Removal of nascent transcripts by TTF2 is required for sister chromatid resolution in human cells

During mitosis, chromosome assembly is accompanied by a global shutdown of transcription. However, how this transcriptional silencing contributes to mitotic fidelity and genome stability remains poorly understood. Here, we used depletion of Transcription Termination Factor 2 (TTF2) – a key factor in mitotic transcriptional inactivation – to investigate the impact of pervasive transcription on mitotic fidelity. TTF2 depletion causes accumulation of elongating transcripts on mitotic chromatin and multiple mitotic defects, including abnormal chromosome alignment, delayed progression, and impaired chromosome compaction. Notably, defects in sister chromatid resolution are particularly prominent, with DNA bridges as the major segregation error, increasing micronuclei formation. These defects are linked to altered chromatin organisation, including R-loops accumulation at mis-segregating DNA. Most anaphase defects are suppressed when transcription is chemically inhibited, establishing a causal link between transcription and the observed mitotic defects. Our findings reveal how abnormal retention of transcriptional activity on mitotic chromatin disrupts mitosis, with impaired sister chromatid resolution linking transcriptional dysregulation to genome instability.