A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers

Summary While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR- Cas9 genetic screens, degronassays, Hi-C, and cryoelectronmicroscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essentialsubunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essentialsubunits increasing structural complexityof the tail module, a primary transcriptionfactor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator’s structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.