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

  1. Laura Tovini
  2. Inês Milagre
  3. Catarina Peneda
  4. Raquel A. Oliveira

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Abstract

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.

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  1. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    During mitosis transcription is silenced (except at centromeres) and the majority of chromatin-bound RNA is removed from chromosomes. Previous work has shown that retention of elongating RNAPII on mitotic chromosomes through a WAPL degron leads to transcription-dependent chromosome segregation errors. Additionally, retention of chromatin bound RNAs through mutation of HNRNPU/SAF-A also leads to chromosome segregation errors. However, the field lacks a complete understanding of the mechanisms that remove RNA from chromatin in mitosis. Previous work from the Oliveira lab (and others) demonstrated that depletion of Lds/TTF2 also leads to chromosome segregation errors, but it was not possible to directly link chromosome segregation defects to persistent mitotic transcription. In this current work the authors examine the role of TTF2 in mitosis in human cells. They nicely show that TTF2 is required to release transcripts from chromatin during mitosis and that retention of transcripts on chromatin leads to chromosome segregation errors. Interestingly the authors find that depletion of TTF2 leads to an increase in the number of R-loops present on mitotic chromosomes and that R-loop-containing DNA is a major component of anaphase bridges. The results presented in the manuscript are high quality and the data support the conclusions. This work is important because it provides further evidence that the removal of RNA and transcription complexes from mitotic chromosomes is important for accurate chromosome segregation. There are a couple of points that the authors should consider prior to publication listed below and some minor issues with data presentation that should be corrected.

    Major points

    1. The authors use RNAi to deplete TTF2 and examine mitosis following depletion. The authors state that TTF2 depletion requires 48 hours, or approximately 2 cell cycles. Since TTF2 is depleted for the entire cell cycle it is possible that transcription termination defects caused by TTF2 depletion during interphase causes defects in mitosis and that the observed phenotypes are not a result of mitotic function of TTF2. This concern is somewhat addressed by the observation where the authors inhibit transcription using triptolide and show that this treatment rescues chromosome segregation defects observed following TTF2 depletion. However, the TRP treatment is for 4 hours, which includes a substantial portion of interphase. The length of TTF2 depletion is a significant concern and I think there are two ways that this could be addressed:

    a. Create a TTF2-AID (or dTAG) cell line and analyze chromosome segregation defects following TTF2 depletion only in mitosis. This is a difficult and time-consuming experiment but is also the most direct test of the role of TTF2 in mitosis. This experimental system would be required for publication in a high-impact journal.

    b. Include a section in their discussion acknowledging that indirect effects could be a cause of the chromosome segregation errors observed following TTF2 depletion.

    1. The authors nicely show that TTF2 depletion leads to a significant retention of EU-labeled RNA on mitotic chromosomes but do not address the nature of these transcripts. Additionally, the authors do not show that TTF2 depletion leads to changes in transcription in interphase cells. This work could be improved by the addition of EU-RNA sequencing data showing that TTF2 depletion leads to transcriptional changes in interphase (e.g. transcription past the normal termination site) and EU-RNA sequencing to identify that transcripts that are retained on mitotic chromosomes. Neither of these experiments are absolutely necessary for publication but would significantly improve the general interest of this work.
    2. The authors show that TTF2 depletion leads to an increase of R-loops on anaphase bridges. Previous work has shown that R-loops are present at mitotic centromeres (29170278) and that activation of ATR through these R-loops is necessary for accurate chromosome segregation. This work is clearly relevant to the authors results and has not been cited or discussed. This previous work should be included in the discussion and interpretation of the authors' work.

    Minor points

    1. In Figure 1 the authors show a comparison of the levels of EU-labeled RNA in control and TTF2-depleted cells at each stage of mitosis. The graphs in B and D would be much easier to compare if these were combined into a single graph.
    2. There are a number of quantitative plots that are lacking statistical comparisons between key groups. These are: Figure 1B and D, Figure 1F, S2B, Figure 4DE, S6C,

    Significance

    This advance is incremental but adds to accumulating evidence that transcription termination is important for normal chromosome segregation.

    The strengths of this work are high quality data, careful analysis, and the fact that conclusions follow directly from the data presented.

    The weaknesses are that the model system in not optimal to address the question being posed and that the authors have not completely characterized their model system.

    This work will primarily be of interest to groups working on mitotic chromatin:RNA and transcriptional regulation.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this original article, authors attempt to define the molecular and cellular consequences of retaining spurious transcriptional activity in mitotic chromosomes. The study uses the depletion of TTF2 (transcription termination factor 4) in tissue culture of HeLa cells as a model. Results show the accumulation of nascent RNAs and R-loops as a product of such aberrant transcription which, strikingly, increases the incidence of chromosome segregation errors. Importantly, authors elegantly show that these errors are corrected by inhibition of RNA Polymerase2 activity with previously reported drugs. This reviewer finds the study very interesting and compelling, very well written and structured. However, I consider that the adjustment of several aspects might improve the manuscript:

    Major comments.

