Cooperativity among clustered κB sites within promoters and enhancers dictates transcriptional specificity of NF-κB RelA along with specific cofactors

Abstract

ABSTRACTThe promoter/enhancer region of a gene often contain clusters of multiple binding sites of a specific transcription factor (TF). The functional role of this multiplicity in gene expression is unclear. Here we report not only the abundance of NF-κB binding (κB) sites in promoters and enhancers of NF-κB regulated genes, but also a striking variability in their affinity with a particular need for the low-affinity (weak) sites for expression in cells. Since the nuclear concentration of RelA, the predominant NF-κB, typically is low in stimulated cells, the weak κB sites are unlikely to function as a direct recruiter of TF. We observed association of a multitude of nuclear factors, including several other TFs to NF-κB bound to κB-sites by proteomic analyses. ChIP-seq, RNA-seq and in vitro phase-separated condensation analyses suggest these other transcription factors (TF), referred to as the cofactors of NF-κB, facilitate recruitment of NF-κB to clustered κB sites of specific target genes. Overall, our findings demonstrate a collective contribution of both strong and weak κB sites in occupancy of NF-κB at the promoters and enhancers, with the recruitment facilitated by a variety of cofactors. This congregation of multiple factors in a transcription condensate is likely to be common to all transcriptional programs.