Author response:
The following is the authors’ response to the original reviews.
Public Reviews:
Reviewer #1 (Public review):
Meiotic recombination at chromosome ends can be deleterious, and its initiation-the programmed formation of DSBs-has long been known to be suppressed. However, the underlying mechanisms of this suppression remained unclear. A bottleneck has been the repetitive sequences embedded within chromosome ends, which make them challenging to analyze using genomic approaches. The authors addressed this issue by developing a new computational pipeline that reliably maps ChIP-seq reads and other genomic data, enabling exploration of previously inaccessible yet biologically important regions of the genome.
In budding yeast, chromosome ends (~20 kb) show depletion of axis proteins (Red1 and Hop1) important for recruiting DSB-forming proteins. Using their newly developed pipeline, the authors reanalyzed previously published datasets and data generated in this study, revealing heretofore unseen details at chromosome ends. While axis proteins are depleted at chromosome ends, the meiotic cohesin component Rec8 is not. Y' elements play a crucial role in this suppression. The suppression does not depend on the physical chromosome ends but on cis-acting elements. Dot1 suppresses Red1 recruitment at chromosome ends but promotes it in interior regions. Sir complex renders subtelomeric chromatin inaccessible to the DSB-forming machinery.
The high-quality data and extensive analyses provide important insights into the mechanisms that suppress meiotic DSB formation at chromosome ends. To fully realise this value, several aspects of data presentation and interpretation should be clarified to ensure that the conclusions are stated with appropriate precision and that remaining future issues are clearly articulated.
(1) To assess the chromosome fusion effects on overall subtelomeric suppression, authors should guide how to look at the data presented in Figure 2b-c. Based on the authors' definition of the terminal 20 kb as the suppressed region, SK1 chrIV-R and S288c chrI-L would be affected by the chromosome fusion, if any. In addition, I find it somewhat challenging to draw clear conclusions from inspecting profiles to compare subtelomeric and internal regions. Perhaps, applying a quantitative approach - such as a bootstrap-based analysis similar to those presented earlier-would be easier to interpret.
The reviewer is correct that we could not simply fuse two ends but had to create translocations that also removed variable amounts of subtelomeric sequence. Targeted translocations require unique sequences, and thus the extent to which telomeric sequences were deleted varied based on the availability of such sequences. As noted by the reviewer this necessarily limits the conclusions that can be drawn. We have expanded the description of this experiment and also explicitly state the limitations of this assay. To improve clarity, we have also included a schematic to better highlight which chromosomal sequences were removed.
To further probe our finding that subtelomeric axis protein enrichment may largely be encoded in cis, we now compared axis protein enrichment between S288c and SK1, as suggested by reviewer 2. For this analysis, we took advantage of a dataset we had produced previously that measures Red1 enrichment in SK1/S288c hybrid strains, which provide a powerful internally controlled setup that eliminates effects caused by differential timing and synchrony between samples. As now shown in Supplementary Fig. 5, SK1 and S288c differ substantially in their subtelomeric architecture at many ends, including extensive differences in the number and distribution of Y’ elements. Importantly, axis protein distribution was very consistent between SK1 and S288c when correcting for the differences in length of individual chromosome ends, supporting the conclusion that axis protein enrichment levels are primarily encoded in cis. This analysis is now shown in Fig. 2c. These data also indicate that the presence of a Y’ element does not affect axis protein levels beyond displacing the axis-recruiting sequences further into the chromosome interior.
(2) The relationship between coding density and Red1 signal needs clarification. An important conclusion from Figure 3 is that the subtelomeric depletion of Red1 primarily reflects suppression of the Rec8-dependent recruitment pathway, whereas Rec8-independent recruitment appears similar between ends and internal regions. Based on the authors' previous papers (referencess 13, 16), I thought coding (or nucleosome) density primarily influences the Rec8-independent pathway. However, the correlations presented in Figure 2d-e (also implied in Figure 3a) appear opposite to my expectation. Specifically, differences in axis protein binding between chromosome ends and internal regions (or within chromosome ends), where the Rec8-dependent pathway dominates, correlate with coding density. In contrast, no such correlation is evident in rec8Δ cells, where only the Rec8-independent pathway is active and end-specific depletion is absent. One possibility is that masking coding regions within Y' elements influences the correlation analysis. Additional analysis and a clearer explanation would be highly appreciated.
