Transcription can be sufficient, but is not necessary, to advance replication timing

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   Review Commons

   May 6, 2025

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   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   The study investigates the relationship between replication timing (RT) and transcription. While there is evidence that transcription can influence RT, the underlying mechanisms remain unclear. To address this, the authors examined a single genomic locus that undergoes transcriptional activation during differentiation. They engineered the Pln locus by inserting promoters of varying strengths to modulate transcription levels and assessed the impact on replication timing using Repli-seq. Key Findings: • Figure 1C and 1D: The data show that higher transcription levels correlate with an …

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   Reply to the reviewers

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   The study investigates the relationship between replication timing (RT) and transcription. While there is evidence that transcription can influence RT, the underlying mechanisms remain unclear. To address this, the authors examined a single genomic locus that undergoes transcriptional activation during differentiation. They engineered the Pln locus by inserting promoters of varying strengths to modulate transcription levels and assessed the impact on replication timing using Repli-seq. Key Findings: • Figure 1C and 1D: The data show that higher transcription levels correlate with an advanced RT, suggesting that transcriptional activity influences replication timing. • Figure 2: To determine whether transcription alone is sufficient to alter RT, the authors inserted an hPGK reporter at different genomic locations. However, given the findings in Figure 1, which suggest that this is not the primary mechanism, • Figure 3: The authors removed the marker to examine whether the observed effects were due to the promoter-driven Pln locus, which has significantly larger then the marker. • Figure 4: The study explores the effect of increased doxycycline (Dox) treatment at the TRE (tetracycline response element), further supporting the role of transcription in RT modulation. • Figure 5: The findings demonstrate that Dox-induced RT advancement occurs rapidly, is reversible, and correlates with transcription levels, reinforcing the hypothesis that transcription plays a direct role in influencing replication timing. • Figure 6. Shows that during differentiation transcription of Pln is not required for RT advancement.

   Overall, the study presents a compelling link between transcription and replication timing, though some experimental choices warrant further clarification. I have no major comments.

   __Minor Comments: __Overall, the results are convincing, and the study appears to be well-conducted. In Figure 2, the authors use the hPGK promoter. However, it is unclear why they did not use the constructs from the previous experiments. Given that the hPGK promoter did not advance RT in Figure 1, the results in Figure 2 may not be entirely unexpected.

   We took advantage of previously published cell lines using a PiggyBac Vector designed to pepper the reporter gene at random sites throughout the genome; the point of the experiment was to acquire supporting evidence for the hypothesis that any vector with its selectable marker driven by the hPGK promoter will not advance RT no matter where it is inserted. Since there are reports concluding that transcription per se is sufficient to advance RT, it was important to confirm that there was nothing unique about the particular vector or locus into which we inserted our panel of vectors.

   ACTION DONE: We have now added the following sentence to the results describing this experiment: “____By analyzing RT in these lines, we could evaluate the effect of a different hPGK vector on RT when integrated at many different chromosomal sites. “

   Additionally, the study does not formally exclude the possibility that Pln protein expression itself influences RT. In Figure 1, readthrough transcription at the Pln locus could potentially drive protein expression. It would be useful to know whether the authors address this point in the discussion.

   NOT DONE FOR NEED OF CLARIFICATION: It is unclear why a secreted neural growth factor would have a direct effect on replication timing in embryonic stem cells and, in particular, only in cis (remember there is a control allele that is unaffected). We would be happy to address this in the Discussion if we understood the reviewers’ hypothesis. We cannot respond to this comment without understanding the hypothesis being tested as we do not know how a secreted protein could affect the RT of one allele without affecting the other.

   Regarding the mechanism, if transcription across longer genomic regions contributes to RT changes, transcription-induced could DNA supercoiling play a role. For instance, could negative supercoiling generated by active transcription influence replication timing?

   Yes, many mechanisms are possible.

   ACTION DONE: ____We have added the following sentence to the discussion, referencing a seminal paper on that topic by Nick Gilbert: “ ____For example, long transcripts could remodel a large segment of chromatin, possibly by creating domains of DNA supercoiling (Naughton et. al., 2013____).____”

   It remains puzzling why Pln transcription does not contribute to replication timing during differentiation. Is there any evidence of chromatin opening during this process? For example, are ATAC-seq profiles available that could provide insights into chromatin accessibility changes during differentiation?

   We thank the reviewer for asking this as we should have mentioned something very important here. Lack of necessity for transcription implies that independent mechanisms are functioning to elicit the RT switch. In other work (Turner et. al., bioRxiv, provisionally accepted to EMBO J.), we have shown that specific cis elements (ERCEs) can function to maintain early replication in the absence of transcription.

   ACTION DONE: We now explicitly state in the Discussion: “____This is not surprising, given that ERCEs can maintain early RT in the absence of transcription (Turner, bioRxiv).”

