Cohesin acts as a transcriptional gatekeeper by restraining pause–release to promote processive elongation

Abstract 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. 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 steps of transcription, offering mechanistic insight into cohesin-related diseases, including cancers and cohesinopathies. Competing Interest Statement The authors have declared no competing interest. Footnotes New in vivo CUT&Tag under ATP→ADP-AlFx conditions supports the physiological existence of the cohesin-loader-Pol II promoter complex identified in vitro and advances our mechanistic understanding of how cohesin regulates Pol II pausing. Additional ChIP-seq analyses enabled a more precise assessment of the effects of cohesin depletion, revealing that cohesin promotes Pol II promoter recruitment rather than transcription initiation. Reanalysis of published datasets (Rao et al.) from the same cell line independently strengthens our conclusions.