Genome-wide recruitment profiling of transcription factor Crz1 in response to high pH stress

Background: Exposure of the budding Saccharomyces cerevisiae to an alkaline environment produces a robust transcriptional response involving hundreds of genes. Part of this response is triggered by an almost immediate burst of calcium that activates the Ser/Thr protein phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor (TF) Crz1, which moves to the nucleus and binds to calcineurin/Crz1 responsive gene promoters. In this work we present a genome-wide study of the binding of Crz1 to gene promoters in response to high pH stress. Results: Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 and 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 140 genes likely responsive to Crz1 regulation. Gene Ontology analysis confirmed the relevant impact of calcineurin/Crz1 on a set of genes involved in glucose utilization, and uncovered novel targets, such as genes responsible for trehalose metabolism. We also identified over a dozen of genes encoding TFs that are likely under the control of Crz1, suggesting a possible mechanism for amplification of the signal at the transcription level. Further analysis of the binding sites allowed refining the consensus sequence for Crz1 binding to gene promoters and the effect of chromatin accessibility in the timing of Crz1 recruitment to promoters. Conclusions: The present work defines at the genomic-wide level the kinetics of binding of Crz1 to gene promoters in response to alkaline stress, confirms diverse previously known Crz1 targets and identifies many putative novel ones. Because of the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involve calcium signaling, such as exposition to sexual pheromones or saline stress

Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study

[EN] Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase.This work was supported by grants BFU2011-30197-C3-01, BFU2014-54591-C2-1-P and EUI2009-04147 (SysMo2) to JA. (Ministry of Industry and Competitivity, Spain, and Fondo Europeo de Desarrollo Regional [FEDER]). JA is the recipient of an Ajut 2014SGR-4 award (Generalitat de Catalunya). DC was recipient of a predoctoral fellowship from the Spanish Ministry of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Petrezsélyová, S.; López-Malo, M.; Canadell, D.; Roque, A.; Serra-Cardona, A.; Marques Romero, MC.; Vilaprinyó, E.... (2016). Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study. PLoS ONE. 11(6):e0158424-e0158424. https://doi.org/10.1371/journal.pone.0158424Se0158424e015842411

Genome-wide recruitment profiling of transcription factor Crz1 in response to high pH stress

Background: Exposure of the budding Saccharomyces cerevisiae to an alkaline environment produces a robust transcriptional response involving hundreds of genes. Part of this response is triggered by an almost immediate burst of calcium that activates the Ser/Thr protein phosphatase calcineurin. Activated calcineurin dephosphorylates the transcription factor (TF) Crz1, which moves to the nucleus and binds to calcineurin/Crz1 responsive gene promoters. In this work we present a genome-wide study of the binding of Crz1 to gene promoters in response to high pH stress. Results: Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 and 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 140 genes likely responsive to Crz1 regulation. Gene Ontology analysis confirmed the relevant impact of calcineurin/Crz1 on a set of genes involved in glucose utilization, and uncovered novel targets, such as genes responsible for trehalose metabolism. We also identified over a dozen of genes encoding TFs that are likely under the control of Crz1, suggesting a possible mechanism for amplification of the signal at the transcription level. Further analysis of the binding sites allowed refining the consensus sequence for Crz1 binding to gene promoters and the effect of chromatin accessibility in the timing of Crz1 recruitment to promoters. Conclusions: The present work defines at the genomic-wide level the kinetics of binding of Crz1 to gene promoters in response to alkaline stress, confirms diverse previously known Crz1 targets and identifies many putative novel ones. Because of the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involve calcium signaling, such as exposition to sexual pheromones or saline stress

Regulation of the Na+/K+-ATPase Ena1 expression by calcineurin/Crz1 under high pH stress : a quantitative study

Regulated expression of the Ena1 Na+-ATPase is a crucial event for adaptation to high salt and/or alkaline pH stress in the budding yeast Saccharomyces cerevisiae. ENA1 expression is under the control of diverse signaling pathways, including that mediated by the calcium-regulatable protein phosphatase calcineurin and its downstream transcription factor Crz1. We present here a quantitative study of the expression of Ena1 in response to alkalinization of the environment and we analyze the contribution of Crz1 to this response. Experimental data and mathematical models substantiate the existence of two stress-responsive Crz1-binding sites in the ENA1 promoter and estimate that the contribution of Crz1 to the early response of the ENA1 promoter is about 60%. The models suggest the existence of a second input with similar kinetics, which would be likely mediated by high pH-induced activation of the Snf1 kinase

Strains used in this study.

<p>Strains used in this study.</p

Impact of the absence of Crz1 on mRNA levels and Ena1 protein accumulation after high pH stress.

<p>A) Time-course of the accumulation of Ena1 mRNA after shifting cells to pH 8.0. Total RNA from wild type (closed circles) and <i>crz1</i> (open circles) cells was extracted and mRNA levels were determined by qRT-PCR and quantified as indicated in Materials and Methods. B) The strains described above were transformed with plasmid pKC201, carrying the <i>lacZ</i> reporter fused to the <i>ENA1</i> promoter. Cells were exposed to pH 8.0 for the indicated times, collected, and β-galactosidase activity measured. C) Strain MLM001 (<i>CRZ1</i>) and MLM002 (<i>crz1</i>), carrying in both cases C-terminally GFP-tagged version of <i>ENA1</i> were shifted to pH 8.0 for the indicated times and the amount of total Ena1 quantified by immunoblot using anti-GFP antibodies and internal standards of recombinant GFP protein. Data is presented as the mean ± SEM from 3 (panel A) or 4 (panels B and C) independent experiments.</p

Relative levels of Crz1 after switching cells to pH 8.0.

<p>The number of Crz1 molecules/cell was calculated as described in Material and Methods and the value just prior alkalinization of the medium was set as the reference (100%). Data correspond to the mean ± SEM from 8 independent experiments.</p

Time-course profile of Crz1 recruitment at the <i>ENA1</i> promoter following alkaline stress.

<p>A) Cumulative counts of reads using a 50-nt window spanning the <i>ENA1</i> promoter region are represented as fold-change over reads counted at time 0. The positions of CDRE deduced from computational analysis using the PWMTools website (<a href="http://ccg.vital-it.ch/pwmtools/pwmscan.php" target="_blank">http://ccg.vital-it.ch/pwmtools/pwmscan.php</a>) of the Swiss Bioinformatics Institute are indicated as colored boxes on the black line. Red boxes denote the CDRE previously characterized in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158424#pone.0158424.ref023" target="_blank">23</a>]. B) Evaluation of relative Crz1 binding to CDREs at positions -813/-821 and -719/-727 (red boxes in panel A). Chromatin immunoprecipitation using HA-tagged Crz1 was carried out and the immunoprecipitate used to amplify the -680/792; -771/-917; and -986/-1120 promoter regions (denoted as thick short lines 1, 2 and 3, respectively, in panel A). Data is presented as the mean ± SEM from 3–4 individual experiments assayed in duplicate and expressed as relative to the maximum value.</p

Genome-wide recruitment of Crz1 to gene promoters in response to high pH stress.

<p>ChIP-seq technology was used to identify intergenic regions immunoprecitating with Crz1 and accumulating at least two-fold reads than the genome average. A) The 150 genes showing peaks of reads above the indicated threshold were ranked according the number of reads at the peak. The relative occupancy with respect the highest value (<i>HOR7</i>) is shown for the top ten genes in the ranking. B) The percentage of genes showing the peak of accumulation of reads at a given time after alkalinization of the medium is shown.</p