A Mediator Switch Tunes Transcription Pausing to Drive Erythropoiesis

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development.

To address whether composition of the Mediator complex undergoes dynamic changes during erythropoiesis, we detected its expression in primary human erythroblasts derived from CD34 ⁺ hematopoietic stem and progenitor cells. We found that while the protein levels of most Mediator subunits decreased substantially in the terminal erythroid differentiation, MED26 remained relatively abundant. A gain-of-function assay in the primary human erythroid culture system showed that MED26 overexpression promoted erythroid differentiation. Furthermore, Med26 conditional knockout (cKO) mice exhibited severe erythroid and hematological defects. Erythroid precursors (especially the Cd71+Ter119+ stage) were absent in the bone marrow of Med26-cKO mice, which phenocopies the cKO mice of Gata1. Prussian blue staining of the spleen section showed more iron deposition in the cKO mice. Moreover, the cKO mice were unable to recover after phenylhydrazine-induced hemolytic anemia.

To dissect the molecular basis underlying MED26 function, we analyzed the chromatin occupancy signals of MED26 by CUT&Tag assay and compared them to those of MED1, which is often regarded as a representative subunit of Mediator, in human CD34 ⁺ erythroid culture cells on Day 4. Intriguingly, we found that ~60% of chromatin sites with MED1 or MED26 occupancy did not colocalize, suggesting these subunits exert context-dependent gene regulation. Notably, MED26 colocalizes with GATA1 and GATA2 better than MED1. PRO-seq analysis further revealed that MED26-enriched loci were associated with RNA polymerase II (Pol II) pausing. To examine how MED26 might mediate transcription pausing, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify interacting proteins of MED1 and MED26. While MED26 and MED1 similarly coimmunoprecipitated with the majority of other Mediator subunits and the elongation complex, MED26 exhibited a substantially higher interaction with transcription pausing related factors (NELF, DSIF, and PAF complexes) compared to MED1. Knocking out MED26 significantly impairs the recruitment of PAF1 to MED26 occupancy sites, indicating the presence of MED26 facilitates the recruitment of PAF1. Our results also revealed that MED26 enrichment is associated with increased transcription pausing during terminal erythroid stages, which can promote erythroid development.

Moreover, we uncovered that MED26 exhibited pronounced biomolecular condensate-forming capability. The intrinsically disordered region (IDR) of MED26 was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors to the MED26-enriched condensates. Taken together, our findings demonstrate that the relative enrichment of MED26 in terminal erythropoiesis leads to a shift of transcription condensates towards a MED26-enriched form. This shift, in turn, facilitates the recruitment of pausing related factors, thereby achieving genome-wide transcription repression during erythropoiesis. This study not only illustrates the indispensable function of MED26 in erythropoiesis but also elucidates the functional coordination of MED1 and MED26 during different erythroid stages. The dynamic switch of Mediator stoichiometry might serve as an important example for understanding the mechanism of the versatile Mediator function during development.