   ACTION TO BE DONE SOON: We will provide a new Figure 6D showing ATAC-seq changes upon differentiation of mESCs to mNPCs and their location relative to the promoter/enhance deletion. As you will see, there is an ATAC-seq site that appears during differentiation, upstream of the deletion. We will hypothesize in the revised manuscript that these are the elements that drive the RT switch and that future studies need to investigate that hypothesis. We have also added the following sentences to the discussion after the sentence above, stating: “____In fact, new sites of open chromatin, consistent with ERCEs appear outside of the deleted Ptn transcription control elements after differentiation (soon to be revised Figure 6D). The necessity and sufficiency of these sites to advance RT independent of transcription will be important to follow up.”

   We also have preliminary data that are part of a separate project in the lab so they are not ready for publication, but are directly relevant to the reviewer’s question. This data shows evidence for a region upstream of the Ptn promoter/enhancer deletion described in Figure 6 that, when deleted, DOES have an effect on the RT switch during differentiation. This deletion overlaps an ATAC-seq site we will show in the new figure 6D.

   Reviewer #1 (Significance (Required)):

   This is a compelling basic single-locus study that systematically compares replication timing (RT) and transcription dynamics while measuring several key parameters of transcription.

   My relevant expertise lies in transcriptional regulation and understanding how noncoding transcription influences local chromatin and gene expression.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   In the manuscript entitled: Transcription can be sufficient, but is not necessary, to advance replication timing", the authors use as they state a "reductionist approach" to address a long-standing question in the replication field on what level the process of transcription within a replication domain can alter the underlying replication timing of this domain. The authors use an elegant hybrid mouse embryonic stem cell line to discriminate the two allelic copies and focus on a specific replication domain harboring the neuronal Ptn gene that is only expressed upon differentiation. The authors first introduce four different promoters in the locus upstream of Ptn gene that drive expression of small transgenes. Only the promoters with highest transcriptional induction could advance RT. If the promoters are placed in such a way that they drive expression of the 96kb Ptn gene, then also some the weaker promoters can drive RT advancement, suggesting that it is a combination of transcriptional strength and size of the transcribed domain important for RT changes. Using a DOX-inducible promoter, the authors show that this happens very fast (3-6h after transcription induction) and is reversible as removal of DOX leads to slower RT again. Finally, deleting the promoter of Ptn gene and driving cells into differentiation still advances RT, allowing the authors to conclude that "transcription can be sufficient but not necessary to advance replication timing."

   Major comments: Overall, this is a well designed study that includes all necessary controls to support the author's conclusions. I think it is a very interesting system that the authors developed. The weakness of the manuscript is that there is no mechanistic explanation how such RT changes are achieved on a molecular basis. But I'm confident that the system could be indeed used to further dissect the mechanistic basis for the transcription dependence of RT advancements.

   Therefore, I support publication of this manuscript if a few comments below can be addressed.

   1. Figure 4 shows a titration of different DOX concentrations and provides clear evidence that the degree of RT advancement tracks well with the level of transcription. As the doses of DOX are quite high in this experiment, have the authors checked on a global scale to what extent transcription might be deregulated in neighbouring genes or genome-wide?

   The DOX concentration that we use for all experiments other than the titration is 2 µg/ml, which is quite standard. The high concentrations (up to 16µg/ml) are only used in the titration experiments shown in Figure 4 to demonstrate that we have reached a plateau. In fact, we stated in Materials and Methods that high doses of Dox led to cell toxicity. Looking at the transcription datasets, there are no significant changes in transcription below 8µg/ml, a few dozen significant changes at 8 and more such changes at 16µg/ml of DOX. The tables of genome wide RT and transcription are provided in the manuscript for anyone wishing to investigate the effects of Dox on cellular physiology but at the concentration used in all other experiments (2µg/ml) there are no effects on transcription.

   __ACTION DONE: We have now modified the statement in the Materials and Methods to read: “ ____Mild toxicity and changes in genome-wide transcription were observed at 8µg/ml and more so at 16µg/ml”. __

   1. One general aspect is that the whole study is only focused on the one single Ptn replication domain. Could the authors extend this rather narrow view a bit and also show RT data in the neighbouring domains. This would be particularly important for the DOX titration experiment that has the potential to induce transcriptional deregulation (see comment above).

