SMURFs regulate mesendoderm differentiation through SMAD2-dependent ubiquitination of CDK9

Precise control of transcriptional timing is essential during embryonic development, where dynamic gene expression programs govern lineage specification. Promoter-proximal pausing of RNA polymerase II (Pol II) and its regulated release into productive elongation constitute a critical checkpoint in transcriptional regulation. Nodal, a transforming growth factor β (TGF-β) family morphogen, promotes mesendoderm differentiation through SMAD2-dependent transcription, in part by promoting cyclin-dependent kinase 9 (CDK9)-mediated release of paused Pol II. While mechanisms of transcriptional activation downstream of Nodal are well characterized, how this transcriptional program is attenuated during gastrulation remains unclear. Here, we show that E3 ubiquitin ligases SMAD Ubiquitination Regulatory Factor (SMURF) 1 and SMURF2 are critical regulators of SMAD2-driven transcription. SMURF1/2 ubiquitinate CDK9 and promote its proteasome-mediated degradation, thereby restraining CDK9-dependent promoter-proximal pause release and productive transcription of SMAD2-dependent genes. Loss of SMURF function stabilizes CDK9, increases its occupancy at SMAD2 target genes, and enhances Pol II pause release, thereby sustaining mesendodermal gene expression. These findings reveal a ubiquitin-dependent mechanism that restricts morphogen-induced gene expression and ensures timely developmental progression.