A. fumigatus wild type strain transcriptome analysis upon 200 mM CaCl2 treatment.
Ontology highlight
ABSTRACT:
ORGANISM(S): Aspergillus fumigatus
PROVIDER: GSE9516 | GEO | 2007/11/09
SECONDARY ACCESSION(S): PRJNA103317
REPOSITORIES: GEO
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Project description:CRZ is a C2H2 zinc finger transcription factor activated by calcineurin, a protein phosphatase activated by calcium-calmodulin, in different stress conditions related with calcium cell accumulation. CRZ binds to a specific element on the promoter region of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for calcium- and calcineurin-dependent gene expression. In order to investigate which pathways are involved under calcium stress conditions, we determined the transcriptional profile of A. fumigatus delta crzA mutant strain upon a time course 200 mM calcium chloride treatment. Conidia from the mutant and wild type strains were incubated at 37°C in complete medium for 16 hours and were challenged with 200 mM calcium chloride for 10 and 30 minutes. At each time point, the mycelia were harvested by centrifugation and used for competitive microarray hybridizations. We were able to observe a decreased mRNA expression of genes encoding inorganic ion transport proteins and lipid transport. In contrast, increased mRNA expression was observed for several genes involved in transcription, energy production and conversion, cell cycle control, cell division, chromosome partitioning, amino acid, lipid and carbohydrate transport and metabolism, nucleotide transport, cell wall and membrane biogenesis, posttranslation modifications, protein turnover, chaperones, inorganic ion transport, signal transduction mechanisms, intracellular trafficking, secretion and vesicular transport. Keywords: gene expression array-based (log2 ratio)
2009-04-02 | GSE15432 | GEO
Project description:CRZ is a C2H2 zinc finger transcription factor activated by calcineurin, a protein phosphatase activated by calcium-calmodulin, in different stress conditions related with calcium cell accumulation. CRZ binds to a specific element on the promoter region of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for calcium- and calcineurin-dependent gene expression. In order to investigate which pathways are involved under calcium stress conditions, we determined the transcriptional profile of A. fumigatus delta crzA mutant strain upon a time course 200 mM calcium chloride treatment. Conidia from the mutant and wild type strains were incubated at 37°C in complete medium for 16 hours and were challenged with 200 mM calcium chloride for 10 and 30 minutes. At each time point, the mycelia were harvested by centrifugation and used for competitive microarray hybridizations. We were able to observe a decreased mRNA expression of genes encoding inorganic ion transport proteins and lipid transport. In contrast, increased mRNA expression was observed for several genes involved in transcription, energy production and conversion, cell cycle control, cell division, chromosome partitioning, amino acid, lipid and carbohydrate transport and metabolism, nucleotide transport, cell wall and membrane biogenesis, posttranslation modifications, protein turnover, chaperones, inorganic ion transport, signal transduction mechanisms, intracellular trafficking, secretion and vesicular transport. Keywords: gene expression array-based (log2 ratio) For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus wild type [CEA17 delta akuB(KU80)] and delta crzA strains were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C. After this time, the cultures were added by calcium chloride (200 mM) and incubated for 10 and 30 more minutes before mycelia recovery and RNA extraction. Hybridization experiments were competitive using probes derived from calcium chloride shock cultures using the wild type strain exposed to calcium (10 or 30 minutes) as the reference RNA for every hybridizantion. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene's in-slide replicates were averaged (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects. These final, processed data are linked below as supplementary files.
2010-06-20 | E-GEOD-15432 | biostudies-arrayexpress
Project description:Gene expression was quantified in S. cerevisae cells over a time course of three days of CaCl2 treatment to activate calcineurin. crz1 and crz1 cnb1 mutant strains were compared to allow the identification of calcineurin-dependent gene expression changes that are independent of the downstream transcription factor Crz1. We show that mitochondrial genes are downregulated in both geneotypes over time. Additionally, genes that promote glutathione biosynthesis are upregulated in cells that express calcineurin.
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Project description:CRZ1 (Calcineurin Responsive Zinc finger 1) is a conserved transcription factor that relays Ca2+/calcineurin signals within the cell. To catalog genome wide locations that MoCRZ1 binds to, we have conducted chromatin immunoprecipitation coupled with microarray (ChIP-chip) in the rice blast fungus, Magnaporthe oryzae. Fungal mycelia were treated with 200mM CaCl2 to activate translocation of MoCRZ1 to the nucleus, thereby binding to the promoter regions of downstream genes, and CaCl2 together with 10ug/ml FK506 to block these phenomena. Using ChIP-chip, we were able to identify over 100 direct targets of MoCRZ1 which include well known downstream genes as well as previously unknown genes. Fungal mycelia expressing MoCRZ1::GFP under its native promoter were treated with 200mM CaCl2. MoCRZ1 bound DNA fragments which were immunoprecipitated with antiGFP-antibody were compared with 30% input DNA. Mycelia treated with 200mM CaCl2 and 10ug/ml FK506 were processed as the same way and used as negative control treatment.
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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.
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Project description:Gene expression was quantified in S. cerevisae cells over a time course of three days of CaCl2 treatment to activate calcineurin. Wild type (WT) and cnb1 mutant strains were compared to allow the identification of calcineurin-dependent gene expression changes. We show that mitochondrial genes are downregulated, and ribosome biogenesis genes are upregulated, in both geneotypes over time.
