Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression timecourse in clock rescue strains

ABSTRACT: The goal of this experiment was to determine whether global circadian gene expression oscillation depends strictly on the presence of rpaA, even when the KaiABC post-translational oscillator is oscillating with a circadian period. Strains deleted for rpaA lack functional KaiABC post-translational oscillators because their reduced kaiBC expression level leads to a non-permissive Kai protein stoichiometry. We restored KaiABC post-translational oscillator function in a M-NM-^TrpaA M-NM-^TkaiBC strain by ectopic expression of kaiBC from the Ptrc promoter and used microarrays to measure the timecourse of gene expression globally. As a control, we used microarrays to measure the gene expression timecourse in a M-NM-^TkaiBC Ptrc::kaiBC strain, in which gene expression was expected to be rhythmic (Y Murayama et al, J. Bac. 198, 2008), as it is in the pure wild-type strain. Cultures were grown in a flasks bubbled with 1% CO2 in air, initially in the absence of IPTG. Cultures were treated with two consecutive light/dark cycles and released into continuous light at time T = 0, at which time IPTG was added to a final concentration of 6 M-BM-5M. Cultures were samples every 4 hours for 44 h between T = 24 h and T = 68 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray.

ORGANISM(S): Synechococcus elongatus PCC 7942

SUBMITTER: Joseph Markson

PROVIDER: E-GEOD-50920 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. Schizosaccharomyces pombe 972 h- cells were starved for inorganic phosphate for 0, 30, 60, 120, or 240 minutes pior to microarray preparation to determine the extent and temporal resolution of the phosphate starvation response. At 120 minutes post-starvation we define a set of genes that are directly and specifically induced by phosphate starvation, providing a time-point at which all other experiments were performed. We characterize the pho7+- and csk1+- dependency of this PHO response at 120 minutes post-starvation in pho7+csk1+, pho7M-NM-^T, csk1M-NM-^T, and pho7M-NM-^Tcsk1M-NM-^T cells. In a seperate set of experiments we characterized the S. pombe stress response to copper limitation, iron limitation, and carbon switching at 120 minutes post-stress and osmotic shift at 20 minutes post-stress in both pho7+ and pho7M-NM-^T cells.

Project description:The cyanobacterium Synechococcus elongatus contains a circadian clock which coordinates circadian changes in gene expression of a large percentage of its genes. The response regulator RpaA has been implicated as an important regulator of many circadian genes, but the role of this protein in regulating changes in gene expression genome-wide is not known. We show that deletion of rpaA abrogates circadian gene expression genome-wide and arrests cells in a gene expression state highly similar to that of wildtype cells in the morning. Furthermore, we show that RpaA binds DNA in an circadian manner that is dependent on phosphorylation of the protein. To demonstrate the sufficiency of phosphorylated RpaA in driving global changes in gene expression, we used RNA sequencing to measure changes in gene expression elicited by a phosphomimetic of RpaA (RpaA D53E) and compared these changes to those that occur during a circadian cycle in wildtype cells. This analysis reveals that induction of RpaA D53E is sufficient to drive all circadian gene expression changes that happen from dawn to dusk in wildtype cells. Interestingly, the dynamics of gene expression elicited by RpaA D53E induction mirror those observed during a circadian cycle in wildtype cells, suggesting that the dynamics of circadian gene expression and hard-wired in the regulon downstream of RpaA. Enriched mRNA was prepared from synchronized wildtype S. elongatus cells every four hours over a circadian cycle and sequenced using the Illumina TruSeq Stranded mRNA Sample Prep Kit and Illumina HiSeq technology. To test the role of phosphorylated RpaA in driving circadian gene expression, we generated a strain which we refer to as OX-D53E that lacks core clock components (M-NM-^TrpaA, M-NM-^TkaiBC) with an RpaA phosphomimetic (RpaA D53E) under the control of an IPTG-inducible promoter (Ptrc::rpaAD53E). We used the same methodology to measure gene expression in OX-D53E before and after induction of RpaA D53E. As a control, we also measured gene expression in the OX-D53E strain over time in the absence of IPTG. Also, we generated a strain similar to OX-D53E in which the only difference was that no gene was inserted downstream of the IPTG inducible promoter (OX-Empty). We measured gene expressio in OX-Empty before and after IPTG addition to test for off-target effects of IPTG.

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Gene expression timecourse in clock rescue strains

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