   __ACTION DONE: We have now added to revised Supplemental Figure 4 a zoom out of 10 Mb surrounding the Ptn gene showing no detectable effects on RT at any of the titration concentrations. __

   __ACTION TO BE DONE SOON: To address the generalization of the findings (length and strength matter), we have repeated the ESC to NPC differentiation and performed both Repli-seq and BrU-seq to evaluate RT changes relative to total genomic nascent transcriptional changes. The sequencing reads for this experiment are in our analyst’s hands so we expect this to be ready within a few weeks. We will provide a new Figure 7 comparing genome-wide changes in RT vs. transcription to determine the significance of length and strength of transcription induction to RT advances and the necessity of transcriptional induction for RT advances. We and other laboratories have performed many integrative analyses of RNA-microarray/RNA-seq data vs. RT changes, but not total genomic nascent transcription and not with a focus on the effect of length and strength of transcription. For example, outcomes that would be consistent with our reductionist findings at the Ptn locus would be if we find domains that are advanced for RT with no induction of transcription (transcription not necessary) and little to no regions showing significant induction of transcription without RT advances. __

   1. Figure 5 shows that the full capacity to advance RT upon DOX induction of the Ptn gene is achieved after 3h to 6h of DOX induction, so substantially less than a full cell cycle in mEScs (12h). This result suggests that origin licensing/MCM loading cannot be the critical mechanism to drive the RT change because only a small fraction of the cells has undergone M/G1-phase where origins are starting to get loaded. As a large fraction of mESCs (60-70%) are S-phase cells in an asynchronous population, the mechanism is likely taking place directly in S-phase. Could the authors try to synchronize cells in G1/S using double-thymidine block, then induce DOX for 3h before allowing cells to reenter S-phase and then check replication timing of the domain? This can be compared to an alternative experiment where transcription is only induced for 3h upon release into S-phase. This could provide more mechanistic insights as to whether transcription is sufficient to drive RT changes in G1 versus S-phase cells.

   We agree that the timing of induction is such that it is very likely that alterations in RT can occur during S phase. The reviewer proposes a reasonable experiment that could be done, but it would require a long delay of this publication to develop and validate those synchronization protocols and we do not have personnel at this time to carry out the experiment. This would be a great initiating experiment for someone to pursue the mechanisms by which transcription can advance RT.

   ACTION DONE: We have added the following sentence to the Discussion section on mechanisms: ____The rapid nature of the RT change after induction of transcription suggests that RT changes can occur after the functional loading of inactive MCM helicases onto chromatin in telophase/early G1 (Dimitrova, JCB, 1999; Okuno, EMBO J. 2001; Dimitrova, J. Cell Sci, 2002), and possibly after S phase begins.

   __Minor comments: __• Figure 1B and Figure 6A. Quality of the genome browser snapshots could be improved and certain cryptic labelling such as "only Basic displayed by default" could be removed

   ACTION DONE: We have modified these figures.

   The genome browser tracks appear a bit small across the figures and could be visually improved.

   ACTION DONE: We have modified the genome browser tracks to improve their presentation

   In figure 1E we see an advancement in RT in Ptn gene caused by nearby enhanced Hyg-TK gene expression induced by mPGK promoter. However, in figure 3D we see mPGK promoter has reduced ability to advance RT of Ptn gene. It would be nice to address this discrepancy in the results.

   The reviewer’s point is well taken. We are not sure of the answer. You can see that the transcription is very low in both cases, while the RT shift is greater in one replicate vs. the other.

   ACTION DONE: We have, rather unsatisfactorily, added the following sentence to the results section describing Figure 3. “____We do not know why the mPGK promoter was so poor at driving transcription in this context.”

   Reviewer #2 (Significance (Required)):

   In my point of view, this is an important study that unifies a large amount of literature into a conceptual framework that will be interesting to a broad audience working on the intertwined fields of gene regulation, transcription and DNA replication, as well as cell fate switching and development.

   __Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ In their manuscript, "Transcription can be sufficient, but is not necessary, to advance replication timing," Vouzas et al. take a systematic and reductionist approach to investigate a late-replicating domain on chromosome VI. Here, they examine the effect of transcribing a single gene locus, Pleiotrophin, on replication timing. When inserting or manipulating promoters or transcript lengths using CRISPR-Cas9, replication timing was altered in mESCs as judged by a combination of Repli-Seq, Bru-Seq, and RNA-Seq. Importantly, they found that transcription can be sufficient to advance replication timing depending on the length and strength of the expression of an ectopically transcribed gene. Taken together, the manuscript presents a compelling argument that transcription can advance replication timing but is not necessary for it.

   __Major comments __• A schematic or conceptual model summarising the major findings of transcription-dependent and independent mechanisms of RT advancement should be included in the discussion to add to the conceptual framework

   NOT DONE: We discussed this at length between the two senior authors and the first author and we do not feel ready to draw a summary model. We do not know what is advancing RT when transcription is induced or not induced, and we are not comfortable choosing one possible model of many. We hope that the added speculations on mechanism in the Discussion will sufficiently convey the future research that we feel needs to be done.

