Unknown,Transcriptomics,Genomics,Proteomics

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A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

ABSTRACT: 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.

ORGANISM(S): Halobacterium sp. NRC-1

SUBMITTER: Amy Schmid

PROVIDER: E-GEOD-58696 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

Tonner Peter D PD Pittman Adrianne M C AM Gulli Jordan G JG Sharma Kriti K Schmid Amy K AK

PLoS genetics 20150108 1

Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bac ...[more]

PMID: 25569531

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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.

Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, AgmR. Like TrmB, its thermophilic archaeal homolog, AgmR regulates glycolytic and gluconeogenic pathways in response to sugar availability. However, using high throughput genome-scale experiments, we find that AgmR directly governs the transcription of nearly 100 additional genes encoding enzymes in diverse metabolic pathways. Genome-scale in vivo binding site location data reveals that >60% of these are direct targets. Integration of these systems-scale datasets with metabolic reconstruction models suggests that AgmR, a sequence-specific bacterial-like regulator, interacts with the general transcription factor machinery to coordinate nitrogen and carbon metabolism with the de novo synthesis of cognate cofactors and reducing equivalents, achieving system-wide redox and energy balance. Halobacterium salinarum NRC-1 (ATCC700922) ura3 parent and VNG1451C strains were grown in rich medium (CM, 250 NaCl, 20 g/L MgSO4?7H2O, 3 g/L sodium citrate, 2 g/L KCl, 10 g/L peptone) with or without varying concentrations of glucose or glycerol at 37ÂºC under full-spectrum white light. The cells were cross-linked with 1.2% formaldehyde, then lysed and DNA sheared via sonication. Next cleared cell lysate was subject to immunoprecipitation against an anti-myc antibody. Protein-DNA complexes were purified and DNA was isolated after protease and RNAase treatment. DNA was amplified using universal linkers, labeled directly using Kreatech 547 and 647 dyes. Samples were grown in biological duplicate. Each DNA sample was hybridized against 2 microarrays as dyefilps. At least two experiments (including biological duplicates) were carried out.

Project description:Oxidative stress (OS) results from genetic defects or stressful environmental challenges that cause unchecked production of reactive oxygen species (ROS). OS has been implicated in many diseases due to its wide ranging damage to nucleic acids, proteins and lipids. Using Halobacterium salinarum NRC-1, an extremophile that thrives under conditions of excessive OS, we have constructed a systems model for oxidative stress response (OSR) by integrating transcriptional changes induced by treatments with H2O2 and paraquat, functional associations inferred through comparative genomics, and de novo discovered cis-regulatory motifs. Together with phenotypic analysis of mutant strains this has revealed a multi-tiered OS management program to transcriptionally coordinate three peroxidase/catalase enzymes, two superoxide dismutases, production of rhodopsins, carotenoids, and gas vesicles, metal trafficking, and various other aspects of metabolism. Remarkably, a significant fraction of this OSR was accurately recapitulated by a model that was previously constructed from cellular responses to diverse environmental perturbations –this constitutes the general stress response component. Notwithstanding this observation, comparison of the two models has identified the coordination of frontline defense and repair systems by regulatory mechanisms that are triggered uniquely by severe OS and not by other environmental stressors, including sub-inhibitory levels of redox-active metals, extreme changes in oxygen tension, and a sub-lethal dose of g rays. Mid-log phase cultures of H. salinarum NRC-1 (2 biological replicates) grown in flasks were treated with either sub-lethal concentrations of 25mM H2O2 or 4mM PQ and incubated with shaking for up to 240 min. For each treatment, two time courses were run to determine the transcriptional responses to (1) constant stress and (2) recovery of H. salinarum NRC-1 to H2O2 and PQ. During constant stress, culture aliquots (~4ml) were collected over a time course (-1, 5, 10, 20, 40, 80 and 160 minutes), centrifuged (16000g, 90 seconds) and flash frozen. For recovery, cells were first treated for 2-hours with either 25 mM H2O2 or 4mM PQ, recovered by centrifugation, washed and re-suspended in CM. Cultures were returned to the incubator with shaking and samples were taken at 0, 10, 20, 30, 40, 50, 60, 120, and 240 minutes and processed as described previously. Analysis of temporal transcriptional changes (-1, 0, 5, 10, 20, 40, 80, 160 and 320m) to sub-inhibitory concentrations of PQ (0.25mM) was also characterized. Total RNA was prepared and labeled with Alexa547 and Alexa647 dyes. Intensities were normalized between the Alexa 546 and Alexa 647 channels by scaling all intensities in one channel such that the 75th percentile in each channel was made equal.

Project description:Approximately 2x106 CL1-5 and H23 cells were seeded on 10-cm dishes with RPMI-1640 medium containing 10% fetal bovine serum. After the cells covered 80% of the dish, cells were serum-starved for 24 h in RPMI-1640 medium. CL1-5 and H23 cell lysates were harvested, trypsinized and analyzed by using LTQ-Orbitrap XL and LTQ-Orbitrap Discovery, respectively. Halobacterium salinarum NRC-1 (ATCC700922) starter culture was prepared by inoculating a single colony in 5 mL of Halobacterium medium containing trace metals (CM+) and grown at 37 °C with shaking at 225 rpm for one week. An aliquot of 300 μL of the culture was spread on 15-cm diameter CM+ plates containing 2% of agarose. After 7~10 days of incubation at 37 °C, the GVs from cells on 10 plates were harvested using the centrifugation facilitated flotation method as previously described except the GVs were washed for a total of 5 times instead of the suggested 3 times to remove nonspecific bound proteins.(29) After collecting the gas vesicles in the primary cell lysate, the cell membrane and residual GV were removed by ultracentrifugation at 53 000× g, 4 °C for 16 h twice to obtain the GV-depleted lysate (GVD). GVD lysate was harvested, trypsinized and analyzed by using LC-MS/MS ( LTQ-Orbitrap XL). The H. salinarum strain NRC-1 was grown in complex medium (CM+) at 37 oC with shaking (200 rpm) to an OD600 of 0.5 (early exponential phase. The cultures were then subjected to an additional hour of incubation at 37 oC for preparation of proteins. The total lysate was harvested, trypsinized and analyzed by using LC-MS/MS (LTQ-Orbitrap Discovery). All MS raw data in Thermo XCalibur (version 2.0.7) binary format were converted to the mzXML open data format using a modified version of the ReAdW program. SEQUEST (version 27) was applied to search the MS/MS spectra against the forward and reverse (decoy) protein sequences of H. salinarum (2,427 proteins; EMBL-EBI Integr8, www.ebi.ac.uk/integr8/EBI-Integr8-HomePage.do) or human (38,425 proteins) plus bovine (22,863 proteins) (GenBank reference sequences, www.ncbi.nlm.nih.gov). SEQUEST results were further processed using the Trans Proteomic Pipeline.

Dataset Information

A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

Publications

A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

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