A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.
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ABSTRACT:
ORGANISM(S): Halobacterium salinarum NRC-1
PROVIDER: GSE58696 | GEO | 2015/02/18
SECONDARY ACCESSION(S): PRJNA253194
REPOSITORIES: GEO
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Project description:Previous work identified the winged helix-turn-helix DNA binding transcription factor (TF) RosR (encoded by VNG0258H gene), which dynamically regulates expression of more than 300 genes in response to oxidative stress in Halobacterium salinarum (Sharma 2012). RosR is required for survival of oxidants from multiple sources (e.g. H2O2 and paraquat), as deletion mutants are impaired for ROS outgrowth. Genes directly and indirectly controlled by RosR in response to ROS encode macromolecular repair functions. In the current study, we ask which of these genes are direct targets of RosR regulation. Dynamic chromatin immunoprecipitation combined with microarray (ChIP-chip) analysis validates that genes encoding these functions are direct targets of RosR binding and control. In addition, new RosR direct target genes are identified, including those encoding central cellular functions and a surprisingly high number of other TFs. The majority of the 252 sites throughout the genome are RosR-bound in the absence of stress and cleared of RosR binding in the presence of H2O2. However, binding is dynamic, with promoter-specific differences in the timing of RosR-DNA release and re-binding relative to ROS exposure. Halobacterium salinarum harboring VNG0258H(rosR)::myc was grown to mid-logarithmic phase (OD600 ~ 0.2 - 0.4) and either left untreated or exposed to 25 mM H2O2 for 10, 20, and 60 minutes. Transcription factor-chromatin complexes from each treated time point and untreated cultures were then cross-linked in vivo with 1% formaldehyde for 30 min at room temperature and subjected to immunoprecipitation (IP) by virtue of the myc epitope tag as described previously (Schmid et al. 2011). One M-BM-5g of each IP sample was hybridized against matched, mock-treated controls on a custom 2 x 105,000 feature 60-mer oligonucleotide microarray (Agilent Technologies, AMADID 026819). On this high-resolution array, the entire H. salinarum genome was tiled every 30 bp in triplicate. Randomly selected regions of the genome were spotted in quadruplicate. Dye swaps were conducted to correct for bias in incorporation. Seven biological replicate experiments were conducted for VNG0258::myc in the absence of H2O2 and three in the presence of H2O2. Four biological replicate experiments in an H. salinarum strain harboring the empty vector control plasmid were conducted to detect background peaks resulting from technical aspects of the protocol. This yielded a total of at least 18 replicate intensity data points per 30-bp genomic region per condition. DNA fragments were directly labeled with Cy3 and Cy5 dyes (Kreatech) as described previously (Facciotti et al., 2007). Microarray slide hybridization and washing protocols were conducted according to the manufacturerM-bM-^@M-^Ys instructions (Agilent technologies) with the exception that hybridization was conducted in the presence of 37.5% formamide at 68M-KM-^ZC to ensure proper stringency.
2015-02-18 | E-GEOD-58696 | biostudies-arrayexpress
Project description:Previous work has shown that the hypersaline-adapted archaeon, Halobacterium salinarum NRC-1, is highly resistant to oxidative stress caused by exposure to hydrogen peroxide, UV and gamma radiation. Genome-wide dynamics alteration of gene the GRN has been implicated in such resistance. However, the molecular function of transcription regulatory proteins involved in this response remains unknown. Here we have leveraged several existing GRN and systems biology datasets for H. salinarum to identify and characterize a novel winged helix-turn-helix transcription factor, VNG0258H, as a regulator required for reactive oxygen species resistance in this organism. This protein appears to be unique to the haloarchaea at the primary sequence level. Quantitative growth assays in a deletion mutant strain implicate VNG0258H in extreme oxidative stress resistance. According to time course gene expression analyses, this transcription factor is required for the appropriate dynamic response of nearly 300 genes to reactive oxygen species damage from paraquat and hydrogen peroxide. These genes are predicted to function in repair of oxidative damage to proteins and DNA. in vivo DNA binding assays (ChIP-qPCR) demonstrate that VNG0258H binds DNA to mediate gene regulation. Together these results suggest that VNG0258H is a novel archaeal transcription regulatory protein that regulates gene expression to enable adaptation to the extremely oxidative, hypersaline niche of H. salinarum. We have therefore renamed VNG0258H as RosR, for reactive oxygen species regulator. Data in this archive are linked to the publication Sharma KS, Gillum NA, Schmid AK 2012 The M-NM-^Tura3 and M-NM-^TrosR strains were grown to mid-logarithmic phase (OD600 ~ 0.5) in CM supplemented with uracil. For the H2O2 time courses, 4 ML culture aliquots were removed for RNA extraction at three time points prior to the addition of H2O2 (-40 min, -20 min, 0min) and five time points following H2O2 addition (10 min, 20 min, 40 min, 60 min, 80 min). Paraquat time courses were prepared similarly with the exception that additional time points were taken at 2h, 8h, and 24 h after the addition of paraquat. RNA from two biological replicate time courses were prepared, along with a dye filip.
