MED26-mediated phase separation regulates erythropoiesis by tuning transcription pausing

The Mediator complex plays a pivotal role in various aspects of transcription and development. However, it remains unclear whether individual Mediator subunits play differential roles during the progression of the developmental process. In this study, we found that the protein level of MED26, a subunit in the core Mediator complex, remained relatively abundant in terminal erythropoiesis, while that of most Mediator subunits drastically diminished. MED26 exhibits pronounced phase separation capability, which is essential for erythroid differentiation of primary human erythroblasts. Interestingly, we found that ~60% of chromatin sites with MED26 or MED1 occupancy did not co-localize. Moreover, MED26-enriched loci were associated with RNA polymerase II pausing, and MED26 preferentially interacted with transcription pausing factors, including PAF1, via its TFIIS domain. During erythropoiesis, both the relative abundance of MED26 and the Pausing Index (PI) increased. Erythroid differentiation was promoted by small molecule-induced PI elevation or overexpression of MED26—full-length or the IDR alone—indicating that MED26-associated pausing is necessary for erythropoiesis. In conclusion, this study revealed that MED26 tunes transcription pausing to facilitate erythroid development via a phase separation mechanism.