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Transcriptional profiling analysis of S. pombe kinase deletin strains treated (60min) or untreated (0min) with nitrogen starvation in comparison with a common reference.

ABSTRACT: Kinase deletion strains treated with or without nitrogen starvation Background: Protein kinase-mediated signaling pathways play a pivotal role in regulation of stress responses. Nitrogen starvation (NS) induces sexual development when mating partner available or entry into a quiescent state (G0) in heterothallic background. However, it is unclear whether transcriptional profiles in response to NS allows distinguishing between kinase deletion strains defective in sexual development and/or G0-arrest. Results: In this study, we used DNA microarray to analyze transcriptional profiles in 68 protein kinase deletion strains prior to and after NS. Microarray analyses indicated that NS-induced transcriptional profiles of all kinase strains plus wild type are highly correlated with one another (i.e., Pearson¡¯s correlation coefficient r > 0.7) except for that of pka1¦¤, which showed no high correlation with other 11 kinase strains. Significantly, 9 of 11 strains were defective in sexual development, suggesting that the NS-induced profile of pka1¦¤ serves as a signature with no high correlation with profiles of kinase strains defective in sexual development. Furthermore, we show that expression of the NS-induced genes is elevated in pka1¦¤ and cyr1¦¤ strains prior to NS. On the other hand, levels of the NS-induced genes expression in pka1¦¤, cyr1¦¤, sty1¦¤, and byr1¦¤ are significantly lower than that of wild type. Conclusions: Taken together, our analyses indicate that the cAMP-dependent Pka1-mediated signaling pathway play a major role in regulation of the NS-induced genes prior to and after NS. In addition, the ROS-activated kinase Sty1 and the pheromone-induced kinase Byr1 are also involved in up-regulation of NS-induced genes upon NS stress.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE47544 | GEO | 2014/05/31

SECONDARY ACCESSION(S): PRJNA206060

REPOSITORIES: GEO

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Project description:Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which subsequently enter meiosis and sporulate. Prior to mating, M cells secrete M-factor and P-cells secrete P-factor, and these diffusible mating pheromone can activate a signal transduction pathway in the opposite cell type. The pheromone response orchestrates mating and is also required for entry into meiosis. Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone stimulation independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. As expected, there was a substantial overlap between the genes induced by M- and P-factor. Surprisingly, we found that pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M-specific genes and one new P-specific gene. Finally, our results support the notion that Ste11 is the key transcription factor activated by pheromone signalling. Keywords: dose response Gene expression profiling experiment in which gene expression in pheromone treated cultures was compared to that of untreated control cells. All cultures were growing exponentially in MSL minimal medium. S. pombe h+ cyr1- cells treated with M-factor mating pheromone for 2, 4 and 6 hrs respectively, were compared to non-treated cells. Similarly, S. pombe h- sax2- cyr1- cells treated with P-factor mating pheromone for 2, 4 and 6 hrs, were compared to non-treated cells. For identiofication of cell-type specific gene expression, h+ cyr1- cells were compaired to h+ sxa2- cyr1- cells. This was done both before pheromone treatment (0h/0h) and after 6 hours of pheromone treatment (6h/6h). RNA was extracted and subjected to cDNA expression profiling analysis using the established protocols (Xue et al, Yeast 21, 25-39, 2004).Data normalized with 'Lowess' per chip per spot normalization.

Project description:The conserved cAMP-dependent protein kinase (PKA) holoenzyme is composed of two catalytic and two regulatory subunits. It plays critical roles in the regulation of many biological processes in eukaryotic organisms. In the human fungal pathogen Candida albicans, the PKA kinase has been extensively investigated for its importance in the regulation of morphological transitions and virulence. It has been long thought that the PKA catalytic subunit is essential for cell viability in C. albicans. Paradoxically, the single adenylyl cyclase-encoding gene, CRY1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. In this study, we successfully generated a null double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1 or t2t1), which encode two isoforms of the PKA catalytic subunit in C. albicans. We reevaluated the roles of the PKA catalytic subunit in cell growth and phenotypic transitions. Inactivation of the PKA catalytic subunit by deletion of both TPK1 and TPK2 blocked filamentation and dramatically attenuated the ability of white-to-opaque switching, but promoted sexual mating in C. albicans. Tpk2 plays a major role in these regulations, while Tpk1 generally functions as a negative regulator in morphological transitions and sexual mating. A comparative transcriptomic analysis demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared to the WT strain, the general transcriptional activity and expression of genes involved in metabolism, translation, biosynthesis, adhesion and filamentation are significantly decreased in both the t2t1 and cyr1/cyr1 mutants. And a portion of stress-response and cell wall-related genes were upregulated in these mutants, which is consistent with their increased ability of anti-stresses. To further explore the global regulatory role of the PKA kinase, we performed quantitative phosphoproteomics analysis. Combining with bioinformatics analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. Cell wall and membrane-related proteins (e.g. Ecm3, Bni1, and Smi1) were enriched in Tpk1-specific targets, while Tpk2-specific substrates include transporters, filamentation and cytoskeleton-related proteins (e.g. Smf3, Sep7, and Mhp1). There were also many Tpk1 and Tpk2 overlapped and coordinately regulated-substrates. Our study clarifies the essentiality of the PKA catalytic subunit and shed new insights into the global regulatory features of the cAMP/PKA pathway in C. ablicans. The t2t1 null mutant generated in this study would also be a new resource for the field to study this important pathway.

Dataset Information

Transcriptional profiling analysis of S. pombe kinase deletin strains treated (60min) or untreated (0min) with nitrogen starvation in comparison with a common reference.

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