Engineering a superactivator of smooth muscle gene expression with the Myocardin condensate forming region [RNA-seq]

Differentiation of smooth muscle cells (SMCs) is accompanied by the expression of a battery of genes controlled by serum response factor (SRF), a ubiquitous transcription factor with weak intrinsic transcriptional activity. Myocardin (MYOCD) is a potent transcriptional coactivator that activates smooth muscle differentiation through its association with SRF. MYOCD forms condensates through its highly disordered transcription activation domain (TAD), whereas SRF displays a diffuse distribution within the nucleus. SRF is recruited to nuclear condensates by the TAD of MYOCD (MYOCD-TAD), thereby triggering the smooth muscle gene program. Swapping the weak TAD of SRF with that of MYOCD enables SRF to form condensates and acquire the ability to reprogram fibroblasts to SMCs. SRF-MYOCD-TAD chimeric protein exhibits stronger transcriptional activity on smooth muscle genes than MYOCD. Using protein proximity labelling, quantitative proteomics and super-resolution confocal microscopy, we show that MYOCD-TAD condensates aggregate chromatin remodelers, RNA polymerases, and mRNA processing factors to drive smooth muscle gene expression. These findings provide insights into the mechanisms whereby nuclear condensates promote tissue-specific gene expression and point toward a strategy for engineering superactivators leveraging transcriptional condensates.