2012-08-12 | E-GEOD-33980 | biostudies-arrayexpress
Project description:Regulation of gene expression by reactive oxygen species (ROS) is an important component of abiotic stress signal transduction mechanisms. Previous genome-wide analysis of chilling-induced changes in gene expression suggested a potential role of H2O2 and other ROS as key signals in the induction of early response genes in japonica rice (cv. Nipponbare). Candidate regulators including bZIP-, Myb- or WRKY-types of transcription factors that were responsive to both chilling (10oC) and exogenous H2O2, and their probable target clusters (as1/ocs/TGA1-like, MybR-like element containing genes) were inferred by integrative analysis of qPCR expression profiles and ab initio promoter motif data. In an effort to define the composition and hierarchical organization of the ROS-mediated chilling response regulatory networks, we examined the effect of exogenous H2O2 (6 hours in 4mM H2O2 at 28oC and then 6 hours of recovery in water at 28oC) on the transcriptome of Nipponbare in a genome-wide scale and compared them with the chilling stress transcriptome. Results of this study were consistent with our earlier model that a ROSbZIP-as1/ocs/TGA1 regulatory module is a direct consequence of chilling-induced elevation of intracellular ROS during the initial 24 hours. Keywords: time course (response to exogenous H2O2)
2009-12-31 | GSE10062 | GEO
Project description:Regulation of gene expression by reactive oxygen species (ROS) is an important component of abiotic stress signal transduction mechanisms. Previous genome-wide analysis of chilling-induced changes in gene expression suggested a potential role of H2O2 and other ROS as key signals in the induction of early response genes in japonica rice (cv. Nipponbare). Candidate regulators including bZIP-, Myb- or WRKY-types of transcription factors that were responsive to both chilling (10oC) and exogenous H2O2, and their probable target clusters (as1/ocs/TGA1-like, MybR-like element containing genes) were inferred by integrative analysis of qPCR expression profiles and ab initio promoter motif data. In an effort to define the composition and hierarchical organization of the ROS-mediated chilling response regulatory networks, we examined the effect of exogenous H2O2 (6 hours in 4mM H2O2 at 28oC and then 6 hours of recovery in water at 28oC) on the transcriptome of Nipponbare in a genome-wide scale and compared them with the chilling stress transcriptome. Results of this study were consistent with our earlier model that a ROSbZIP-as1/ocs/TGA1 regulatory module is a direct consequence of chilling-induced elevation of intracellular ROS during the initial 24 hours. Keywords: time course (response to exogenous H2O2) This experiment describes the analysis of the temporal changes in gene expression in response to exogenous H2O2 in Nipponbare rice. A total of 5 treatment regimes were examined (1, 3, and 6 hours at 4 mM H2O2 and then 3 and 6 hours of recovery in water after 6 hours of exposure to H2O2). The microarray platform used in this study was the 45K oligonucleotide microarray (www.ricearray.org) which comes in pairs of slides A and B (each containing about 50% of the total number of features). The data described in this series include all hybridizations with slide A and with slide B. All experiments were performed with two independent biological replicates (R1 and R2) for each time point.
2010-06-24 | E-GEOD-10062 | biostudies-arrayexpress
Project description:GAPDHs from human pathogens S. aureus and P. aeruginosa can be readily inhibited by ROS-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 induces rapid metabolic reroute from glycolysis to pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces a metabolic reroute from glycolysis to PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Given that H2O2 can be produced constitutively by the host immune response, exposure to the steady-state stress of H2O2 recapitulates more accurately bacterial responses to host immune system in vivo. RNA-seq in Pseudomonas aeruginosa and Staphylococus aureus under steady state of H2O2
2014-05-08 | E-GEOD-55528 | biostudies-arrayexpress
Project description:Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H202, as well as biotic and abiotic induced redox signals. Inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Overexpressor (oe) lines are impaired in root and shoot development, light sensitive and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors which scavenge ROS. More than 850 stress-, redox-, ROS regulated-, ROS scavenging-, defense-, cell death- and senescence-related genes are regulated by RRTF1, ~ 30% of them have ROS related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box and GCC-box like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli as well as H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains GCC-box like sequene in its promoter, but RAP2.6 oe lines do not accumulate higher ROS levels. RRTF1 stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the highly conserved RRTF1 rapidly, transiently and systemically induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals. Necrotrophs stimulate RRTF1 expression, while symbiotic interactions of Arabidopsis with (hemi)-biotrophs and P. indica do not affect or repress RRTF1 expression. The seedlings were grown on MS medium with 1.37% sucrose under short day conditions and low light intensity (30 µmol m-2s-1) at 20˚C for 14 days. Transcriptome analysis was performed for the rrtf1 knock-out line and a RRTF1-overlexpressor line (called oe18) in comparison to wild-type seedlings.
