Abstract
Mitosis poses major challenges to transcription, as gene expression is silenced and must be restored upon mitotic exit. These processes are regulated by Cdk1-dependent phosphorylation. Additional mechanisms, including transcription termination factor 2 (TTF2)–mediated removal of nascent transcripts, reinforce transcriptional shutdown. How these regulatory layers control different RNA polymerases and influence reactivation at mitotic exit remains poorly understood. Here, we probed how TTF2 controls transcription of distinct RNA classes, using polymerase-specific perturbations and nascent RNA labelling across mitosis. Loss of TTF2 led to accumulation of chromatin-associated transcripts during metaphase, predominantly RNA polymerase II–derived, consistent with its role in transcriptional clearance. Unexpectedly, TTF2 depletion caused premature RNA polymerase I reactivation during anaphase, resulting in unscheduled rRNA synthesis and early nucleolar protein recruitment. These findings place TTF2 as a novel regulator of RNA polymerase I reactivation at mitotic exit. Disruption of this control persists beyond mitosis, resulting in nucleolar fragmentation in interphase. These findings reveal TTF2 as an important regulator that interfaces with multiple RNA polymerases through distinct modes, coordinating transcriptional shutdown and timely reactivation across mitosis.