   ACTIONS DONE: In addition to the speculation on mechanism that already was in our Discussion section, we have added: On mechanisms of rapid induction of RT change, we have added to the Discussion: “____The rapid nature of the RT change after induction of transcription suggests that RT changes can occur after the functional loading of inactive MCM helicases onto chromatin in telophase/early G1 (Dimitrova, JCB, 1999; Okuno, EMBO J. 2001; Dimitrova, J. Cell Sci, 2002), and possibly after S phase begins.” And “For example, long transcripts could remodel a large segment of chromatin, possibly by creating domains of DNA supercoiling (Naughton et. al., 2013, PMID ____23416946).____ “ On mechanisms of RT advance in the absence of transcription, we have added the following to the Discussion: “____This is not surprising, given that ERCEs can maintain early RT in the absence of transcription (Turner, bioRxiv). In fact, chromatin features with the properties of ERCEs do appear outside of the deleted Ptn transcription control elements after differentiation (soon to be revised Figure 6C). The necessity and sufficiency of these new chromatin features to advance RT independent of transcription will be important to follow up.”

   Vouzas et al. spend a substantial part of the manuscript to delve into the requirements to advance RT and even use a Doxycycline-based titration for temporal advancement of RT. Yet, all conclusions come from the use of hybrid-genome mouse embryonic stem cells (mESCs). Therefore, it remains speculative if and whether findings can be generalized to other cell types or organisms. The authors could include another organism/ cell type to strengthen the relevance of their findings to a broader audience, particular as they identified promoters that drive ectopic gene expression without affecting RT. Showcasing this in other model organisms would be of great interest.

   NOT DONE: To set this system up in another cell type or species would take a very long time. We also do not have personnel to carry that approach.

   ACTION TO BE DONE SOON: As an alternative approach that partially addresses this reviewer’s concern, we will provide a new Figure 7 with an analysis of RT changes vs. transcriptional changes when mESCs are differentiated to neural precursor cells. As described above in response to Revier #2s criticism #2, we have repeated the ESC to NPC differentiation and performed both Repli-seq and BrU-seq to evaluate RT changes relative to total genomic nascent transcriptional changes. The sequencing reads for this experiment are in our analyst’s hands so we expect this to be ready within a few weeks. We will compare genome-wide changes in RT vs. transcription to determine the significance of length and strength of transcription induction to RT advances and the necessity of transcriptional induction for RT advances. We and other laboratories have performed many integrative analyses of RNA-microarray/RNA-seq data vs. RT changes, but not total genomic nascent transcription and not with a focus on the effect of length and strength of transcription. For example, outcomes that would be consistent with our reductionist findings at the Ptn locus would be if we find domains that are advanced for RT with no induction of transcription (transcription not necessary) and little to no regions showing significant induction of transcription without RT advances.

   OPTIONAL: as with the previous point, the authors went to great depth and length to show how ectopic manipulations affect RT changes on a single locus using genome-wide methods. In addition, the manuscript would benefit from the inclusion of other loci, particularly as transcription of the Ptn locus wasn't needed during differentiation to advance RT at all.

   NOT DONE: This rigorous reductionist approach is laborious and to set it up at one gene at a time at additional loci would be a huge effort taking quite a long time.

   ACTION TO BE DONE SOON: (same as response above) As an alternative approach that partially addresses this reviewer’s concern, we will provide a new Figure 7 with an analysis of RT changes vs. transcriptional changes when mESCs are differentiated to neural precursor cells. As described above in response to Reviewer #2s criticism #2, we have repeated the ESC to NPC differentiation and performed both Repli-seq and BrU-seq to evaluate RT changes relative to total genomic nascent transcriptional changes. The sequencing reads for this experiment are in our analyst’s hands so we expect this to be ready within a few weeks. We will compare genome-wide changes in RT vs. transcription to determine the significance of length and strength of transcription induction to RT advances and the necessity of transcriptional induction for RT advances. We and other laboratories have performed many integrative analyses of RNA-microarray/RNA-seq data vs. RT changes, but not total genomic nascent transcription and not with a focus on the effect of length and strength of transcription. For example, outcomes that would be consistent with our reductionist findings at the Ptn locus would be if we find domains that are advanced for RT with no induction of transcription (transcription not necessary) and little to no regions showing significant induction of transcription without RT advances.

   The same point of Ptn not needing to be transcribed to advance RT of the respective domain, albeit being a very interesting observation, disturbs the flow of the manuscript, as the whole case was built around transcription and this particular locus-containing domain. Maybe one can adapt the storytelling to fit better within the overall framework.

   We would argue that demonstrating induction of Ptn, the only gene in this domain, is sufficient to induce early RT is a logical segway to asking whether, in the natural situation, induction is correlated with advance in RT. Our results show that transcription is sufficient but not necessary, which is expected if there are other mechanisms that regulate RT.

   __ACTION DONE: To make this transition more smooth, we have added the following sentence to the beginning of the results section describing Figure 6: “ ____This raises the question as to whether the natural RT advance that accompanies Ptn induction during differentiation requires Ptn transcription, or whether other mechanisms, such as ERCEs (Sima / Turner) can advance RT independent of transcription. “ __

   ACTION TO BE DONE SOON:____ To finish the work flow in a way that ties length and strength and sufficiency but not necessity in to the theme of natural cellular differentiation, we will provide a new Figure 7 with an analysis of RT changes vs. transcriptional changes when mESCs are differentiated to neural precursor cells, as described above.

