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

The functional role of weak DNA binding sites for transcription factor (TF) recruitment and gene expression remains largely unknown. Our study reveals that the weak NF-κB DNA binding sites, which are abundant in gene promoters and enhancers, appear in clusters and exhibit minimal to undetectable NF-κB binding activity in isolation in vitro, yet they play prominent roles in gene regulation within native context in cells. We found nuclear concentration of RelA/p65, the predominant NF-κB, is approximately 0.2 µM in stimulated cells, challenging the idea that these weak κB sites operate through mass action- dependent binding mechanisms. Through proteomic analysis, we identified a range of nuclear factors, including various other TFs, interacting with RelA at these κB-sites. ChIP-seq, RNA-seq and phase- separated condensation analyses suggest these additional TFs, referred to as the cofactors of NF-κB, facilitate dynamic recruitment of NF-κB to clustered κB sites of specific target genes. Overall, our findings demonstrate the 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 forming larger dynamic complexes appearing as a transcription condensate is likely to be common to all transcriptional programs.