Mediator is a multi-subunit complex organized into four modules (head, middle, tail and kinase module). It is involved in several steps of PolII-dependent transcription and enhancer–promoter (E–P) interactions. Mutations in different subunits (SU) are associated with cancer and developmental diseases. Mediator's function and structure have been mainly studied in yeast. Earlier studies suggest significant differences with the mammalian complex in terms of composition and structure. Additionally, the chromatin architecture between both species is different, particularly E–P interactions. We aimed to comprehensively characterize the mammalian complex.

We screened for cell essentiality of Mediator subunits, in a murine B-lymphoma cell line, CH12, in embryonic stem cells and in primary CD4T cells, using CRISPR-Cas9 technology. We generated knockout clones (KO) whenever possible in CH12 cells, and a degron-based conditional KO of the essential Mediator's backbone, Med14.

We analyzed the impact on gene expression (RNAseq), on PolII and Mediator recruitment at promoters and enhancers (ChIPseq), on chromatin accessibility (ATACseq), on histone modifications (MNase ChIPseq), on chromatin architecture (in situ HiC) and on structure (cryo-EM).

Depletion of non-essential SUs induces transcriptional deregulation of only a subset of genes, whereas depleting the essential Med14 induces global decrease in transcription associated with a decrease in PolII recruitment. Interestingly, the ablation of the whole tail module is viable. With this tailless model and the Med14-degron we do not observe an impact on chromatin architecture. Preliminary cryo-EM results show a conserved location of common SUs between yeast and mammalian cells. We are able to locate mammalian specific SUs and describe the interactions between the different SUs in the mammalian complex.

Our study provides new insights into mammalian Mediator structure and a comprehensive view on how Mediator and its tail module are involved in transcription and chromatin architecture. This data is essential for a better understanding of transcriptional regulation and Mediator related diseases.