Dissecting the regulatory strategies of NFkB RelA target genes in the inflammatory response reveals differential transactivation logics (RNA-seq.WT.p53ko.MEFs_4)

NFkB RelA is the potent transcriptional activator of inflammatory response genes. We stringently defined a list of direct RelA target genes by integrating physical (ChIPseq) and functional (RNAseq in knockouts) datasets. We then dissected each gene's regulatory strategy by testing RelA variants in a novel primary-cell genetic complementation assay. All endogenous target genes required that RelA makes DNA-base-specific contacts, and none could be activated by the DNA binding domain alone. However, endogenous target genes differed widely in how they employ the two transactivation domains. Through model-aided analysis of the dynamic timecourse data we reveal gene-specific synergy and redundancy of TA1 and TA2. Given that post-translational modifications control TA1 activity and intrinsic affinity for coactivators determines TA2 activity, the differential TA logics suggests context-dependent vs. context-independent control of endogenous RelA-target genes. While some inflammatory initiators appear to require co-stimulatory TA1 activation, inflammatory resolvers are a part of the NFkB RelA core response. Total RNA extracted from two biological replicates of p53-deficient and 1 WT control replicate from primary mouse embryonic fibroblasts were subjected to stimulation with IL-1beta, TNF, and LPS in 8-hour time-course analyis (0, 0.5H, 1H, 3H, 8H). In order to prolong the lifespan of primary MEFs, cells deficient in p53 could be propagated sufficiently longto allow retroviral transduction, selection, and expansion for transcriptional time-course studies. Examining gene expression profiles of inflammatory response genes in p53 MEFs was a control experiment for studying inflammatory response genes. Two biological replicates of p53-deficient MEFs and their WT littermate control MEFs were used for pairwaise comparisons to show reproducibility in inducible gene expression between p53-deficient and WT MEF cells in response to three stimuli known to activate NF-kappaB signaling pathway namely, IL-1beta, TNF, and LPS.