Article activity feed

  1. Author Response

    Reviewer #1 (Public Review):

    This manuscript investigates a role for YAP in replication. Previous work from this group has shown that Yap knock-down leads to accelerated S-phase and an abnormal progression of DNA replication in the frog eye. Here they extend this to show that YAP depletion accelerates S-phase and DNA replication in the frog embryo, and that YAP binds a DNA replication regulator called Rif1. Combing assays suggest that YAP acts on origin firing. This is an interesting new aspect of YAP function. I am not an expert on DNA replication, however, I feel that the manuscript would have been improved if more mechanistic insight was gained into how Rif1 and YAP interact, and how that interaction influences replication timing.

    In the revised version of the manuscript, we have strengthened our conclusion that Yap regulates the dynamics of DNA replication. We now provide additional experiments in addition to DNA combing and nascent strand analysis by agarose gel electrophoresis: Rhodamine-dUTP incorporation/nucleus, 32P-dCTP incorporation, western blotting for replication fork proteins. All show that DNA synthesis and origin activation is increased after Yap depletion.

    Moreover, in the revised manuscript we also directly compared the effects of YAP depletion to those of Rif1 depletion alone (page 7, New Figure 4). As for Yap depletion, we first quantified rhodaminedUTP incorporation after Rif1 depletion by direct fluorescence microscopy that demonstrated a clear increase of DNA synthesis, consistent with Alver et al. 2017. Second, we performed DNA combing experiments after Rif1 depletion in egg extracts that show a marked increase in DNA replication and fork density like those seen after Yap depletion, spanning from very early to mid S-phase. We therefore found that Rif1 depletion and Yap depletion qualitatively show the same main effects: an increase of DNA synthesis and fork density, that are more pronounced in early S-phase. We also noticed quantitative differences in the direct fluorescence after rhodamine incorporation of whole nuclei and fork density, with stronger effects after Rif1 depletion compared to Yap depletion. This suggests that there might be an additional mechanism for Rif1 in regulating origin activation.

    The title of the manuscript is "A non-transcriptional function of YAP orchestrates the DNA replication program". It is not clear that YAP "orchestrates" DNA replication - for this to be true, it would have to be signal responsive. Since the authors did not reveal any links to YAP activity (such as YAP phosphorylation or nuclear/cytoplasmic distribution) it is not "orchestrating" DNA replication.

    We have replaced “orchestrates” by “regulates”.

    Figure 1 shows that YAP is recruited onto chromatin after MCM2 and MCM7 and at the same time as PCNA and the start of DNA synthesis. Addition of geminin, an inhibitor of Cdt and MCM loading inhibits YAP loading onto chromatin. YAP immuno depletion leads to premature DNA synthesis or replication. Fig 1 B is quite confusing- the labeling in Figure 1B is likely incorrect.

    We apologize for this confusion. This has been corrected and the Figure 1B is now properly labelled.

    Figure 2 investigates if YAP depletion affects origin firing or fork speed, using DNA combing. Fig 2A shows that there is increased activated replication origins and decreased distance between origins. The authors say that the increase of fork density is more pronounced than the decreased distance, suggesting YAP is regulating the activation of origins. The number of replicates is low. This is especially true for the conclusion that eye length is unaltered -it appears that there is a subset of eye length that is increased in 2F, which might reach significance if triplicates were performed.

    As the referee points out, both the observed increase of fork density and decrease of origin distances argues that origin activation is increased after Yap depletion. The fact that the increase of the fork density seems more pronounced than the local decrease of neighbouring origins allows a more detailed interpretation, explicitly that whole clusters of origins are activated on top of origins inside already active clusters. This can be observed in the two independent experiments probing many fibers for eye distances and eyes numbers.

