Discovering and Dissecting the Function of Conserved NF-κB Binding Events in the Human Genome

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor plays a prominent role in inflammation and contributes to the development of atherosclerosis. Genome-wide DNA binding assays of the human NF-κB subunit RELA (p65) have revealed tens of thousands of NF-κB binding sites and hundreds of target genes. However, the function of individual RELA binding sites and the extent to which NF-κB occupancy and function is conserved across mammals are not well understood. To better understand the function of NF-κB we characterized the genome-wide binding of RELA in primary vascular endothelial cells (ECs) isolated from the aortas of human, mouse and cow. ECs were stimulated acutely with the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA) and we profiled RELA occupancy, open chromatin, select histone modifications, and RNA expression. We found ~5000 RELA binding events conserved across all three species and these highly conserved human binding events were enriched for genes controlling vascular development, apoptosis, and pro-inflammatory responses. Approximately 2000 of these highly conserved RELA binding events were also shared across multiple human cell types, revealing a conserved core of robustly bound NF-κB sites. These NF-κB binding sites were also prominent components of ~40 inflammation-induced super-enhancers (SE) common to several tissues. To gain insight into the function of individual conserved NF-κB binding sites we focused on the inflammation-induced SE proximal to the monocyte recruiting chemokine CCL2 , which we detected as a SE in all three species and across multiple cell types. We tested the functional significance of six conserved RELA binding sites comprising this SE using CRISPR/Cas9 genome editing. We found that only deletion of the most proximal upstream RELA binding site could abolish the induction of CCL2 upon TNFA treatment. This site also contains a disease associated variant that can modulate CCL2 induction. Overall, our comparative genomics assessment of NF-κB binding gives new insight into NF-κB biology and the function of conserved transcription factor binding events within mammalian super-enhancers.