Pedro, C.; Tovini, L.; Peneda, C.; Krapinec, M. M.; Oliveira, R. A.

Mitosis poses major challenges to cellular transcription. As cells enter mitosis, transcription is globally silenced and must be precisely restored upon mitotic exit. These processes are primarily regulated by Cdk1-dependent phosphorylation. In parallel, additional mechanisms, including Transcription Termination Factor 2 (TTF2)-mediated removal of nascent transcripts, reinforce transcriptional shutdown. How these layers of regulation control individual RNA polymerases and influence transcriptional reactivation at mitotic exit remains poorly understood. Here, we probed how TTF2 differentially controls transcription of distinct RNA classes, using polymerase-specific perturbations and nascent RNA labelling across mitosis. Loss of TTF2 led to the accumulation of chromatin-associated transcripts during metaphase, predominantly RNA Polymerase II-derived, consistent with its established role in transcriptional clearance. More unexpectedly, TTF2 depletion caused premature RNA Polymerase I reactivation during anaphase, resulting in unscheduled rRNA synthesis and early recruitment of nucleolar proteins. 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 increased nucleolar fragmentation in interphase. Together, these findings reveal TTF2 as a conserved regulator that interfaces with multiple RNA polymerases through functionally distinct modes of control, coordinating both transcriptional shutdown and timely reactivation across mitosis.