    Concerning Figure 2F, the scatter plot makes it look like that the impression that there are more eyes with larger sizes after Yap depletion, but please note that there are also more EL measured as stated in the legend (Mock n=182 versus Yap n=311). To highlight this parameter, we added these numbers below the scatter plot in the revised Figure 2F, as we have done consistently for all of the experiments presented in the revised Figures. The means of the two EL distributions are numerically different but since both distributions are not Gaussian (tested by d'Agostino and Pearson test), only non-parametric tests can apply (Mann-Whitney or Kolmogorov Smirnow test). The results of the two non-parametric tests show that the distributions are not significantly different, as mentioned in the legend. However, we cannot rule out that after Yap depletion some larger eyes may arise from fusions of forks or from a higher fork speed, but again, the tests, applied to a high number of measurements, show no significant statistical differences.

    The authors conducted AP-MS on egg extracts to identify proteins that co-IP with YAP. One of many proteins identified was RIF1 Figure 3 shows a co-IP with RIF1 and YAP. It is a very weak co-IP.

    We agree that the Rif/Yap co-IP is weak, but it is reproducible in several independent experiments with different extracts. There could be many reasons for this. Co-IPs with a high molecular weight partner like Rif1 (250 kDa) are generally tedious (poor gel migrations and WB transfer). Further, Rif1 has been described as having a subnuclear localisation and to associate with the nuclear lamina and heterochromatin. These characteristics are known to make the proteins highly insoluble. These technical limitations have been reported for the mouse Rif1 for instance (Sukackaite R; et al. Sci Rep 2017 May 18;7(1):2119). In fact, similar “weak co-IPs” were also obtained between Rif1 and Nanog (Wang J. et al. Nature 2006 (444), 364–368 ) as well as with PPI (Hiraga S. et al. EMBO Rep. 2017 Mar;18(3):403-419). Finally, it could also be that this interaction is not permanent but dynamic, making it difficult to capture in a Co-IP. Taken together, these parameters mean that the identification of the interaction is in itself challenging. What we did manage to provide is a reciprocal co-IP using the endogenous proteins, which we believe best reflects native conditions.

    Figure 4 shows that YAP levels increase during development and that depletion of YAP or RIF1 leads to increased cell division. The authors use Trim-away to deplete YAP and RIF1 and find that depletion of either leads to an increased number of small cells. The YAP depletion shown in Fig 4B is clear, as is the increased number of small cells in YAP depletion or RIF1 depletion.

    Figure 4 supplement 1 is arguing that trim away and morpholino combined are more effective. Quantitation of the western blots in panel A is needed for this to be convincing.

    The quantification is now presented in new Figure 5-figure supplement 1A. At the 2-cell stage, we observe some fluctuations in the amounts of Yap between samples, the origin of which we do not fully understand. At the 4-cell stage, a reduction in Yap is observed regardless of the depletion strategy used. It is from the 8-cell stage onwards that differential effects between the depletion methods can be appreciated. From this stage onwards, the quantifications confirm that the TRIM-Away and morpholino combined are more effective than taken separately.

    Figure 5 shows that RIF1 is expressed in the eye in RSC and that loss of RIF1 leads to a small eye. Panel B shows that by western blot analysis RIF1 antibody is specific. However, antibodies can have very different abilities in western vs staining. The RIF1 and YAP antibodies should be validated in staining. Also, the staining in Fig5C is at low resolution for both YAP and RIF1 and the identification of foci is unclear.

    This is indeed an important issue. To address this point, we performed immunostaining on retinal sections from embryos depleted with the target protein and compared the fluorescent signal obtained in control versus depleted samples. We show that upon depletion of Yap or Rif, the signal from the immunostaining is severely reduced for Yap or Rif1, respectively, which attests the specificity of the antibodies used in this study. We have added an additional supplementary Figure to show this control (Figure 6-figure supplement 1).

    We agree with the reviewers that the quality of the images could be improved. We now provide confocal images with a better resolution (Figure 6C).

