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Mediator complex interaction partners organize the transcriptional network that defines neural stem cells

neural stem cells nsc mediator complex

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#1 Engadin

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Posted 17 June 2019 - 08:34 PM


F U L L   T E X T :    Nature

 

 

 

 

ABSTRACT

 

The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density defines super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here, we purify Mediator from neural stem cells (NSCs) and identify 75 protein-protein interaction partners. We identify super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalize with Mediator at enhancers and super enhancers. Transcription factor families with high affinity for Mediator dominate enhancers and super enhancers and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator of NSCs. Tcf4 interacts with Mediator, colocalizes with Mediator at super enhancers and regulates neurogenic transcription factor genes with super enhancers and broad H3K4me3 domains. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity.

 

 

 

INTRODUCTION

 

The Mediator complex is a complex of ~30 subunits that is important for transcriptional regulation and is conserved from yeast to human1,2,3,4. The Mediator complex provides communication between active enhancers and promoters by interacting with proteins that bind to either of these two classes of regulatory DNA elements2,3,5. Accordingly, identified Mediator-interacting proteins include many transcription factors2,5, RNA polymerase II (RNApol2) and transcription elongation factors6. Recently, Mediator content was used to rank enhancers in embryonic stem cells (ESCs) and enhancers with the highest Mediator content were postulated as super enhancers (SEs)7, a class of enhancers that regulates key genes in cell identity and oncogenes7,8,9. Related enhancer types such as stretch enhancers and anti-pause enhancers were described independently10,11. There is debate on whether SEs act mechanistically different from typical enhancers12. Arguments in favor of the functional distinction of SEs is their ability to drive high levels of transcription and their selective sensitivity to inhibitors of Brd4, a chromatin-binding protein enriched at SEs9,10,13. Besides Mediator and Brd4, chromatin modifiers such as Ep300 and Kdm1a (LSD1 complex), chromatin remodelers such as Chd7, Brg1 (SWI-SNF complex) and Chd4 (NuRD complex) and Smc1a (Cohesin complex) were found to be enriched at SEs8. In a recently proposed model, the constituent enhancers of an SE and their regulated promoter(s) would group together to form a phase-separated assembly14. Such an assembly would rely on interactions between transcriptional and chromatin regulators14.

 

Cell-type specific master TFs colocalize with Mediator at SEs7,8. However, evidence for interactions between master TFs and Mediator, which would underpin their role in recruiting Mediator to SEs, is scarce. For example, among SE-binding master TFs Oct4, Sox2 and Nanog (ESCs), Pu.1 (pro-B cells), MyoD (Myotubes) and C/EBPα (Macrophages)7, Mediator interactions were only detected in immunoprecipitations of Sox2 and C/EBPα and these were with single Mediator subunits15,16. Also our understanding of the recruitment of the above chromatin modifiers to enhancers and SEs and their subsequent maintenance at high levels at SEs is far from complete. Mediator was shown to interact with SE-enriched chromatin modifier Crebbp17 and the Cohesin complex18, suggesting that Mediator could, in principle, provide an anchoring role at enhancers, SEs and the proposed phase-separated assemblies.

 

To investigate the relevance of Mediator interactors in defining enhancers and SEs, here we describe the purification of the Mediator complex from neural stem cells (NSCs) and identify its protein–protein interaction partners by mass spectrometry. To prevent recording interactions that are mediated via DNA/chromatin, we purify Mediator from non-treated nuclear extracts, nuclear extracts treated with nuclease benzonase and nuclear extracts treated with ethidium bromide to disrupt protein-DNA interactions and only take interactions with the Mediator complex that are not affected by these treatments. Our resulting Mediator interactome contains 95 proteins of which 75 have not been, to the best of our knowledge, previously characterized as Mediator-interacting proteins. Subsequently, we perform Mediator ChIP-seq in NSCs and define SEs in NSCs by their Mediator content. Remarkably, we find that the three most frequent motifs in SEs are bound by multiple members of the small set of TFs that we identify as Mediator interactors in NSCs. We show that one of these TFs, Tcf4, regulates a set of key NSC transcription factor genes with SEs and broad H3K4me3 domain-containing promoters. High-Mediator affinity therefore appears an important characteristic of master TFs. Our Mediator interactome contains many known enhancer-binding chromatin modifiers and we show that Mediator-interacting chromatin modifiers Jmjd1c and Carm1 bind genome-wide to enhancers and SEs. Together this suggests that high-Mediator-binding affinity selects proteins that play important roles in establishing and maintaining enhancers and SEs to facilitate the regulation of cell identity.

 

 

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Also tagged with one or more of these keywords: neural stem cells, nsc, mediator complex

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