Teamwork of clustered low-affinity ?B sites and accessory factors regulates transcriptional strength of NF-?B RelA dimers [ChIP-seq]

Non-consensus binding sites of transcription factors are often observed within the promoters and enhancers of various genes; however, their effect on transcriptional strength is unclear. Within the promoters and enhancers of NF-?B-responsive genes, we identified clusters of non-consensus ?B DNA sites, many exhibiting low affinity for NF-?B in vitro. Deletion of these sites demonstrated their collective critical role in transcription. We explored how these “weak” ?B sites exert their influence, especially given the typically low nuclear concentration of NF-?B. Using proteomics approaches, we identified additional nuclear factors, including other DNA-binding TFs, that could interact with ?B site-bound NF-?B RelA without binding to DNA directly. ChIP-seq and RNA-seq analyses suggest that these accessory TFs, referred to as the cofactors of NF-?B, facilitate dynamic recruitment of NF-?B to the clustered ?B sites. Overall, the occupancy of NF-?B at promoters and enhancers appears to be defined by a collective contribution from all ?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. Overall design: Gene expression and RelA genome binding/occupancy profiling by high-throughput sequencing in control and knockdown mouse embryonic cell lines.