The cohesin protein complex is essential for forming topologically associating domains (TADs) and chromatin loops in interphase chromosomes. Loading cohesin onto chromosomes requires the cohesin loader complex, comprising NIPBL and MAU2. Cohesin associated with NIPBL adopts an ATPase-active form, which drives chromatin loop formation through a loop extrusion mechanism.

In mammalian cells, cohesin localizes with NIPBL at enhancers and gene promoters, forming stable enhancer-promoter loops through interactions with the transcriptional mediator complex. However, its functions in transcriptional regions remain unclear, and there is no direct evidence that cohesin-formed loops are essential for gene expression.

Cohesin inactivation affects transcription in only a subset of genes. We conducted genome-wide analyses of RNA polymerase II (Pol II) dynamics and its accessory factors to address this paradox. Acute cohesin depletion caused premature Pol II release from promoter-proximal pausing, resulting in defective elongation complex formation. While Pol II recruitment to promoters was unaffected, elongation-phase Pol II levels decreased by 30%. This disrupted elongation was accompanied by reduced Pausing Duration (PD), showing proper pausing is crucial for elongation. Cohesin depletion also significantly reduced Paf1C/Cdc73 levels associated with elongating Pol II, destabilizing Pol II progression on DNA. Notably, ATPase-active cohesin specifically binds to elongating Pol II. Both in vitro transcription reconstitution and in vivo studies revealed that Cdk9 regulates cohesin ATPase activity, and ATPase inhibition caused Pol II complex accumulation in a transition state. Our findings demonstrate that cohesin regulates Pol II pausing and release to ensure efficient elongation, RNA processing, and mRNA quality. These results provide the first evidence of cohesin’s essential role in transcriptional elongation and RNA integrity maintenance.