   __Minor comments __• While citations are thorough, some references (e.g., "need to add Wang, Klein, Mol. Cell 2021") are incomplete.

   __ACTION TO BE DONE SOON: We apologize that some references seemed to not be incorporated into the reference manager Mendely. Since we are still planning to add one more figure soon and we will need to add some references for the datasets that will be shown in future Figure 6D, after that draft is ready, we will comb the manuscript for any references that were not entered and correct them. __

   The text corresponding to Figure 1C could use more explanation for readers not familiar with the depiction of Repli-Seq data.

   ACTION DONE: “____Repli-seq labels nascent DNA with BrdU, followed by flow cytometry to purify cells in early vs. late S phase based on their DNA content, then BrdU-substituted DNA from each of these fractions is immunoprecipitated, sequenced and expressed as a log2 ration of early to late synthesized DNA (log2E/L). BrU-seq labels total nascent RNA, which is then immunoprecipitated an expressed as reads per million per kilobase (RPMK).”

   Figure 1C needs labelling of the x-axes.

   ACTION DONE: We have now labeled the X axes.

   Statistical analyses should be used consistently throughout the manuscript and explained in more detail, i.e. significance levels, tests, instead of "Significant differences....calculated using x".

   We used the same analysis for all the Repliseq data and the same analysis for all the Bruseq data. We agree that we did not present this consistently in the figure legends and methods.

   ACTION DONE:____ To correct the confusion we have clarified the statistical methods in the methods section and referred to methods in the figure legends as follows:

   The methods description of statistical significance for RT now reads: “____Statistical significance of RT changes for all windows in each sample, relative to WT, were calculated using RepliPrint (Ryba et al., 2011), with a p-value of 0.01 used as the cut-off for windows with statistically significant differences.”

   The methods description of statistical significance for transcription now reads: “____Differential expression analysis, including the calculation of statistically significant differences in expression, was conducted using the R package DESeq2____. In Figure 1, statistical significance was calculated relative to HTK expression in the parental cell line, which is expected to be zero, since the parental line does not have an HTK insertion. In all other Figures significance was calculated relative to Ptn expression in the parental line, which is expected to be zero, since the parental line does not express Ptn.____”

   The legend to Figure 1C now reads: The red shading indicates 50kb windows with statistically significant differences in RT between WT casteneus and modified 129 alleles, determined as described in Methods.

   The legend to Figure 1E now reads: “The asterisks indicate a significant difference in the levels of HTK expression relative to HTK expression in the parental cell line as described in Methods. ____There are no asterisks for the RT data, as statistical significance was calculated for individual 50kb windows as shown in panel (C).”

   Each time significance is measured in the subsequent legends, it is followed by the phrase “, determined as described in Methods” or “presented as in Figure 1C” or “presented as in Figure 1E” as appropriate.

   __ __ __**Referees cross-commenting** __ Comment on Reviewer#1's review, comment mentioning ATAC-Seq: Another way to look at this could be to investigate for origin usage changes (BrdU-Seq or GLOE-Seq) of chromosome 6 during differentiation.

   NOT DONE: Unfortunately we could not find any studies comparing origin mapping in mESCs and mNPCs.

   Comment on Reviewer#2's review, major comment 3: I do agree with their statement that origin loading cannot be the driver of RT change, as MCM2-7 double hexamer loading is strictly uncoupled from origin firing. Hence, any mechanism responsible for RT advance must happen at the G1/S phase transition or during S-phase, most likely due to the regulated activity of DDK/CDK or the limitation and preferred recruitment of firing factors to early origins. This could be tested through overexpression of said factors.

   NOT DONE: We agree that manipulating these factors would be a reasonable next approach to sort out mechanism. Due to limited resources and personnel, we will not be able to do this in a short period of time. We also argue that these are experiments for the next chapter of the story, likely requiring an entire PhD thesis (or multiple) to sort out.

   ACTION DONE: We have added the following sentence to the Discussion section on mechanisms: ____The rapid nature of the RT change after induction of transcription suggests that RT changes can occur after the functional loading of inactive MCM helicases onto chromatin in telophase/early G1 (Dimitrova, JCB, 1999; Okuno, EMBO J. 2001; Dimitrova, J. Cell Sci, 2002), and possibly after S phase begins.

   Reviewer #3 (Significance (Required)):

   General: This manuscript presents a compelling study investigating the relationship between transcription and replication timing (RT) using a reductionist approach. The authors systematically manipulated transcriptional activity at the Ptn locus to dissect the elements of transcription that influence RT. The study's strengths lie in its rigorous experimental design, clear results, and the reconciliation of seemingly contradictory findings in the existing literature. However, some aspects could be improved, particularly in exploring the mechanistic details of transcription-independent RT regulation at the investigated domain, the generalisability of the findings to other cells/organisms, and enhancing the presentation of certain data (explanation of e.g. Figure 1c, dense figure arrangement, lack of a summary figure illustrating key findings (e.g., correlation between transcription rate, readthrough effects, and RT advancement)).