2015-04-23 | E-GEOD-64783 | biostudies-arrayexpress
Project description:Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H202, as well as biotic and abiotic induced redox signals. Inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Overexpressor (oe) lines are impaired in root and shoot development, light sensitive and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors which scavenge ROS. More than 850 stress-, redox-, ROS regulated-, ROS scavenging-, defense-, cell death- and senescence-related genes are regulated by RRTF1, ~ 30% of them have ROS related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box and GCC-box like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli as well as H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains GCC-box like sequene in its promoter, but RAP2.6 oe lines do not accumulate higher ROS levels. RRTF1 stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the highly conserved RRTF1 rapidly, transiently and systemically induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals. Necrotrophs stimulate RRTF1 expression, while symbiotic interactions of Arabidopsis with (hemi)-biotrophs and P. indica do not affect or repress RRTF1 expression. Mature leaves of 5 weeks-old Col-0 wild type and RRTF1-overexpressor Arabidopsis (called oe18), which were grown on soil under short-day condition at 20˚C, were subjected to RNA extraction and Affymetrix microarray analysis. Three biological independent experiments for both Col-0 and oe18 were performed.
2015-04-23 | E-GEOD-64853 | biostudies-arrayexpress
Project description:GAPDHs from human pathogens S. aureus and P. aeruginosa can be readily inhibited by ROS-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 induces rapid metabolic reroute from glycolysis to pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces a metabolic reroute from glycolysis to PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Given that H2O2 can be produced constitutively by the host immune response, exposure to the steady-state stress of H2O2 recapitulates more accurately bacterial responses to host immune system in vivo.
2014-05-08 | GSE55528 | GEO
Project description:In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis. Keywords: Gene Expression, C2C12 Myotubes, Oxidative Stress, Adaptation
2005-08-11 | GSE3078 | GEO
Project description:Aims: We explore the role of elevated O2-:H2O2 ratio as a prosurvival signal in glioma-propagating cells (GPCs). We hypothesize that depleting this ratio sensitizes GPCs to apoptotic triggers. Results: We observed that elevated O2-:H2O2 ratio conferred enhanced resistance in GPCs, and depletion of this ratio by pharmacological and genetic methods sensitized cells to apopotic triggers. We established the ROS Index as a quantitative measure of normalized O2-:H2O2 ratio and determined its utility in predicting chemosensitivity. Importantly, mice implanted with GPCs of reduced ROS Index demonstrated extended survival. Analysis of tumor sections revealed effective targeting of CD133- and nestin-expressing neural precursors. Furthermore, we established the Connectivity Map to interrogate a gene signature derived from varied ROS Index for patterns of association with individual patient gene expression in 2 clinical databases. We showed that patients with reduced ROS Index demonstrate better survival. These data provide clinical evidence for the viability of our O2-:H2O2-mediated chemosensitivity profiles. Innovation and Conclusion: Gliomas are notoriously recurrent and highly infiltrative, and have been shown to arise from stem-like cells. We implicate elevated O2-:H2O2 ratio as a prosurvival signal in GPC self-renewal and proliferation. The ROS Index provides quantification of O2-:H2O2-mediated chemosensitivity, an advancement in a previously qualitative field. Intriguingly, glioma patients with reduced ROS Index correlate with longer survival and the Proneural molecular classification, a feature frequently associated with tumors of better prognosis. These data emphasize the feasibility of manipulating the O2-:H2O2 ratio as a therapeutic strategy. Total RNA from primary neurosphere culture of brain tumor specimens treated with DPI and DDC as well as non-treated CD133+ and CD133- fractions were compared. Specimens were obtained from 3 patients and replicate arrays were performed for all 3 neurosphere cultures.
2013-03-13 | E-GEOD-32335 | biostudies-arrayexpress