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

Non-consensus binding sites of transcription factors are often observed within the regulatory elements of genes; however, their effect on transcriptional strength is unclear. Within the promoters and enhancers of NF-{kappa}B-responsive genes, we identified clusters of non-consensus {kappa}B DNA sites, many exhibiting low affinity for NF-{kappa}B in vitro. Deletion of these sites demonstrated their collective critical role in transcription. We explored how these "weak" {kappa}B sites exert their influence, especially given the typically low nuclear concentrations of NF-{kappa}B. Using proteomics approaches, we identified additional nuclear factors, including other DNA-binding TFs, that could interact with {kappa}B site-bound NF-{kappa}B RelA. ChIP-seq and RNA-seq analyses suggest that these accessory TFs, referred to as the TF-cofactors of NF-{kappa}B, facilitate dynamic recruitment of NF-{kappa}B to the clustered weak {kappa}B sites. Overall, the occupancy of NF-{kappa}B at promoters and enhancers appears to be defined by a collective contribution from all {kappa}B sites, both weak and strong, in association with specific cofactors. This congregation of multiple factors within dynamic transcriptional complexes is likely a common feature of transcriptional programs. SIGNIFICANCEThe NF-{kappa}B RelA dimers undergo rapid activation by cytokines and pathogens, driving expeditious expression of target genes upon binding to DNA elements known as {kappa}B sites, located in the regulatory regions. We find that promoter and enhancer regions of RelA target genes harbor multiple {kappa}B sites, most being non-consensus with minimal affinity to NF-{kappa}B in vitro. Recruitment of RelA dimer in vivo depend on these {kappa}B sites, weak and strong, and appears to be regulated by various accessory factors, including other DNA-binding transcription factors. Overall, this study points to a coordinated network of factors communicating with both weak and strong {kappa}B sites to recruit RelA dimers, enabling rapid gene activation.