    For Rif1, we observe a clear nuclear staining, rather non-homogenous which is consistent with data reported in the literature. Indeed, Rif1 localisation has been shown to be highly dynamic during the cell cycle and also during S-phase (Cornacchia D. et al. EMBO J. 2012). Some brighter foci could be observed at specific phases (such as G1-phase) but overall, the general pattern appears rather “granular” and restricted to the nucleus. This is what we are also observing. Interestingly, Rif1 does not appear to colocalize with the replication fork or with the replicative helicase MCM3 (Cornacchia D. et al. EMBO J. 2012). The replication foci observed in this study are therefore to be understood independently of the Rif1 localisation pattern.

    For Yap, we do not detect any granular expression but observe rather homogeneous nuclear and cytoplasmic staining, which is also consistent with reported data showing YAP nucleo-cytoplasmic shuffling (see for instance Manning S.A. et al. Curr Biol. 2018). STED microscopy might be necessary for higher resolution.

    It is difficult to see the points the authors wish to communicate in Figure 6. There is almost no Edu in the YAP-MO, which questions the ability to recognize the different patterns in this region of the eye.

    Our observations show that there are fewer EdU positive cells in the Yap-MO but not “no EdU”. The fluorescence intensity in the green-labelled nuclei in Figure 7C after Yap MO does not appear different from that in the control-MO. Under these conditions, there is no reason to think that one pattern is more difficult to recognise than the other one.

    Reviewer #2 (Public Review):

    This paper is of potential interest within the field of DNA replication, as it identifies a novel role for YAP protein in DNA replication dynamics. However, the conclusions are not supported by properly controlled data. Several aspects of data analysis and representation need to be revised.

    In this manuscript, the authors characterized YAP function in the control of DNA replication dynamics, taking advantage of the Xenopus laevis system.

    They found that YAP is recruited to replicating-chromatin and showed that its chromatin enrichment depends on the assembly of pre-RC proteins. In addition, they show that the immuno-depletion of YAP leads to increased DNA synthesis and origin activation, revealing YAP's possible role in the regulation of replication dynamics.

    The authors were also interested in finding YAP potential partners that could mediate its function. They identified Rif1, a major regulator of replication timing, as a novel YAP interactor during DNA replication.

    As RIF1 expression in vivo is restricted to the stem cell compartment of the Xenopus retina, similar to YAP, the authors assessed whether Rif1 could regulate the spatial-temporal program of DNA replication in stem cells. They showed that depletion of Rif1 at early stages of Xenopus embryos development leads to alterations in replication foci of retinal stem cells, resembling the effect observed following YAP down-regulation.

    Finally, they studied the impact of YAP and RIF1 down-regulation at early stages of development, showing that their absence results in the acceleration of cell division rate of Xenopus embryos, where RNA transcription is absent. Based on these results they concluded that YAP has a role in S-phase independent from transcription.

    The higher rate of DNA synthesis observed in the absence of Yap in Figure 1D is not very evident from the gels in Figure 1, supplement 3B. The timing of the experiments is continuously changing throughout the figures. It is therefore difficult to compare them. Also, comparisons across different gels are difficult to interpret. Most importantly, relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of YAP. To accurately quantify the replication of DNA added to the extract, the total amount of DNA synthesized must be quantified.

    Although we do not agree that relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of Yap, we thank the reviewer for his suggestion since we now provide additional data clearly strengthening our conclusion.

    Many studies, published in high standards journals and coming from different Xenopus replication laboratories have quantified DNA synthesised after 32P-dCTP incorporation and separation by agarose gel electrophoresis (Shechter et al, 2004; Trenz et al, 2008; Guo et al, 2015; Walter & Newport, 1997; Suski et al, 2022, Nature). Nevertheless, as the referee suggested, we quantified the total amount of DNA synthesized in three new independent experiments. These new results, presented page 5, lines 34-39 and shown in Figure 1G, support our conclusion, as they also show that Yap depletion increases total DNA synthesis. Please note that the DNA combing results presented in Figure 2 also show that replication is increased after Yap depletion. Finally, we also added another set of experiments to Figure 1 to further confirm these findings. We used the incorporation of Rhodamine-dUTP followed by the quantification of the fluorescence intensity within nuclei. This nuclei-fluorescence based method is frequently used in proliferation assays to assess nucleotide incorporation resulting from the DNA replication process in other organisms. Our new results demonstrate that DNA synthesis is increased 1.5-fold in six biological replicates and represent a third independent method, in addition to DNA combing and 32P-dCTP incorporation, showing that DNA synthesis is increased upon YAP depletion. These new results are now presented page 5, lines 27-24 and shown in Figure 1D-F.

