Unknown,Transcriptomics,Genomics,Proteomics

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Pervasive genomic transcription during oxidative stress generates thousands of previously uncharacterized lncRNAs (ChIP-Seq)

ABSTRACT: Oxidative stress (OS) is caused by an imbalance between pro-oxidant and antioxidant reactions which leads to accumulation of reactive oxygen species (ROS) within cells. ROS can be harmful, due to their damaging effects on several cellular components, but are at the same time essential components of signaling cues. We investigate the effect of OS on the immediate early transcriptional response at a genomic level, by deep sequencing of nuclear and cytosolic RNA fractions, in in human fibroblasts treated for 30 or 120 minutes with sub-lethal doses of H2O2. In contrast to most of the protein-coding transcriptome, OS induces de novo transient transcription of thousands of previously uncharacterized genomic loci. We classify these stress induced long non coding RNAs (lncRNA) based on their genomic annotation and strand orientation relative to their nearest gene: distal, overlapping, terminal-associated or promoter-associated. The latter class of stress-induced promoter associated antisense lncRNAs (si-paancRNAs), is preferentially transcribed by PolII, which elongates from bidirectional promoters, enriched for specific transcription factor-binding sites (ZFP161, ZFX, MEF2A and divergent-FOS motives). Interestingly the associated sense-coding genes belong to specific functional categories (cellular response to stress and others). We finally demonstrate that a subset of si-paancRNAs associate better with the translation machinery, which is stalled, upon OS. Most studies to date have focused on the repertoire of lncRNAs present in physiological conditions. We here report the surprising result that thousands of lncRNAs are transcriptionally upregulated upon OS from specific loci possibly playing a role in physiological or pathological response to stress. Chromatin immunoprecipitation coupled sequencing of 2 histone modifications in BJ (hTert/sT) and total levels of RNA polymerase II after 0, 30 and 120 minutes of treatment with H2O2 in MRC5 cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Jingxian Zhang

PROVIDER: E-GEOD-55171 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Purpose: Excess oxidative stress (OS) impairs endothelial function and plays an important role in vascular diseases, diabetes, and neuronal disorders. Several consequences of OS including cell recovery and apoptosis have been described previously. In this study, we report systems model of the temporal dynamics of the oxidative stress response in Human Umbilical Vein Endothelial Cells (HUVECs) and characterize HMOX1 as a master regulator in orchestrating the response to oxidative stress. Methods: Following stress induction by hydrogen peroxide (HP), we carried out dose-dependent phenotypic assays and time series measurements of transcripts in HUVECs. To explore the role of HMOX1, HUVECs were transfected with scrambled siRNA or si-HMOX1 and then treated with hydrogen peroxide. Total RNA was collected from Scrambled-untreated, scrambled-H2O2, and si-HMOX1-H2O2 samples for RNAseq. Novel findings were validated by qPCR and functional assays. Results: In this study we have identified three phases of stress response (acute response from 1-2 hours, the transition from 4-6 hours, and the chronic stress response from 8-16 hours). We observe stable cell cycle arrest and impaired DNA replication/repair from 8-16 hours along with and increased resistance to apoptosis from 2-16 hours. Knockdown of HMOX1 prior to HP treatment releases the resistance to apoptosis. Similarly, many cellular processes switch their direction of regulation in HMOX1 deficient cells upon HP treatment (i.e. autophagy, mitochondria, cell surface signaling, expression of histone modifying enzymes and long-noncoding RNAs etc). Conclusions: Endothelial cell fate decisions are critically important for vascular health. In this study we provide a temporal model characterizing the transition from the acute stress response to the chronic response. The nexus of cell function and dysfunction is characterized by the concurrent activation of survival and death pathways. We explore the master regulatory role of HMOX1 and identify novel mechanisms by which HMOX1 may regulate cell fate decisions.

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Pervasive genomic transcription during oxidative stress generates thousands of previously uncharacterized lncRNAs (ChIP-Seq)

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