    1. The main limitation of the study relies on the approach followed to deplete TTF2. Authors used siRNAs in order to decrease the levels of this factor, which implies an incubation time of 48h. This might represent a limitation. Regarding this aspect, this referee request to show the proof of TTF2 depletion in the main figure with accurate quantification when possible.
    2. Most of the main conclusions are very well supported by quantification of imaging data. However, this referee suggests the generation of superplots (ref) where values and average for each replicate can be clearly visualized. Statistical analyses comparing the median from each different conditions by t-student (unpaired test) will better support the outcome.
    3. Authors show a beautiful correlation between the presence of R-loops and chromosome segregation errors (Figure 6). I request the authors to replicate the experiment with cn or TTF2 siRNAs in combination with the triptolide treatment. Additionally, and to provide further evidence about the functional consequences of retaining R-loops, I wonder if authors can drive specific R-loop depletion by using RNase H activity. This will definitely reveal whether transcription activity or/and its product underlies the chromosome segregation defect.

    Minor comments.

    1. I highly recommend to include the value of all the statistic tests in each plot.
    2. I would suggest the incorporation of a final paragraph at the end of the introduction summarizing the most important results of the study.
    3. In the introduction, and based on wide evidences showing transcriptional activity at centromere regions (Chan 2012, Liu 2016, Perea-Resa 2024), and to a much lower extend, at the chromosome arms of mitotic chromosomes (Palozola, 2017), I would rephrase to make clear that transcription is generally repressed rather than globally silenced in mitosis.

    Referees cross-commenting

    After reading the comments from the other two referees, I maintain my view about the quality/interest of the manuscript. I endorse the potential publication of this work, after addressing the recommendations, in a reasonable period of time.

    Significance

    Authors provide a compelling study addressing the functional relevance of repressing transcription early in mitosis to properly segregate chromatids to daughter cells. In addition, the study also pursuits to illuminate the molecular and cellular consequences of inactivating TTF2 function and to provide insight into the chromosome segregation defects found under TTF2 misfunction. The study considers and discusses the most important aspects of the literature relevant for the proposed questions. The results are very encouraging and mostly confirmed by several orthogonal approaches.

    The major strength is the direct correlation found between R-loop retention and chromosome segregation defects. The major limitation is, however, the usage of slow siRNA-based strategies to deplete TTF2. The use of degron-protac alternatives would be very beneficial although I do not consider this an essential aspect.

    The study is sound and address very interesting still open questions. The publication of the results will influence and benefit a wide audience specially researches working on the mitosis and transcription fields. Research on R-loops, a field full of open questions, would also acknowledge the insights from this study.

    Overall, I consider the study very interesting and compelling. I support the evaluation and envision that its publication, once addressed the above described comments, will be feasible within roughly 3-6 months of revision.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Tovini and colleagues investigate the role of transcriptional silencing during mitosis using depletion of TTF2, a factor implicated in mitotic transcription termination. The authors show that TTF2 depletion leads to retention of elongating transcripts on mitotic chromatin, defects in chromosome organization and compaction, delayed mitotic progression, and increased chromosome segregation errors, particularly DNA bridges and UFBs. They further report accumulation of R-loops and demonstrate that transcription inhibition largely suppresses the observed segregation defects.

    Overall, this manuscript is interesting and potentially important, and the study is technically sound. In particular, I liked the experiments showing continued transcript elongation during mitosis in TTF2-depleted cells. The rescue experiments with triptolide also support the conclusion that transcription contributes significantly to the observed phenotypes. The methods and statistical analyses appear generally appropriate and sufficient for reproducibility.

    At the same time, I think that several aspects of the mechanistic interpretation are somewhat overstated, and some important alternative explanations remain insufficiently addressed. For these reasons, I believe the manuscript would benefit from substantial revision before publication.

