P118 IFN-A transcriptional response in endothelial cells: Key modulators and gene regulatory modules

Abstract

Introduction Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. Interferon-α (IFN-α) is a pleiotropic cytokine endowed with potent biological activities, achieved through the up-regulation of hundreds of interferon-stimulated genes (ISGs). Aim of this study was to extend previous work on IFN-α transcriptional response in endothelial cells, by identifying IFN-α modulators and inferring putative gene regulatory modules involving them. Methods Primary endothelial cells (HUVEC) were pulsed in vitro with human recombinant IFN-α under different perturbation conditions: siRNA inactivation of target genes. A panel of 96 transcripts, including ISGs belonging to IFN-α top signature and genes related to the IFN-α signalling pathway, were screened by custom TaqMan Low-Density Arrays. Stealth siRNA were used for RNAi-mediated knockdown of seven candidate IFN-α modulators: STAT1, IRF1, IRF7, GBP1, OAS2, IFIH1 and USP18. The effects of each siRNA were evaluated with respect to a calibrator siRNA using the comparative threshold cycle method (ΔΔCT method). A selection procedure, testing the null hypothesis of no difference in the effects of a siRNA targeting a candidate modulator and the calibrator siRNA, was used to assign a significance value (p-value) to each regulation. Significant regulations were defined fixing a cut-off of 0.05 on the Bonferroni corrected p-values. Regulatory modules, built by different feed-forward loops (FFLs), the basic three-node building blocks of biological networks, were then inferred by combining the significant regulations, induced by different couples of IFN-α modulators. Results Our analysis showed both mainly positive (STAT1, IFIH1) and mainly negative (USP18, GBP1, IRF1) modulators of IFN-α transcriptional response, while IRF7 and OAS2 were found to act equally in both directions. STAT1 was confirmed as the primary positive modulator of IFN-α; following its silencing several genes were significantly down-regulated and only few genes up-regulated. Conversely, USP18 was found to be the strongest negative modulator and its silencing caused a massive up-regulation of genes, including some involved in cell-to-cell adhesion. An interesting regulatory module in which STAT1 activates IFIH1 and they both regulate six genes, including IFNα-R1, was reconstructed by our inference method. Some of the inferred FFLs are currently being validated. Conclusion The investigation of IFN-α transcriptional modulators through our method has led to a clear picture, depicting the transcriptional impact of each considered modulator in terms of sign of regulation, strength and target genes. The inferred gene regulatory modules, involving key modulators, may help to unravel the mechanisms of action of IFN-α and discover future target of intervention in knock-out experiments.