Differential cofactor dependencies define functionally distinct types of human enhancers (STARR-seq)

Development and homeostasis of all multicellular organisms rely on differential cell-type-specific gene expression that is regulated by non-coding genomic enhancer elements. Enhancers function through the transcription-factor-mediated recruitment of cofactors, a structurally and functionally diverse set of proteins that activate RNA polymerase II transcription at promoters. Despite the important roles of enhancers and cofactors in transcriptional regulation, it is still not clear if all cofactors are required for all enhancers or if different enhancers have distinct cofactor dependencies and whether differential cofactor dependencies could be used to functionally categorize enhancers. Here, we quantified enhancer activities along the entire human genome using the massively parallel enhancer-activity assay STARR-seq in HCT116 cells, following the rapid auxin-inducible degradation of eight different cofactors. We identified groups of enhancers with distinct cofactor requirements, including enhancers whose activity is insensitive to the depletion of the core Mediator subunit MED14 or the bromodomain protein BRD4, respectively. In particular, Mediator seemed dispensable for P53-responsive enhancers and MED14-depleted cells in which transcription globally failed were still able to induce endogenous P53 target genes such as p21. Similarly, BRD4 was not required for genes with a CCAAT- and TATA-box proximal enhancer, including histone genes and LTR12 family retrotransposons, and for the induction of TATA-box containing heat-shock genes. Taken together, we show that different types of enhancers with distinct cofactor dependencies exist, including enhancer types that function in the absence of well-known cofactors and are employed to regulate specific gene classes and transcriptional programs. This represents the first functional categorization of enhancers by their cofactor dependencies that improves our understanding of alternative ways to activate transcription, which can aid in developing more precise interventions to modulate gene expression. " Overall design: Enhancer activity measured by STARR-seq upon AID-mediated depletion of individual transcriptional cofactors (COFs) in HCT116 cells. In total 11 COF-AID-tagged cell lines and the Parental cell line (control) were assayed, either with a genome-wide (8 tagged cell lines and Parental) or with a focused BAC-derived STARR-seq library (3 tagged cell lines). Each cell line was treated with auxin (IAA) or water (mock) and enhancer activity was assessed by STARR-seq after 6h of treatment, with 2-4 biological replicates performed per COF and condition.