    Major comments

    1. The authors favor a model where persistent transcription and R-loop accumulation interfere with Top2A-mediated sister chromatid resolution. While this is certainly possible, I do not think the presented data fully support such a specific interpretation. The authors demonstrate a pronounced chromosome organization phenotype. TTF2 depletion causes broadened metaphase plates and approximately 1.5-fold increased chromosome volume. These observations suggest a substantial defect in mitotic chromosome compaction. An important alternative possibility should be considered, namely that persistent mitotic transcription interferes with Condensin I and/or Condensin II loading and/or retention. Given the current understanding that chromosome condensation and loop organization are highly dynamic processes, it is conceivable that ongoing transcription could impair Condensin-mediated chromosome assembly. At minimum, the authors should assess chromosomal localization of Condensin I and II in mitotic cells after TTF2 depletion (for example CAP-H/CAP-D2 and CAP-H2/CAP-D3). Such analysis, ideally also including triptolide rescue, would substantially strengthen the mechanistic interpretation and help distinguish between a primarily transcription/R-loop-mediated defect and a more global chromosome assembly defect.
    2. The authors conclude that kinetochore-microtubule attachments are largely normal, based on Mad2 localization, inter-centromere distance, and the absence of strongly uncongressed chromosomes. In my opinion, these measurements are indirect and do not sufficiently demonstrate robust mature end-on attachments. Given the observed metaphase spreading, mild congression defects, and SAC-dependent delay, it remains possible that TTF2 depletion causes more subtle defects in k-fiber stability or attachment robustness. I think a cold-stability assay of spindle microtubules would be important here. Such experiment would directly address whether cold-stable k-fibers are normally formed and maintained in TTF2-depleted cells. Ideally, this should be combined with kinetochore markers and quantification of k-fiber intensity. Without such analysis, the statement that KT-MT attachments are unaffected should be toned down.
    3. The manuscript generally frames mitotic transcription as detrimental to chromosome segregation. However, several previous studies, including work from Hongtao Yu's lab, reported that localized centromeric/kinetochore transcription during mitosis contributes positively to chromosome segregation, including correct Sgo1 localization and centromeric function (this literature is not discussed, despite appearing conceptually relevant to the present study). This is not necessarily a contradiction; it seems possible that a limited and spatially restricted mitotic transcription program at centromeres may be beneficial, whereas persistent chromosome-wide elongation caused by TTF2 depletion becomes pathological. However, this distinction should be discussed explicitly. As currently written, the manuscript risks giving the impression that mitotic transcription is generally deleterious, which would not be fully consistent with the literature. The authors should check whether Sgo1 is localized correctly after TTF2 depletion in both Noc-arrested and metaphase (MG132- or ProTAME-arrested) cells.
    4. I was somewhat confused by the interpretation of RPA70-positive fibers. The authors state that they "did not detect a major increase" in RPA70-coated UFBs, arguing against an important contribution of replication-associated intermediates. However, in the next sentence, they report that approximately 20% of TTF2-depleted anaphases display an RPA70-positive fiber and, importantly, this phenotype is largely reverted by triptolide. In my opinion, this is not a trivial observation and appears difficult to fully reconcile with the conclusion that replication-associated events contribute minimally to the phenotype. In principle, RPA70 positivity should not necessarily be interpreted exclusively as evidence of replication stress. Persistent transcription, R-loops, or transcription-associated topological stress could generate ssDNA intermediates that can recruit RPA. Do RPA-positive fibers co-localize with R-loops? Therefore, the Discussion would benefit from a more balanced interpretation of these findings.
    5. The rescue experiments with triptolide are convincing and support a transcription-dependent contribution to the phenotype. However, because triptolide treatment was performed over several hours, these experiments do not formally distinguish between ongoing transcription elongation during mitosis and perturbations arising earlier in the cell cycle, including possible transcription-replication conflicts during late S/G2. This limitation should be acknowledged more clearly in the Discussion, especially given the recently described role of TTF2 in replisome eviction. It would be informative to perform a time-course analysis of triptolide rescue. Demonstration that the phenotype can be substantially reverted within ~30 minutes of treatment would strongly support the interpretation that persistent transcription during mitosis, as opposed to earlier cell-cycle perturbations, is the major contributor to the observed defects.

    Minor comment:

    It would be useful to check whether RHINO-positive/R-loop-associated structures preferentially localize to centromeric versus chromosome arm regions. Such information may help distinguish physiological mitotic transcription from pathological transcript retention.

    Referees cross-commenting

    I have carefully read the other reviews and they do not change my overall assessment of the manuscript or my recommendations.

    Significance

    General assessment:

    This is an interesting and potentially important study addressing how transcriptional silencing contributes to mitotic fidelity. The strongest aspect of the work is the convincing demonstration that transcription elongation persists during mitosis after TTF2 depletion and contributes to chromosome segregation defects. The main limitation is that the mechanistic interpretation currently appears somewhat narrower than supported by the data.

    Advance:

    The study extends previous work on TTF2 by linking defective mitotic transcriptional silencing to chromosome organization and chromosome segregation defects. The demonstration of transcription elongation during mitosis after TTF2 depletion is particularly interesting.

    Audience

    The work will likely be of interest to researchers studying mitosis, chromosome biology, transcription, genome stability, and chromosome organization.

    Expertise:

    mitosis, chromosome organization, nuclear organization, cell biology, biochemistry. I do not consider myself an expert in R-loop biology or transcription-coupled repair.

  5. Version published to 10.64898/2026.01.26.701825 on bioRxiv