Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

NFkB perturbation followed by genome-wide allelic-expression data assessment

ABSTRACT: We developed a method that perturbs transcription factor followed by monitoring genome-wide allelic expression measurements in living cells. As a model for this novel approach, we chose the factor NFÎºB. We performed TNF-Î± induction coupled to inhibition of NFÎºB in LCLs followed by Allelic Expression (AE) analysis on Illumina HumanOmni5-Quad BeadChips. Samples used included two HapMap trios: one from the CEU (GM12891, GM12892, and GM12878) and one from the YRI (GM19239, GM19238, and GM19240) population. NF-ÎºB knockdown was validated using RT-PCR of known gene targets for NFkB. Differential AE was assessed on Illumina HumanOmni5-Quad BeadChips for each experimental condition: (1) inhibition with Helenanin followed 1 hour later by activation by TNF-Î± (TN); (2) activation by TNF-Î± (tnfalpha); (3) DMSO; (4) no treatment (notmt).

ORGANISM(S): Homo sapiens

SUBMITTER: Tomi Pastinen

PROVIDER: E-GEOD-61254 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs.

Adoue Veronique V Schiavi Alicia A Light Nicholas N Almlöf Jonas Carlsson JC Lundmark Per P Ge Bing B Kwan Tony T Caron Maxime M Rönnblom Lars L Wang Chuan C Chen Shu-Huang SH Goodall Alison H AH Cambien Francois F Deloukas Panos P Ouwehand Willem H WH Syvänen Ann-Christine AC Pastinen Tomi T

Molecular systems biology 20141016

Most complex disease-associated genetic variants are located in non-coding regions and are therefore thought to be regulatory in nature. Association mapping of differential allelic expression (AE) is a powerful method to identify SNPs with direct cis-regulatory impact (cis-rSNPs). We used AE mapping to identify cis-rSNPs regulating gene expression in 55 and 63 HapMap lymphoblastoid cell lines from a Caucasian and an African population, respectively, 70 fibroblast cell lines, and 188 purified mon ...[more]

PMID: 25326100

Similar Datasets

Project description:Acute respiratory distress syndrome (ARDS) is a catastrophic form of acute lung injury (ALI). The necessity for mechanical ventilation (MV) renders patients at risk for ventilator induced lung injury (VILI). Exposure to repetitive cyclic stretch (CS) and/or over-inflation exacerbates injury. Reducing tidal volume (VT) is the only therapeutic strategy shown to mitigate morbidity and mortality. Cyclic stretch has been shown to differentially regulate gene expression in part through the activation of mammalian mitogen-activated protein kinase (MAPK). Although these studies have shown both molecular and cellular alterations, no unifying hypothesis to explain MV-induced lung injury has emerged. In the current study, we hypothesized that coordinated expression of cyclic stretch (CS)-responsive genes relies on the presence of common CS-sensitive regulatory elements. To identify CS-responsive genes, we undertook a comparative examination of the gene expression profile of human bronchial epithelial airway (Beas-2B) cells in response to various injurious stimuli involved in the pathogenesis of acute lung injury (ALI)/Ventilator induced lung injury (VILI): cyclic stretch, tumor necrosis factor alpha (TNF-a), and lipopolysaccharide (LPS). Experiment Overall Design: Human Bronchial Epithelial Cells (Beas-B2) cells grown on silicon elastic plates coated with Type I collagen (Flexercell International, McKeesport, PA) were exposed to six regiments for 4 h: 1) control (static, [control]); 2) mechanical stretch (25 PKa, 30 cycles per min, [stretch]); 3) LPS (1 mcg/ml [LPS]); 4) TNF-Î± (20 ng/ml; [TNF]); 5) mechanical stretch plus LPS [LPS+S], and 6) mechanical stretch plus TNF-Î± [TNF+S]. Total RNA (duplicate experiments) was extracted using TRIZOL reagent (as per manufactures specifications) and purified using Qiagen mRNA purification Kit (as per manufacturers specifications). mRNA was hybridized to Affymetrix Human U133plus2.0 chips. Probe based analysis, background reduction, and quantile data normalization was performed in MeV 4.0 of TM4 using Robust Multi-array Average (RMA).

