Multi-species RELA, JUN, and histone modification ChIP-seq of mammalian aortic endothelial cells before and after TNFa stimulation
TNFa has an evolutionary conserved role in mediating inflammation via activation of the transcription factor NF-?B. The functions of individual NF-?B binding sites are not well understood. To identify conserved and functionally important NF-?B binding sites in mammals, we performed ChIP-seq to map the genome-wide binding of RELA, JUN, and select histone modifications in primary vascular endothelial cells (ECs) isolated from the aortas of human (HAEC), mouse (MAEC) and cow (BAEC), before and after TNFa. The conserved RELA binding sites show strong epigenetic changes in response to TNFa and enrich near genes controlling vascular development and pro-inflammatory responses. Our method identifies novel modes of RELA-chromatin interactions that are conserved in mammals and shared between multiple cell-types. Particularly, genomic regions bound by RELA prior to stimulation are important responders during TNFa stimulation. We use CRISPR/Cas9 genome editing to validate the roles of the conserved RELA pre-bound sites near pro-inflammatory genes such as CCL2 and PLK2. Our evolutionary approach describes new aspects of mammalian NF-?B biology including its role within super-enhancers and relevance in inflammatory disorders.