Abstract
BackgroundThe Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. The assembly, regulation, and functions of these nuclear condensates remain to be further understood.ResultsWe found that Med15, a subunit in the tail module of the Mediator complex, formed nuclear condensates through a novel mechanism. Nuclear foci of Med15 were detected by both immunostaining of endogenous proteins and live cell imaging. Like Med1 foci and many other biomolecular condensates, Med15 foci were sensitive to 1, 6-Hexanediol and showed rapid recovery during fluorescence recovery after photobleaching. Interestingly, overexpressing DYRK3, a dual-specificity kinase that controls the phase transition of membraneless organelles, appeared to disrupt Med1 foci and Med15 foci. We identified two regions that are required to form Med15 nuclear condensates: the glutamine-rich intrinsically disordered region (IDR) and a short downstream hydrophobic motif. The optodroplet assay revealed that both the IDR and the C-terminal region of Med15 contributed to intracellular phase separation.ConclusionsWe identified that the Mediator complex subunit Med15 formed nuclear condensates and characterized their features in living cells. Our work suggests that Med15 plays a role in the assembly of transcription coactivator condensates in the nucleus and identifies Med15 regions that contribute to phase separation.
Highlights
The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation
We suggest that prominent GFP-Med15 foci in the stable cell line that were colocalized with anti-Med1 (Fig. 2c) may be more consistent markers of Mediator condensates reported in previous studies [13, 14]
Our work revealed that Med1 foci and GFP-Med15 foci can be dissolved by overexpressing DYRK3 kinase, which provides a likely explanation for the dissolution of Med1 foci and GFP-Med15 foci in mitotic cells
Summary
The Mediator complex is an evolutionarily conserved multi-subunit protein complex that plays major roles in transcriptional activation and is essential for cell growth, proliferation, and differentiation. Recent studies revealed that some Mediator subunits formed nuclear condensates that may facilitate enhancer-promoter interactions and gene activation. Much remains to be discovered on the formation, regulation, and functions of membraneless organelles in cells. Recent findings on biomolecular condensates formed by eukaryotic transcription machineries suggested a role of phase separation in gene expression [10]. Diverse TFs can form phase-separated condensates with Mediator, suggesting that nuclear condensates may function in gene activation [15]. These new findings complement the conventional view of eukaryotic gene regulation that Mediator transduces signals from enhancer-bound TFs to the core transcriptional machinery [16]. Further studies are needed to better understand the mechanisms of condensate formation and their proposed functions in gene expression
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