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Genetic screen of the yeast environmental stress response dynamics uncovers a succession of regulatory phases

ABSTRACT: Cells respond to environmental fluctuations by regulating multiple transcriptional programs. This response can be studied by measuring the effect of environmental changes on the transcriptome or the proteome of the cell at the end of the response. However, the dynamics of the response reflect working of the regulatory mechanisms in action. Here we utilized a fluorescent stress reporter gene to track the dynamics of protein production in yeast responding to environmental stress. The response is modulated by changes in both the duration and rate of transcription. We probed the underlying molecular pathways controlling these two dimensions using a library of ~1600 single and double mutant strains. Dissection of the effects of these mutants and the interactions between them identified distinct modulators of response duration and response rate. Using a combination of mRNA-seq and live-cell microscopy we uncover mechanisms by which Msn2/4, Mck1, Msn5, and the cAMP/PKA pathway modulate the response of a large module of stress-induced genes in two discrete regulatory phases. Our results and analysis show that transcriptional stress response is regulated by multiple mechanisms that overlap in time and cellular location.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE127851 | GEO | 2019/09/01

REPOSITORIES: GEO

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Project description:Exposure of Saccharomyces cerevisiae to alkaline pH represents a stress condition that generates a compensatory reaction. Here we examine a possible role of the protein kinase-A (PKA) pathway in this response. The phenotypic analysis reveals that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation develops tolerance to this stress. We observe that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding to STRE sequences in many promoters. Consequently, a synthetic STRE-LacZ reporter shows a rapid induction in response to alkaline stress. An msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 minutes of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated gene remodeling. However, the relevance of attenuation of PKA in high pH tolerance is not restricted to regulation of Msn2 function. Eight samples were analyzed: WT and the MCY5278 mutant strain, lacking both Msn2 and Msn4, in the presence of 20 mM KOH (pH 8) and in the presence of 20 mM KCl (non-induced conditions) for 10 and 30 min of stress. 2 biological replicates were analyzed for each condition, and dye-swapping was carried out for each comparison of samples. We compared the expression profiles of: 1) WT +KOH vs. WT +KCl after 10 min 2) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 10 min 3)WT +KOH vs. WT +KCl after 30 min 4) msn2 msn4 mutant +KOH vs. msn2 msn4 +KCl after 30 min Total number of chips analyzed: 16.

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Genetic screen of the yeast environmental stress response dynamics uncovers a succession of regulatory phases

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