Thank you for pointing this out. We now also included Y’ elements in the analysis in Fig 2d. Including the Y’ elements yielded an increase in average coding density near the very ends of the chromosomes. This increase matches the higher level of axis protein binding seen in rec8 mutants in Fig. 3a and is consistent with the previously noted link between coding density and axis protein deposition. We now provide further description in the text and the figure legends.
We do not have an explanation for why there is no correlation with coding density in the EARs but assume that this reflects the unique regulation of this region (as also implied by Supplementary Fig. 4d). At present, the signals that establish the EARs remain unknown although our data indicate that the Hop1-CBR as well as Dot1 are important for axis protein enrichment in the EARs.
(3) The Dot1-Sir3 section staring from L266 should be clarified. I found this section particularly difficult to follow. It begins by stating that dot1∆ leads to Sir complex spreading, but then moves directly to an analysis of Red1 ChIP in sir3∆ without clearly articulating the underlying hypothesis. I wonder if this analysis is intended to explain the differences observed between dot1∆ and H3K79R mutants in the previous section. I also did not get the concluding statement - Dot1 counteracts Sir3 activity. As sir3Δ alone does not affect subtelomeric suppression, it is unclear what Dot1 counteracts. Perhaps, explicitly stating the authors' working model at the outset of this section would greatly clarify the rationale, results, and conclusions.
Thank you for this comment. We reworked the introduction to this paragraph to be more focused on Sir3 rather than Dot1. We hope that this introduction is less confusing and more in line with the data presented in this paragraph. We also expanded the conclusion to suggest the alternative possibility that the Sir complex only becomes a regulator of axis proteins in the absence of Dot1.
Reviewer #2 (Public review):
Summary:
In this manuscript, Raghavan and his colleagues sought to identify cis-acting elements and/or protein factors that limit meiotic crossover at chromosome ends. This is important for avoiding chromosome rearrangements and preventing chromosome missegregation.
By reanalyzing published ChIP datasets, the researchers identified a correlation between low levels of protein axis binding - which are known to modulate homologous recombination - and the presence of cis-acting elements such as the subtelomeric element Y' and low gene density. Genetic analyses coupled with ChIP experiments revealed that the differential binding of the Red1 protein in subtelomeric regions requires the methyltransferase Dot1. Interestingly, Red1 depletion in subtelomeric regions does not impact DSB formation. Another surprising finding is that deleting DOT1 has no effect on Red1 loading in the absence of the silencing factor Sir3. Unlike Dot1, Sir3 directly impacts DSB formation, probably by limiting promoter access to Spo11. However, this explains only a small part of the low levels of DSBs forming in subtelomeric regions.
Strengths:
(1) This work provides intriguing observations, such as the impact of Dot1 and Sir3 on Red1 loading and the uncoupling of Red1 loading and DSB induction in subtelomeric regions.
(2) The separation of axis protein deposition and DSB induction observed in the absence of Dot1 is interesting because it rules out the possibility that the binding pattern of these proteins is sufficient to explain the low level of DSB in subtelomeric regions.
(3) The demonstration that Sir3 suppresses the induction of DSBs by limiting the openness of promoters in subtelomeric regions is convincing.
Weaknesses:
(1) The impact of the cis-encoded signal is not demonstrated. Y' containing subtelomeres behave differently from X-only, but this is only correlative. No compelling manipulation has been performed to test the impact of these elements on protein axis recruitment or DSB formation.