    As explained in the MM section page 14 in the original manuscript, the replication extent (percent of replication) differs for a specific time point from one extract to another, because each egg extract prepared from one batch of eggs replicates nuclei with its own replication kinetics. To overcome this problem and to compare different independent experiments performed using different egg extracts, the data points of each sample were normalized to maximum incorporation value.

    It is also necessary to analyze the dynamics and the abundance of chromatin-bound replication proteins associated with the active replication fork after Yap depletion using chromatin binding assays. This would further confirm the increase in the fork density observed by DNA combing experiments.

    We thank the referee for this suggestion and we added a western blot of chromatin bound proteins after Yap depletion. This shows that two replication proteins associated with the active replication fork, namely Cdc45 and PCNA, are enriched after Yap depletion compared to the control at the beginning of S-phase. This observation further supports the DNA combing results showing that more forks are active after YAP depletion. This new data is now presented page 6 lines 25-32 and displayed in Figure 2H.

    We would like to stress here that with these additional methods added to the revised version, five different methods in total (Rhodamine-dUTP incorporation/nucleus, 32P-dCTP incorporation - total synthesis, 32P-dCTP incorporation - nascent strand analysis, DNA combing, western blotting for replication fork proteins) show that DNA synthesis and origin activation is increased after Yap depletion.

    The quantification of the amount of YAP in Figure 1B is confusing. The legend of the chart states "Control in light grey and presence of geminin in black", but the bar colors are of different shades of grey. It is not clear how to evaluate them.

    We apologize for this confusion. This has been corrected and the Figure 1B is now properly labelled.

    The efficiency of depletion for both Rif1 and YAP is different in Figure 4B and Figure 4A, supplement 1.

    We agree with the referee that the efficiency of depletion is different in both figures. This is explained by the fact that the extent of the depletion varies from experiment to experiment. We work with different batches of in vitro fertilized embryos and extracts, so these differences simply reflect the technical/biological variability.

    Moreover, the combined use of the TRIM-away approach with injections of MO led to a stronger and prolonged YAP depletion but also triggered toxicity in the tadpoles, which display severe abnormalities.

    It is important to point out that abnormal development is not always attributable to a toxic effect. Many losses of gene function result in malformations without being ascribed to toxicity or unspecific effects. However, we agree with the reviewers on the need to present a rescue experiment, which is now shown in new Figure 5C and new Figure 5-figure supplement 1B. In addition, we also provide gain-of-function (GOF) data for YAP in early embryos. In brief, we find that the Yap GOF leads to opposite outcomes than those of its depletion with embryos at the same stage of development, having fewer and larger cells than the control. Furthermore, we show that the effects of Yap depletion, i.e. embryos with more and smaller cells than the control at the same developmental stage, are rescued by the injection of MO-resistant Yap mRNA to restore the protein level. This is true for both embryonic divisions (new Figure 5C) and development, as we obtained normal-looking neurula after Yap rescue (new Figure 5-figure supplement 1B). Overall, these data now clearly show that Yap is both sufficient and necessary to maintain the rate of embryonic divisions and that this phenotype is specific since it can be rescued by expressing Yap alone. These new data are presented page 8, lines 2-10.

    Reviewer #3 (Public Review):

    The article by Garcia et al clearly describes a set of experiments establishing Yap as a novel regulator of DNA replication dynamics. Its characterization as both a RIF1 interaction partner as well as playing its own role in replication initiation will likely have a significant impact on the field, as currently little is known about how DNA replication during early embryonic cell divisions is regulated.

