Classification of IRF3- vs ISGF3-dependent gene induction in murine fibroblasts
To investigate the dependency on gene expression on the PRR-activated transcription factor IRF3 versus the type I IFN receptor (IFNAR)-activated transcription factor ISGF3, we extracted primary murine embryonic fibroblasts (MEF) of wild-type, IRF3-knockout or IFNAR-knockout mice (strain C57BL/6J, protocol of Podlech et al., 2002). We stimulated the 3 different cell lines by transfection of immunostimulatory DNA (ISD, 4 hours) for activation of IRF3, or by treatment with murine IFNbeta (3 hours) for activation of the IFNAR-ISGF3 cascade. Mock-transfected (4 hours) or untreated cells served as respective controls. In the last 2 hours before harvest, cells were additionally treated with 200µM 4-thiouridine (4sU) for metabolic labelling of nascent transcripts, and total mRNAs were processed accordingly by SLAM-sequencing procedures for gene expression profiling of newly synthesized transcripts. Overall design: Comparative gene expression profiling analysis of nascent transcripts from SLAM-seq data for gene induction by PRR-IRF3 vs. by IFNAR-ISGF3 signalling.