoform-deleted strains following exposure to extreme and mild oxidative stress, respectively. Survival and Hsp12-GFP expression levels were normalized to the levels of the msn4-deleted strain. All gene deletions were generated on the background of the W303 msn4-deleted strain.

FIGURE 7:

Whi2 is a central Msn2/4 regulator controlling Msn2 nuclear localization and transcription activity. (A) Changes in Hsp12-GFP and Tps2-GFP in the whi2, psr1, and psr2 deletion strains relative to the msn4-deleted strain examined under nonstress conditions. (B) Nuclear localization of Msn2-GFP is significantly reduced in a whi2-deleted strains following exposure to heat or osmotic stress. The msn5-deleted strain served as a positive control. The percentage of cells containing nuclear-localized Msn2-GFP are highlighted above each column following exposure to osmotic stress (left) or heat stress (right). For each strain, 200 cells were analyzed, and the percentage of cells containing at least a twofold increase in nuclear fluorescence was counted as positive. Arrows indicate representative nuclear localization of Msn2-GFP. All strains analyzed in A and B were generated on the W303 background. (C) Relationships between Whi2 and several Msn2 suppressors indicating that Whi2 activity is independent of the PKA or the TOR pathway. The analyzed strains were generated on the S288c background.

FIGURE 8:

Deletion of UMP1 increases Hsp12-GFP expression levels in the absence of stress and reduces Msn2 degradation and yeast growth rates. (A) A significant increase in Hsp12-GFP expression is seen in the ump1-deleted strain in the absence of stress relative to the WT* strain containing only the msn4 deletion. (B) The slow growth rate of the ump1-deleted strain is suppressed by deletion of the MSN2 gene under nonstress growth conditions. Comparison of the generation times of the different strains indicates significant difference between the ump1 msn4 double-deleted strain and the ump1 msn2 msn4 triple-deleted strain. (C) Reduced degradation rate of Msn2-GFP in the ump1-deleted strain relative to the wild-type or msn5-deleted strains. The decrease in the Msn2-GFP fluorescence level was monitored following cycloheximide addition using flow cytometry. (D) Msn2 is in physical interaction with Ump1, as indicated by yeast two-hybrid analysis. MSN2 was fused to the DNA-activating domain (AD), whereas UMP1 was fused to the DNA-binding domain (BD). All strains, except the Y2H strains, are W303 derivatives. All data presented in A and C represent the average of four independent repeats of each experiment. The data presented in B represent an average of eight independent repeats.