   Advance: The manuscript directly addresses and reconciles contradictory findings in the literature regarding the effect of ectopic transcription on RT. Previous studies have reported varying effects, with some showing that transcription advances RT (Brueckner et al., 2020; Therizols et al., 2014), while others have shown no effect or only partial effects depending on the insertion site (Gilbert & Cohen, 1990; Goren et al., 2008). The current study conceptually advances the field by systematically testing different promoters and transcript lengths at a single locus (mechanistic insight), demonstrating that the length and strength of transcription, as well as promoter context, influence RT. This presents a unifying concept on how RT can be influenced. The authors also present a tunable system (technical advance) that allows rapid and reversible alterations of RT, which will certainly be useful for future studies and the field.

   Audience: The primary audience will be specialised researchers in the fields of replication timing, epigenetics, and gene regulation. This study may be of interest beyond the specific field of replication timing, such as cancer biology, developmental biology, particularly if a more broader applicability of its tools and concepts can be shown.

   Expertise: origin licensing, origin activation, MCM2-7, yeast and human cell lines

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   May 6, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #3

   Evidence, reproducibility and clarity

   In their manuscript, "Transcription can be sufficient, but is not necessary, to advance replication timing," Vouzas et al. take a systematic and reductionist approach to investigate a late-replicating domain on chromosome VI. Here, they examine the effect of transcribing a single gene locus, Pleiotrophin, on replication timing. When inserting or manipulating promoters or transcript lengths using CRISPR-Cas9, replication timing was altered in mESCs as judged by a combination of Repli-Seq, Bru-Seq, and RNA-Seq. Importantly, they found that transcription can be sufficient to advance replication timing depending on the length and …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   In their manuscript, "Transcription can be sufficient, but is not necessary, to advance replication timing," Vouzas et al. take a systematic and reductionist approach to investigate a late-replicating domain on chromosome VI. Here, they examine the effect of transcribing a single gene locus, Pleiotrophin, on replication timing. When inserting or manipulating promoters or transcript lengths using CRISPR-Cas9, replication timing was altered in mESCs as judged by a combination of Repli-Seq, Bru-Seq, and RNA-Seq. Importantly, they found that transcription can be sufficient to advance replication timing depending on the length and strength of the expression of an ectopically transcribed gene. Taken together, the manuscript presents a compelling argument that transcription can advance replication timing but is not necessary for it.

   Major comments

   • A schematic or conceptual model summarising the major findings of transcription-dependent and independent mechanisms of RT advancement should be included in the discussion to add to the conceptual framework
   • Vouzas et al. spend a substantial part of the manuscript to delve into the requirements to advance RT and even use a Doxycycline-based titration for temporal advancement of RT. Yet, all conclusions come from the use of hybrid-genome mouse embryonic stem cells (mESCs). Therefore, it remains speculative if and whether findings can be generalized to other cell types or organisms. The authors could include another organism/ cell type to strengthen the relevance of their findings to a broader audience, particular as they identified promoters that drive ectopic gene expression without affecting RT. Showcasing this in other model organisms would be of great interest.
   • OPTIONAL: as with the previous point, the authors went to great depth and length to show how ectopic manipulations affect RT changes on a single locus using genome-wide methods. In addition, the manuscript would benefit from the inclusion of other loci, particularly as transcription of the Ptn locus wasn't needed during differentiation to advance RT at all.
   • The same point of Ptn not needing to be transcribed to advance RT of the respective domain, albeit being a very interesting observation, disturbs the flow of the manuscript, as the whole case was built around transcription and this particular locus-containing domain. Maybe one can adapt the storytelling to fit better within the overall framework.

   Minor comments

   • While citations are thorough, some references (e.g., "need to add Wang, Klein, Mol. Cell 2021") are incomplete.
   • The text corresponding to Figure 1C could use more explanation for readers not familiar with the depiction of Repli-Seq data.
   • Figure 1C needs labelling of the x-axes.
   • Statistical analyses should be used consistently throughout the manuscript and explained in more detail, i.e. significance levels, tests, instead of "Significant differences....calculated using x".

   Referees cross-commenting

   Comment on Reviewer#1's review, comment mentioning ATAC-Seq: Another way to look at this could be to investigate for origin usage changes (BrdU-Seq or GLOE-Seq) of chromosome 6 during differentiation.

