Mediator complex and transcriptional control of cellular identity and plasticity

Abstract

[eng] Our understanding of the transcriptional control of cellular identity is rapidly emerging. Mediator complex enriches at active enhancers, forms a protein bridge to target genes, and recruits RNA Polymerase II (RNA-Pol II). Thus, Mediator plays an essential role in transcriptional regulation. However, precisely how Mediator complex completes these critical steps remains unclear. In this study, we delve into the intricacies of Mediator interactions with newfound transcriptional regulators. For this, we establish and employ a proximity-ligation assay (PLA) to characterize protein interactions in the nucleus by immunofluorescence and quantitative imaging. We detect changes in the Mediator-Pol II interaction between different cell types. Moreover, we show that small molecule manipulation of the Mediator-Pol II interaction correlates with subsequent changes in cell identity. This suggests an important role for the Mediator-Pol II interaction in establishing cell identity transitions. We chose Mediator ́s biggest subunit: MED1 (Mediator of RNA polymerase II transcription subunit 1) and performed immunoprecipitation followed by mass spectrometry (IP-MS) analysis of mouse ESCs with the aim of identifying its protein interactome. We observe a strong overlap between the proteins and biological functions of our mouse Mediator interactome and previously reported Mediator interactomes. Beyond the already know interactors. We present an updated interaction network of MED1 with novel proteins connected with Mediator. We further explored the particularly strong interaction with all the constitutive subunits of the Pyruvate Dehydrogenase complex (PDH). We provide evidence that the nuclear PDH is not randomly distributed in the nucleus, but instead tightly associates with Mediator and RNA-Pol II. Chromatin Immunoprecipitation combined with DNA Sequencing (ChIP-Seq) of the DLAT subunit of PDH complex confirms that the nuclear PDH complex is enriched at enhancers and super- enhancers, it coincides with highly acetylated regions of the DNA and is involved in biological processes that regulate cellular identity and chromatin maintenance and regulation. Experimental manipulation of the PDH complex reflects the functional significance of this nuclear complex in the transcriptional regulation of cell identity and viability independently of the mitochondrial PDH activity. Taken together, our data provide novel insights about how the Mediator-PDH axis controls cell identity.