A CDK11-dependent RNA polymerase II pause-release checkpoint precedes CDK9-mediated transition to transcriptional elongation [PRO-seq and TT-seq]

Controlled gene expression is achieved through the intricate regulation of RNA polymerase II (Pol II) progression through transcription-cycle checkpoints. The pausing checkpoint, where Pol II temporarily stalls prior to release into transcriptional elongation, is a key molecular mechanism that fine-tunes gene expression. While the critical contribution of cyclin-dependent-kinase 9 (CDK9) for Pol II pause-release is well established, the requirement for other CDKs has not been fully elucidated. Here we identify a new role for CDK11 in the Pol II pausing-to-elongation transition at a checkpoint that is distinct and independent from CDK9. Selective CDK11 inhibition results in genome-wide stalling of Pol II at transcription-start-site (TSS) proximal regions and acute RNA synthesis ablation near the beginning of transcriptional units. High-resolution chromatin-immunoprecipitation and precision-nuclear-run-on assays reveals spatial differences between CDK11- and CDK9-dependent Pol II pause sites, with CDK11 regulating Pol II closer to the TSS. Cancer cells exhibit profound reliance on functional CDK11, with selective CDK11 inhibition providing therapeutic efficacy in pre-clinical in vivo models of leukaemia and lymphoma, demonstrating the importance of CDK11-dependent Pol II regulation for aggressive haematological malignancies. To investigate the role of CDK11 for Pol II transcriptional activity human AML cells were treated with CDK11 or CDK9 inhibitors, either at steady-state or following synchronisation with DRB, and precision-nuclear-run on experiments were performed.