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Project description:The Ca2+/calcineurin signaling pathway is a central conduit regulating growth, development, and virulence of fungal pathogens infecting plants and human. We have analyzed global gene expression profiles during Ca2+ treatment in the rice blast fungus, Magnaporthe oryzae. An immunosuppressive drug, FK506, and the knock-out mutant of a transcription factor, MoCRZ1, were included to analyze calcineurin- and/or CRZ1-dependent gene expression, respectively. About 1,400 genes were up or down regulated by Ca2+ treatment, while about 200 genes seemed to be up-regulated in a calcineurin/CRZ1-dependent manner. This study analyzes global gene expression dynamics during Ca2+ treatment in the rice blast fungus. We have used Agilent M. grisea 2.0 4×44K Microarrays using a single channel hybridization design. An immunosuppressive drug, FK506, and the knock out mutant of a transcription factor, MoCRZ1, were included to analyze calcineurin- and/or CRZ1-dependent gene expression, respectively. To know the time point at which the gene expression fluctuates most significantly, we treated mycelia with CaCl2 for 0, 15, 30, and 60 min. PMC1 expression level was checked by RT-PCR with the highest expression at 30 min time point. Four biological replicates of wild type and mutant mycelia were harvested after 30 min treatment with chemicals. Pairwise comparisons between Ca2+ treated vs. control and Ca2+ vs. Ca2+/FK506 in wild type (WT), and Ca2+ treated in WT vs. in mutant were conducted.
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Project description:Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae, to investigate the mechanism of action of this compound. PFA elicited a transcriptome response indicative of a Ca2+ imbalance, affecting the expression of genes known to be responsive to altered cellular calcium levels. Several additional lines of evidence obtained supported a role for Ca2+ in PFA's activity. First, mutants lacking calcineurin and various Ca2+ transporters including pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1) showed increased sensitivity to PFA. In addition, the calcineurin inhibitors FK506 and cyclosporine A strongly enhanced PFA activity in wild-type cells. Furthermore, PFA activated the transcription of a lacZ reporter driven by the calcineurin-dependent response element. Finally, elemental analysis indicated a significant increase in intracellular calcium levels in PFA-treated cells. Collectively, our results demonstrate that PFA mediates its antifungal activity by perturbing Ca2+ homeostasis, thus representing a potentially novel mechanism distinct from that of currently used antifungal agents. DNA microarray analysis of gene expression response to the antifungal compound plakortide F acid in yeast S. cerevisiae S288C cells at OD 0.2 were treated with either plakortide F acid (PFA) at IC50 concentration (0.62 ug/ml), or solvent (0.25% DMSO), allowed to grow to OD 0.5, then harvested and frozen. Three biological replicate samples were analyzed for each treatment.
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Project description:Plakortide F acid (PFA), a marine-derived polyketide endoperoxide, exhibits strong inhibitory activity against the opportunistic fungal pathogens Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model organism Saccharomyces cerevisiae, to investigate the mechanism of action of this compound. PFA elicited a transcriptome response indicative of a Ca2+ imbalance, affecting the expression of genes known to be responsive to altered cellular calcium levels. Several additional lines of evidence obtained supported a role for Ca2+ in PFA's activity. First, mutants lacking calcineurin and various Ca2+ transporters including pumps (Pmr1 and Pmc1) and channels (Cch1 and Mid1) showed increased sensitivity to PFA. In addition, the calcineurin inhibitors FK506 and cyclosporine A strongly enhanced PFA activity in wild-type cells. Furthermore, PFA activated the transcription of a lacZ reporter driven by the calcineurin-dependent response element. Finally, elemental analysis indicated a significant increase in intracellular calcium levels in PFA-treated cells. Collectively, our results demonstrate that PFA mediates its antifungal activity by perturbing Ca2+ homeostasis, thus representing a potentially novel mechanism distinct from that of currently used antifungal agents. DNA microarray analysis of gene expression response to the antifungal compound plakortide F acid in yeast
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Project description:In Saccharomyces cerevisiae, the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, is activated by specific environmental conditions, including exposure to Ca2+ and Na+, and induces gene expression by regulating the Crz1p/Tcn1p transcription factor. We used DNA microarrays to perform a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca2+ (200 mM) or Na+ (0.8 M) to yeast. 163 genes exhibited increased expression that was reduced 50% or more by calcineurin inhibition. These calcineurin dependent genes function in signaling pathways, ion/small molecule transport, cell wall maintenance, vesicular transport, and include many open reading frames of heretofore-unknown function. Three distinct gene classes were defined: 28 genes displayed calcineurin-dependent induction in response to Ca2+ and Na+, 125 showed calcineurin-dependent expression following Ca2+ but not Na+ addition, and 10 were regulated by calcineurin in response to Na+ but not Ca2+. Analysis of crz1D cells established Crz1p as the major effecter of calcineurin-regulated gene expression in yeast. We identified the Crz1p binding site as 5-GNGGC(G/T)CA-3 by in vitro site selection. A similar sequence, 5-GAGGCTG-3, was identified as a common sequence motif in the upstream regions of calcineurin/Crz1p-dependent genes. This finding is consistent with direct regulation of these genes by Crz1p.
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