   Comment on Reviewer#2's review, major comment 3: I do agree with their statement that origin loading cannot be the driver of RT change, as MCM2-7 double hexamer loading is strictly uncoupled from origin firing. Hence, any mechanism responsible for RT advance must happen at the G1/S phase transition or during S-phase, most likely due to the regulated activity of DDK/CDK or the limitation and preferred recruitment of firing factors to early origins. This could be tested through overexpression of said factors.

   Significance

   General: This manuscript presents a compelling study investigating the relationship between transcription and replication timing (RT) using a reductionist approach. The authors systematically manipulated transcriptional activity at the Ptn locus to dissect the elements of transcription that influence RT. The study's strengths lie in its rigorous experimental design, clear results, and the reconciliation of seemingly contradictory findings in the existing literature. However, some aspects could be improved, particularly in exploring the mechanistic details of transcription-independent RT regulation at the investigated domain, the generalisability of the findings to other cells/organisms, and enhancing the presentation of certain data (explanation of e.g. Figure 1c, dense figure arrangement, lack of a summary figure illustrating key findings (e.g., correlation between transcription rate, readthrough effects, and RT advancement)).

   Advance: The manuscript directly addresses and reconciles contradictory findings in the literature regarding the effect of ectopic transcription on RT. Previous studies have reported varying effects, with some showing that transcription advances RT (Brueckner et al., 2020; Therizols et al., 2014), while others have shown no effect or only partial effects depending on the insertion site (Gilbert & Cohen, 1990; Goren et al., 2008). The current study conceptually advances the field by systematically testing different promoters and transcript lengths at a single locus (mechanistic insight), demonstrating that the length and strength of transcription, as well as promoter context, influence RT. This presents a unifying concept on how RT can be influenced. The authors also present a tunable system (technical advance) that allows rapid and reversible alterations of RT, which will certainly be useful for future studies and the field.

   Audience: The primary audience will be specialised researchers in the fields of replication timing, epigenetics, and gene regulation. This study may be of interest beyond the specific field of replication timing, such as cancer biology, developmental biology, particularly if a more broader applicability of its tools and concepts can be shown.

   Expertise: origin licensing, origin activation, MCM2-7, yeast and human cell lines

   Read the original source
3. 

   Review Commons

   May 6, 2025

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   Referee #2

   Evidence, reproducibility and clarity

   In the manuscript entitled: Transcription can be sufficient, but is not necessary, to advance replication timing", the authors use as they state a "reductionist approach" to address a long-standing question in the replication field on what level the process of transcription within a replication domain can alter the underlying replication timing of this domain. The authors use an elegant hybrid mouse embryonic stem cell line to discriminate the two allelic copies and focus on a specific replication domain harboring the neuronal Ptn gene that is only expressed upon differentiation. The authors first introduce four different promoters …

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   Referee #2

   Evidence, reproducibility and clarity

   In the manuscript entitled: Transcription can be sufficient, but is not necessary, to advance replication timing", the authors use as they state a "reductionist approach" to address a long-standing question in the replication field on what level the process of transcription within a replication domain can alter the underlying replication timing of this domain. The authors use an elegant hybrid mouse embryonic stem cell line to discriminate the two allelic copies and focus on a specific replication domain harboring the neuronal Ptn gene that is only expressed upon differentiation. The authors first introduce four different promoters in the locus upstream of Ptn gene that drive expression of small transgenes. Only the promoters with highest transcriptional induction could advance RT. If the promoters are placed in such a way that they drive expression of the 96kb Ptn gene, then also some the weaker promoters can drive RT advancement, suggesting that it is a combination of transcriptional strength and size of the transcribed domain important for RT changes. Using a DOX-inducible promoter, the authors show that this happens very fast (3-6h after transcription induction) and is reversible as removal of DOX leads to slower RT again. Finally, deleting the promoter of Ptn gene and driving cells into differentiation still advances RT, allowing the authors to conclude that "transcription can be sufficient but not necessary to advance replication timing."

   Major comments:

   Overall, this is a well designed study that includes all necessary controls to support the author's conclusions. I think it is a very interesting system that the authors developed. The weakness of the manuscript is that there is no mechanistic explanation how such RT changes are achieved on a molecular basis. But I'm confident that the system could be indeed used to further dissect the mechanistic basis for the transcription dependence of RT advancements. Therefore, I support publication of this manuscript if a few comments below can be addressed.

