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Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

ABSTRACT: To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-? (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ?-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.

SUBMITTER: Baril M

PROVIDER: S-EPMC3681753 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

Baril Martin M Es-Saad Salwa S Chatel-Chaix Laurent L Fink Karin K Pham Tram T Raymond Valérie-Ann VA Audette Karine K Guenier Anne-Sophie AS Duchaine Jean J Servant Marc M Bilodeau Marc M Cohen Eric E Grandvaux Nathalie N Lamarre Daniel D

PLoS pathogens 20130613 6

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-β (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of β-catenin (CTNNB1) upon virus ...[more]

PMID: 23785285

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Project description:BACKGROUND:Polycomb group (PcG) and trithorax group (trxG) proteins contribute to the specialization of cell types by maintaining differential gene expression patterns. Initially discovered as positive regulators of HOX genes in forward genetic screens, trxG counteracts PcG-mediated repression of cell type-specific genes. Despite decades of extensive analysis, molecular understanding of trxG action and regulation are still punctuated by many unknowns. This study aimed at discovering novel factors that elicit an anti-silencing effect to facilitate trxG-mediated gene activation. RESULTS:We have developed a cell-based reporter system and performed a genome-wide RNAi screen to discover novel factors involved in trxG-mediated gene regulation in Drosophila. We identified more than 200 genes affecting the reporter in a manner similar to trxG genes. From the list of top candidates, we have characterized Enoki mushroom (Enok), a known histone acetyltransferase, as an important regulator of trxG in Drosophila. Mutants of enok strongly suppressed extra sex comb phenotype of Pc mutants and enhanced homeotic transformations associated with trx mutations. Enok colocalizes with both TRX and PC at chromatin. Moreover, depletion of Enok specifically resulted in an increased enrichment of PC and consequently silencing of trxG targets. This downregulation of trxG targets was also accompanied by a decreased occupancy of RNA-Pol-II in the gene body, correlating with an increased stalling at the transcription start sites of these genes. We propose that Enok facilitates trxG-mediated maintenance of gene activation by specifically counteracting PcG-mediated repression. CONCLUSION:Our ex vivo approach led to identification of new trxG candidate genes that warrant further investigation. Presence of chromatin modifiers as well as known members of trxG and their interactors in the genome-wide RNAi screen validated our reverse genetics approach. Genetic and molecular characterization of Enok revealed a hitherto unknown interplay between Enok and PcG/trxG system. We conclude that histone acetylation by Enok positively impacts the maintenance of trxG-regulated gene activation by inhibiting PRC1-mediated transcriptional repression.

Project description:BackgroundDysfunctional CFTR in the airways is associated with elevated levels of NFkappaB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.Methodology/principal findingsWe tested the hypothesis that wt-CFTR down-regulates NFkappaB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFkappaB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1beta induced IL-8, and NFkappaB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-beta-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFkappaB reporter activities as compared to control cells suggesting a negative regulation of NFkappaB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFkappaB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1beta in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IkappaB, an inducer of NFkappaB mediated signaling in the CFKO mice.Conclusions/significanceOur data suggest that CFTR is a negative regulator of NFkappaB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.

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Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

Publications

Genome-wide RNAi screen reveals a new role of a WNT/CTNNB1 signaling pathway as negative regulator of virus-induced innate immune responses.

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