Dissecting the regulatory activity and sequence content of loci with exceptional numbers of transcription factor associations.

DNA associated proteins (DAPs) regulate gene expression by binding to regulatory loci such as enhancers or promoters. An understanding of how DAPs cooperate at regulatory loci is essential to deciphering how these regions contribute to normal development and disease. In this study, we aggregated publicly available ChIP-seq data from 469 human DNA-associated proteins assayed in three cell lines and integrated these data with an orthogonal dataset of 352 non-redundant, in vitro-derived motifs mapped to the genome within DNase hypersensitivity footprints in an effort to characterize regions of the genome that have exceptionally high numbers of DAP associations. We subsequently performed a massively parallel mutagenesis assay to discover the key sequence elements driving transcriptional activity at these loci and explored plausible biological mechanisms underlying their formation. We establish a generalizable definition for High Occupancy Target (HOT) loci and identify putative driver DAP motifs, including HNF4A, SP1, SP5, and ETV4, that are highly prevalent and exhibit sequence conservation at HOT loci. We also found the number of DAP associations is positively associated with evidence of regulatory activity and, by systematically mutating 245 HOT loci in our massively parallel reporter assay, localize regulatory activity in these loci to a central core region that is dependent on the motif sequences of our previously nominated driver DAPs. In sum, our work leverages the increasingly large number of DAP motif and ChIP-seq data publicly available to explore how DAP associations contribute to genome-wide transcriptional regulation.