    The authors aim to identify a non-transcriptional function of YAP through the use of the Xenopus in vitro replication system and Yap depletion. Strengths of the paper include the particularly appropriate use of the Xenopus in vitro replication system, as well as the combined use of Trim-Away and morpholino oligonucleotides to deplete Yap and Rif1. Moreover, these experiments were elegantly complemented by single-molecule molecular combing and in vivo studies. Identifying Yap as a novel regulator of DNA replication dynamics, the authors achieved their aim. Through characterization of Yap as both playing a role in replication initiation and as a Rif1 interaction partner will likely have a significant impact on the field, as currently little is known about how DNA replication during early embryonic cell divisions is regulated. A weakness of the paper is that some of the representative data does not appear to be very representative of the entire data set.

    We replaced representative data in Figure 2 A, which we think better reflects the main conclusions of the entire data set.

    Was this evaluation helpful?
  2. Evaluation Summary:

    This manuscript is of interest within the fields of DNA replication, developmental biology and oncology. Focusing on the YAP protein, a major regulator of tissue growth and repair, it identifies an interesting new role in DNA replication dynamics, beyond its known role in gene transcription regulation. A series of experimental manipulations support the key claims of the paper. Additional control experiments, as well as mechanistic insight into how RIF1 and YAP interact, and insight into how that interaction influences replication timing would make the paper stronger.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

    Was this evaluation helpful?
  3. Reviewer #1 (Public Review):

    This manuscript investigates a role for YAP in replication. Previous work from this group has shown that Yap knock-down leads to accelerated S-phase and an abnormal progression of DNA replication in the frog eye. Here they extend this to show that YAP depletion accelerates S-phase and DNA replication in the frog embryo, and that YAP binds a DNA replication regulator called Rif1. Combing assays suggest that YAP acts on origin firing. This is an interesting new aspect of YAP function. I am not an expert on DNA replication, however, I feel that the manuscript would have been improved if more mechanistic insight was gained into how Rif1 and YAP interact, and how that interaction influences replication timing.

    The title of the manuscript is "A non-transcriptional function of YAP orchestrates the DNA replication program". It is not clear that YAP "orchestrates" DNA replication - for this to be true, it would have to be signal responsive. Since the authors did not reveal any links to YAP activity (such as YAP phosphorylation or nuclear/cytoplasmic distribution) it is not "orchestrating" DNA replication.

    Figure 1 shows that YAP is recruited onto chromatin after MCM2 and MCM7 and at the same time as PCNA and the start of DNA synthesis. Addition of geminin, an inhibitor of Cdt and MCM loading inhibits YAP loading onto chromatin. YAP immuno depletion leads to premature DNA synthesis or replication. Fig 1 B is quite confusing- the labeling in Figure 1B is likely incorrect.

    Figure 2 investigates if YAP depletion affects origin firing or fork speed, using DNA combing. Fig 2A shows that there is increased activated replication origins and decreased distance between origins. The authors say that the increase of fork density is more pronounced than the decreased distance, suggesting YAP is regulating the activation of origins. The number of replicates is low. This is especially true for the conclusion that eye length is unaltered -it appears that there is a subset of eye length that is increased in 2F, which might reach significance if triplicates were performed.

    The authors conducted AP-MS on egg extracts to identify proteins that co-IP with YAP. One of many proteins identified was RIF1 Figure 3 shows a co-IP with RIF1 and YAP. It is a very weak co-IP.

    Figure 4 shows that YAP levels increase during development and that depletion of YAP or RIF1 leads to increased cell division. The authors use Trim-away to deplete YAP and RIF1 and find that depletion of either leads to an increased number of small cells. The YAP depletion shown in Fig 4B is clear, as is the increased number of small cells in YAP depletion or RIF1 depletion.

    Figure 4 supplement 1 is arguing that trim away and morpholino combined are more effective. Quantitation of the western blots in panel A is needed for this to be convincing.

    Figure 5 shows that RIF1 is expressed in the eye in RSC and that loss of RIF1 leads to a small eye. Panel B shows that by western blot analysis RIF1 antibody is specific. However, antibodies can have very different abilities in western vs staining. The RIF1 and YAP antibodies should be validated in staining. Also, the staining in Fig5C is at low resolution for both YAP and RIF1 and the identification of foci is unclear.