Project description:Schistosoma mansoni is the major causative agent of schistosomiasis in the Americas. This parasite takes advantage from host signaling molecules such as cytokines and hormones to complete its development inside the host. TNF-Î± is the most important cytokine involved in the inflammatory response when cercaria, the infective stage, penetrates the human skin and a severe inflammatory response is started. In this work the authors describe the complete sequence of a possible TNF-Î± receptor in S. mansoni and detect that the receptor is most highly expressed in cercaria among all life cycle stages. In an attempt to mimic the situation at the site of skin penetration cercariae have been mechanically transformed in vitro into schistosomula and immediately exposed to human TNF-Î± . Exposure of these early schistosomula to the human hormone caused a large-scale change in the expression of parasite genes. Exposure of adult worms to human TNF-Î± caused gene expression changes as well, although the set of parasite altered genes was entirely different from that of schistosomula. This work increases the number of known signaling pathways of the parasite, and opens new perspectives into understanding the molecular components of TNF-Î± response as well as possibly interfering with parasite-host interaction. A platform previously designed by our group (PMID: 17517391) was used. 300 ng of RNA from adult schistosomes treated with TNF and its control were used for the hybridization. RNA amplification and labeling kit (Agilent Technologies) were used according to manufacturerÂ´s instructions. Each sample was separately labeled with either Cy3 or Cy5. 825 ng cRNA from each amplification were used for hybridization; self-self experiments were performed. Washing and scanning procedures were according to the manufacturerÂ´s instructions using GenePix 4000B scanner (Molecular Devices, Sunnyvale, CA, USA). Data was extracted using Feature Extraction software (Agilent Technologies). We calculated a virtual Log2 ratio between intensities of TNF-Î± Treated/Control, for each gene. With these ratios, we used two different approaches for SAM statistical analyses. SAM one-class approach was used to identify genes with sustained changes in their expression levels along the entire time period of observation (1 and 24 h); SAM two-class approach was used to identify genes with transient changes in their expression levels. In both cases, genes were considered differentially expressed at q-value â?¤ 0.05. Hierarchical clustering of selected genes was generated using Spotfire Decision Site software (TIBCO Software Inc., Palo Alto, CA, USA). For a gene that was represented in the array by multiple probes, we picked a single representative probe by selecting the probe with the highest absolute value of the Log2 ratio between intensities of TNF-Î± Treated/Control.

Project description:Activation of the vascular endothelium with cytokines such as TNF is widely used to study the role of the vasculature in proinflammatory disease. To gain insight into mechanisms of prolonged vascular endothelial activation we compared changes in gene expression induced by continuous activation in stable tmTNFalpha expressing cells with changes due to acute TNFalpha challenge in vitro. Affymetrix GenechipÂ® analysis was performed on RNA from control, acute and continuous TNFalpha-activated endothelial cells. Only 36% of the significant changes in gene expression were convergent between the acute and continuously activated endothelial cells in comparison to the control. From the divergently regulated genes, for example the cytokine ENA-78 was specifically induced in chronically activated cells, while E-Selectin, a cell adhesion molecule, was upregulated only in acutely activated endothelial cells. Antioxidant SOD gene induction was noted in acute activation while NADPH oxidase was selectively upregulated in continuous activated endothelium in accordance with significant ROS induction occurred only in these cells. In addition, the anti-angiogenic genes PAI-1 and thrombospondin were upregulated only in acutely activated endothelium, consistent with reduced angiogenic activity observed in acute versus chronically activated endothelial cells in vitro. These data suggest that continuous activation of endothelial cells leads to specific expression and functional changes, consistent with alterations observed in dysfunctional endothelium exposed to or involved in chronic inflammation. Experiment Overall Design: This experiment compares the response of endothelial cells from newborn mice that were either stably transfected with a non-cleavable transmembrane mutant form of murine TNFalpha, or treated with soluble TNFalpha (20ng/ml) for 4 hours. Each treatment was compared to mock-transfected control cells. Four replicates for each condition were used. Five micrograms of total RNA was assayed per Genechip using standard Affymetrix protocols.

Dataset Information

NFkB perturbation followed by genome-wide allelic-expression data assessment

Publications

Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure