A pliable mediator acts as a functional, rather than an architectural bridge, between promoters and enhancers

Our mechanistic understanding of Mediator derives largely from studies of the 25-subunit yeast complex. Here we combine CRISPR-Cas9 genetic screens, degron assays, in situ Hi-C, and cryo-EM to dissect the function and structure of the 33-subunit mammalian Mediator (mMED). Deletion analyses in B, T and ES cells reveal that depletion of the entire complex blocks PolII recruitment genome-wide, while loss of non-essential subunits, including the Tail module, primarily affects promoters linked to multiple enhancers. Contrary to current models, we find that mMED is not required to tether regulatory DNA, a topological activity controlled predominantly by architectural proteins. Structurally, we show that alterations in the Tail module, particularly at the core-Tail interphase, effect crucial mMED-PolII contacts, providing a rationale for how TFs stabilize the mMED-PolII holoenzyme and promote gene expression. Our studies therefore reveal key insights into how Mediator functionally bridge promoters and enhancers to regulate transcription initiation in higher eukaryotes. Overall design: ChIP-seq, ATACseq, Hi-C,PromterCaputre Hi-C, CRISPR screen and RNAseq from mouse ESC, CD4+ T cell or CH12 cell line and/or human HCT-116 cell line The processed data files generated from multiple samples are indicated in the corresponding sample description field, and linked to the Series records or to the corresponding 'r1' sample records.