Cohesin acts as a transcriptional gatekeeper by restraining pause–release to promote processive elongation [TT-seq]

Cohesin organizes 3D chromatin architecture, including promoter–enhancer loops, yet its loss has surprisingly modest effects on steady-state gene expression. We address this paradox by demonstrating that cohesin acts at different stages of transcription in human cells. First, it promotes Pol II promoter recruitment by facilitating enhancer–promoter communication that maintains active promoter chromatin states. Second, it delays pause release by transiently associating with the transcriptional machinery during the pause–release transition. Kinetic modelling suggests that reduced Pol II recruitment and enhanced pause release have compensatory effects, contributing to minimal changes in steady-state gene expression across genes upon cohesin loss. In contrast, cohesin depletion impairs robust transcriptional induction in response to external stimuli. Moreover, cohesin ensures sufficient pausing duration as a quality-control-like step to promote elongation complex assembly and transcription processivity. Here, we show that cohesin regulates multiple transcriptional steps, offering mechanistic insight into cohesin-related diseases, including cancers and cohesinopathies.