Mediator Forms Clusters with RNA Polymerase II in Live Stem Cells

Abstract

Transcription in eukaryotic cells is regulated not only by promoters but also enhancer elements. Mediator is the key transcription factor that links enhancers and promoters. Here, we labelled endogenous Mediator using CRISPR/Cas9 gene editing in mouse embryonic stem cells (mESCs). Using live cell super-resolution imaging we observed that Mediator forms transient sub-diffractive clusters in the nucleus of live stem cells. We also observed stable and large Mediator clusters. A rank-ordered size distribution of Mediator clusters was similar to a rank-ordered distribution of Mediator ChIP-seq data. By orthogonal labeling of Pol II we could observe that stable Mediator clusters co-localize with stable Pol II clusters, and the enhancer associated transcription factors Sox2 and Oct3/4 in live mESCs. Using Lattice Light Sheet Microscopy we directly observed Mediator and Pol II clusters at high temporal resolution and followed their 3D dynamics over extended periods of time. Upon incubation with the BRD4 inhibitor JQ1, known to dissolve super-enhancer signatures in ChIP-seq data, stable Mediator clusters disappeared. Based on our data we hypothesize that super-enhancer domains with high Mediator signal observed in ChIP-seq result from interactions of chromatin stretches with the large protein clusters observed in this study. Co-localization of many other key transcription factors, namely Pol II, Sox2, and Oct3/4, suggests that the entities observed here play an important role in the transcriptional program of mESC.