   1. Figure 4 shows a titration of different DOX concentrations and provides clear evidence that the degree of RT advancement tracks well with the level of transcription. As the doses of DOX are quite high in this experiment, have the authors checked on a global scale to what extent transcription might be deregulated in neighbouring genes or genome-wide?
   2. One general aspect is that the whole study is only focused on the one single Ptn replication domain. Could the authors extend this rather narrow view a bit and also show RT data in the neighbouring domains. This would be particularly important for the DOX titration experiment that has the potential to induce transcriptional deregulation (see comment above).
   3. Figure 5 shows that the full capacity to advance RT upon DOX induction of the Ptn gene is achieved after 3h to 6h of DOX induction, so substantially less than a full cell cycle in mEScs (12h). This result suggests that origin licensing/MCM loading cannot be the critical mechanism to drive the RT change because only a small fraction of the cells has undergone M/G1-phase where origins are starting to get loaded. As a large fraction of mESCs (60-70%) are S-phase cells in an asynchronous population, the mechanism is likely taking place directly in S-phase. Could the authors try to synchronize cells in G1/S using double-thymidine block, then induce DOX for 3h before allowing cells to reenter S-phase and then check replication timing of the domain? This can be compared to an alternative experiment where transcription is only induced for 3h upon release into S-phase. This could provide more mechanistic insights as to whether transcription is sufficient to drive RT changes in G1 versus S-phase cells.

   Minor comments:

   • Figure 1B and Figure 6A. Quality of the genome browser snapshots could be improved and certain cryptic labelling such as "only Basic displayed by default" could be removed
   • The genome browser tracks appear a bit small across the figures and could be visually improved.
   • In figure 1E we see an advancement in RT in Ptn gene caused by nearby enhanced Hyg-TK gene expression induced by mPGK promoter. However, in figure 3D we see mPGK promoter has reduced ability to advance RT of Ptn gene. It would be nice to address this discrepancy in the results.

   Significance

   In my point of view, this is an important study that unifies a large amount of literature into a conceptual framework that will be interesting to a broad audience working on the intertwined fields of gene regulation, transcription and DNA replication, as well as cell fate switching and development.

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4. 

   Review Commons

   May 6, 2025

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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   Referee #1

   Evidence, reproducibility and clarity

   The study investigates the relationship between replication timing (RT) and transcription. While there is evidence that transcription can influence RT, the underlying mechanisms remain unclear. To address this, the authors examined a single genomic locus that undergoes transcriptional activation during differentiation. They engineered the Pln locus by inserting promoters of varying strengths to modulate transcription levels and assessed the impact on replication timing using Repli-seq.

   Key Findings:

   • Figure 1C and 1D: The data show that higher transcription levels correlate with an advanced RT, suggesting that transcriptional …

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   Referee #1

   Evidence, reproducibility and clarity

   The study investigates the relationship between replication timing (RT) and transcription. While there is evidence that transcription can influence RT, the underlying mechanisms remain unclear. To address this, the authors examined a single genomic locus that undergoes transcriptional activation during differentiation. They engineered the Pln locus by inserting promoters of varying strengths to modulate transcription levels and assessed the impact on replication timing using Repli-seq.

   Key Findings:

   • Figure 1C and 1D: The data show that higher transcription levels correlate with an advanced RT, suggesting that transcriptional activity influences replication timing.
   • Figure 2: To determine whether transcription alone is sufficient to alter RT, the authors inserted an hPGK reporter at different genomic locations. However, given the findings in Figure 1, which suggest that this is not the primary mechanism,
   • Figure 3: The authors removed the marker to examine whether the observed effects were due to the promoter-driven Pln locus, which has significantly larger then the marker.
   • Figure 4: The study explores the effect of increased doxycycline (Dox) treatment at the TRE (tetracycline response element), further supporting the role of transcription in RT modulation.
   • Figure 5: The findings demonstrate that Dox-induced RT advancement occurs rapidly, is reversible, and correlates with transcription levels, reinforcing the hypothesis that transcription plays a direct role in influencing replication timing.
   • Figure 6. Shows that during differentiation transcription of Pln is not required for RT advancement.

   Overall, the study presents a compelling link between transcription and replication timing, though some experimental choices warrant further clarification. I have no major comments.

   Minor Comments:

   Overall, the results are convincing, and the study appears to be well-conducted. In Figure 2, the authors use the hPGK promoter. However, it is unclear why they did not use the constructs from the previous experiments. Given that the hPGK promoter did not advance RT in Figure 1, the results in Figure 2 may not be entirely unexpected.

   Additionally, the study does not formally exclude the possibility that Pln protein expression itself influences RT. In Figure 1, readthrough transcription at the Pln locus could potentially drive protein expression. It would be useful to know whether the authors address this point in the discussion.

   Regarding the mechanism, if transcription across longer genomic regions contributes to RT changes, transcription-induced could DNA supercoiling play a role. For instance, could negative supercoiling generated by active transcription influence replication timing?

   It remains puzzling why Pln transcription does not contribute to replication timing during differentiation. Is there any evidence of chromatin opening during this process? For example, are ATAC-seq profiles available that could provide insights into chromatin accessibility changes during differentiation?

   Significance

   This is a compelling basic single-locus study that systematically compares replication timing (RT) and transcription dynamics while measuring several key parameters of transcription.

   My relevant expertise lies in transcriptional regulation and understanding how noncoding transcription influences local chromatin and gene expression.

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5. 

   Version published to 10.1101/2025.02.04.636516 on bioRxiv

   Feb 5, 2025