Enhancer activation requires trans-recruitment of a mega transcription factor complex

Enhancers provide critical information directing cell-type-specific transcriptional programs, regulated by binding of signal-dependent transcription factors and their associated cofactors. Here, we report that the most strongly activated estrogen (E₂)-responsive enhancers are characterized by trans-recruitment and in situ assembly of a large 1-2 MDa complex of diverse DNA-binding transcription factors by ERα at ERE-containing enhancers. We refer to enhancers recruiting these factors as mega transcription factor-bound in trans (MegaTrans) enhancers. The MegaTrans complex is a signature of the most potent functional enhancers and is required for activation of enhancer RNA transcription and recruitment of coactivators, including p300 and Med1. The MegaTrans complex functions, in part, by recruiting specific enzymatic machinery, exemplified by DNA-dependent protein kinase. Thus, MegaTrans-containing enhancers represent a cohort of functional enhancers that mediate a broad and important transcriptional program and provide a molecular explanation for transcription factor clustering and hotspots noted in the genome.