Coactivator condensation drives lineage specification

During development, cells make switch-like decisions to activate the expression of new gene programs leading to lineage specification1. The mechanisms underlying these decisive choices remain unclear2. Here we find that Myocardin (MYOCD), a cardiomyocyte and smooth muscle cell-specific transcriptional coactivator3-6, activates lineage-specific gene programs by concentration-dependent and switch-like formation of nuclear condensates. While compartmentalization of the transcriptional machinery by condensates has been associated with gene activation7,8, directly linking the two processes has been a major challenge for the field9,10. By modeling the natural changes in MYOCD concentration during development, coupled with quantitative fluorescence microscopy, single-cell resolution reporter assays, and cellular differentiation assays, we demonstrate that condensate formation is directly linked to transcriptional activation and lineage specification. During cardiomyocyte and smooth muscle cell differentiation, the formation of MYOCD condensates precedes activation of cell identity genes and these condensates are present at sites of cell identity gene transcription. MYOCD condensates form, activate gene expression, and specify cell state at critical concentration thresholds, dependent upon the C-terminal disordered region of MYOCD. Disrupting condensate formation by manipulating the sequence of this region impairs gene activation which can be rescued by replacing this region with condensate-forming disordered regions from functionally unrelated proteins. These results demonstrate that coactivator condensation at critical concentrations enables switch-like changes in gene expression programs crucial for lineage specification. Overall design: Comparative gene expression profiling analysis of RNA-seq data and MYOCD chromatin occupancy by ChIP-seq data for undifferentiated hiPSCs and meso-stage differentiation of smooth muscle (D8) and cardiomyocytes (D5)