    It is difficult to see the points the authors wish to communicate in Figure 6. There is almost no Edu in the YAP-MO, which questions the ability to recognize the different patterns in this region of the eye.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    This paper is of potential interest within the field of DNA replication, as it identifies a novel role for YAP protein in DNA replication dynamics. However, the conclusions are not supported by properly controlled data. Several aspects of data analysis and representation need to be revised.

    In this manuscript, the authors characterized YAP function in the control of DNA replication dynamics, taking advantage of the Xenopus laevis system.
    They found that YAP is recruited to replicating-chromatin and showed that its chromatin enrichment depends on the assembly of pre-RC proteins. In addition, they show that the immuno-depletion of YAP leads to increased DNA synthesis and origin activation, revealing YAP's possible role in the regulation of replication dynamics.
    The authors were also interested in finding YAP potential partners that could mediate its function. They identified Rif1, a major regulator of replication timing, as a novel YAP interactor during DNA replication.

    As RIF1 expression in vivo is restricted to the stem cell compartment of the Xenopus retina, similar to YAP, the authors assessed whether Rif1 could regulate the spatial-temporal program of DNA replication in stem cells. They showed that depletion of Rif1 at early stages of Xenopus embryos development leads to alterations in replication foci of retinal stem cells, resembling the effect observed following YAP down-regulation.

    Finally, they studied the impact of YAP and RIF1 down-regulation at early stages of development, showing that their absence results in the acceleration of cell division rate of Xenopus embryos, where RNA transcription is absent. Based on these results they concluded that YAP has a role in S-phase independent from transcription.

    The higher rate of DNA synthesis observed in the absence of Yap in Figure 1D is not very evident from the gels in Figure 1, supplement 3B. The timing of the experiments is continuously changing throughout the figures. It is therefore difficult to compare them. Also, comparisons across different gels are difficult to interpret.

    Most importantly, relative quantification on gel images cannot support the claim of increased DNA synthesis in the absence of YAP. To accurately quantify the replication of DNA added to the extract, the total amount of DNA synthesized must be quantified.

    It is also necessary to analyze the dynamics and the abundance of chromatin-bound replication proteins associated with the active replication fork after Yap depletion using chromatin binding assays. This would further confirm the increase in the fork density observed by DNA combing experiments.

    The quantification of the amount of YAP in Figure 1B is confusing. The legend of the chart states "Control in light grey and presence of geminin in black", but the bar colors are of different shades of grey. It is not clear how to evaluate them.

    The efficiency of depletion for both Rif1 and YAP is different in Figure 4B and Figure 4A, supplement 1. Moreover, the combined use of the TRIM-away approach with injections of MO led to a stronger and prolonged YAP depletion but also triggered toxicity in the tadpoles, which display severe abnormalities.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The article by Garcia et al clearly describes a set of experiments establishing Yap as a novel regulator of DNA replication dynamics. Its characterization as both a RIF1 interaction partner as well as playing its own role in replication initiation will likely have a significant impact on the field, as currently little is known about how DNA replication during early embryonic cell divisions is regulated.

    The authors aim to identify a non-transcriptional function of YAP through the use of the Xenopus in vitro replication system and Yap depletion. Strengths of the paper include the particularly appropriate use of the Xenopus in vitro replication system, as well as the combined use of Trim-Away and morpholino oligonucleotides to deplete Yap and Rif1. Moreover, these experiments were elegantly complemented by single-molecule molecular combing and in vivo studies. Identifying Yap as a novel regulator of DNA replication dynamics, the authors achieved their aim. Through characterization of Yap as both playing a role in replication initiation and as a Rif1 interaction partner will likely have a significant impact on the field, as currently little is known about how DNA replication during early embryonic cell divisions is regulated. A weakness of the paper is that some of the representative data does not appear to be very representative of the entire data set.

    Was this evaluation helpful?