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

ABSTRACT: 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 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. Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 to 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 139 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 transcription factors 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. 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 the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involves calcium signaling, such as exposition to sexual pheromones or saline stress.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE79342 | GEO | 2016/06/27

SECONDARY ACCESSION(S): PRJNA315547

REPOSITORIES: GEO

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Project description: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 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. Environmental alkalinization promoted a time-dependent recruitment of Crz1 to 152 intergenic regions, the vast majority between 1 to 5 min upon stress onset. Positional evaluation of the genomic coordinates combined with existing transcriptional studies allowed identifying 139 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 transcription factors 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. 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 the relevance of calcineurin/Crz1 in signaling diverse stress conditions, our data will contribute to understand the transcriptional response in other circumstances that also involves calcium signaling, such as exposition to sexual pheromones or saline stress. ChIP-Seq from five time points after alkalinization (t=0, 1, 2, 5 and 20 min) was collected from two independent experiments (A and B) and the ratio Tx/T0 was calculated.

Project description:Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that Crz1 is dephosphorylated by calcineurin, and crz1Δ mutants display phenotypes intermediate between wild-type and calcineurin mutants. RNA-sequencing revealed 102 genes that are regulated in a calcineurin/Crz1-dependent manner at 37°C. 99 genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin/Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. Surprisingly, only five genes have orthologs known to be calcineurin/Crz1-dependent in S. cerevisiae. 393 genes are independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Taken together, these results indicate that the calcineurin/Crz1-dependent pathway controls a transcriptional circuit that has been extensively rewired in C. neoformans compared to S. cerevisiae. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output has been extensively rewired to promote fungal virulence.

Project description:Gene regulation in response to intracellular calcium is mediated by the calcineurin-activated transcription factor Prz1 in the fission yeast Schizosaccharomyces pombe. Genome-wide studies of the Crz1 and CrzA fungal orthologs have uncovered numerous target genes involved in conserved and species-specific cellular processes. In contrast, very few target genes of Prz1 have been published. This paper identified an extensive list of genes using transcriptome and ChIP-chip analyses under inducing conditions of Prz1, including CaCl2, and tunicamycin treatment, as well as a âpmr1 genetic background. We identified 165 upregulated putative target genes of Prz1 in which the majority contained a calcium-dependent response element in their promoters, similar to that of the Saccharomyces cerevisiae ortholog Crz1. These genes were functionally enriched for Crz1-conserved processes such as cell wall biosynthesis. Overexpression of prz1+ increased resistance to the cell wall degradation enzyme zymolyase, likely from upregulation of the O-mannosyltransferase encoding gene omh1+. Loss of omh1+ abrogates this phenotype. We uncovered a novel inhibitory role in flocculation for Prz1. Loss of prz1+ resulted in constitutive flocculation and upregulation of genes encoding the flocculins Gsf2 and Pfl3, as well as the transcription factor Cbf12. The constitutive flocculation of the âprz1 strain was abrogated by the loss of gsf2+ or cbf12+. This study reveals that Prz1 functions as a positive and negative transcriptional regulator of genes involved in cell wall biosynthesis and flocculation, respectively. Moreover, comparison of target genes between Crz1/CrzA and Prz1 indicate some conservation in DNA-binding specificity, but also substantial rewiring of the calcineurin-mediated transcriptional-regulatory network. We generated 3 overexpression microarrays and 2 deletion microarrays with dye swaps, and 2 deletion microarrays with drug treatments with dye swaps, each with a biological replicate performed as a dye swap. The effect of the mutant strains were all compared to wild type or empty vector strains, except for one experiment where the prz1 deletion mutant was compared to the pmr1 deletion mutant.

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

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