Thank you for this comment. Our data indeed appeared contradictory because XY’ ends showed overall lower axis protein enrichment, yet our analysis of chromosome fusions, which also eliminated Y’ elements at some the fused ends, provided no evidence for an effect of Y’ elements at those ends. As also noted in the response to reviewer 1, we now compared axis protein enrichment between S288c and SK1, which differ substantially in their number and distribution of Y’ elements (Supplementary Fig. 5). We found that axis protein distribution and enrichment was very consistent between SK1 and S288c when correcting for the displacement caused by the presence of Y' elements and other subtelomeric sequences (now shown in Fig. 2d). These data support the conclusion that axis protein enrichment levels are primarily encoded in cis and indicate that the presence of Y’ elements does not affect axis protein levels beyond displacing the axis-recruiting sequences further into the chromosome interior (giving rise to the apparently lower axis protein enrichment on XY’ ends).
(2) The mechanism by which Dot1 and Sir3 impact Red1 loading is missing.
Although we do not yet understand the precise molecular details of these effects, we nevertheless believe we have obtained several important insights into this mechanism. First, our data indicate that the suppressive effect of the ends primarily impacts the Rec8-dependent loading of Red1, whereas loading via the Hop1-CBR is largely unaffected. The effect of Dot1 thus likely occurs via the Rec8-Red1 interaction. Second, the increase in Red1 recruitment is fully rescued by deletion of Sir3, suggesting that Sir3 becomes a promoter of axis protein recruitment in the absence of Dot1. These dependencies are now outlined in the model in Fig. 9. We would also like to note that the Sir complex was previously shown to impact cohesin in mitotic cells. Thus, a connection between the Sir complex and cohesin is not without precedent.
(3) Sir3's impact on DSB induction is compelling, yet it only accounts for a small proportion of DSB depletion in subtelomeric regions. Thus, the main mechanisms suppressing crossover close to the ends of chromosomes remain to be deciphered.
Thank you, we absolutely agree. We had discussed this point in the discussion but now also explicitly state this point in the abstract and expanded the discussion of these findings in the results and discussion.
Reviewer #3 (Public review):
Summary:
The paper by Raghavan et. al. describes pathways that suppress the formation of meiotic DNA double-strand breaks (DSBs) for interhomolog recombination at the end of chromosomes. Previously, the authors' group showed that meiotic DSB formation is suppressed in a ~20kb region of the telomeres in S. cerevisiae by suppressing the binding of meiosis-specific axis proteins such as Red1 and Hop1. In this study, by precise genome-wide analysis of binding sites of axis proteins, the authors showed that the binding of Red1 and Hop1 to sub-telomeric regions with X and Y' elements is dependent on Rec8 (cohesin) and/or Hop1's chromatin-binding region (CBR). Furthermore, Dot1 functions in a histone H3K79 trimethylation-independent manner, and the silencing proteins Sir2/3 also regulate the binding of Red1 and Hop1 and also the distribution of DSBs in sub-telomeres.
Strengths:
The experiments were conducted with high quality and included nice bioinformatic analyses, and the results were mostly convincing. The text is easy to read.
Weaknesses:
The paper did not provide any new mechanistic insights into how DSB formation is suppressed at sub-telomeres.
We respectfully disagree with this assessment. We show that the Sir complex suppresses DSB formation at a number of cryptic hotspots in the X elements and the adjacent subtelomeric sequences by causing chromatin compaction. The role of the Sir complex in transcriptional silencing, chromatin accessibility, and DSB formation had not previously been analyzed in the meiotic subtelomeres. That being said, Sir-dependent suppression is clearly not the only mechanism that suppresses DSBs in the subtelomeres, as we only observed DSB formation at a small number of hotspots. This was in and of itself a surprise, in particular given the large scale effect on chromatin compaction. We made an effort to more strongly emphasize the fact that additional layers of regulation must exist in the abstract and in the discussion.
Recommendations for the authors:
Reviewer #1 (Recommendations for the authors):
(1) Evidence for cis-acting suppression by Y' elements requires further support. The authors propose that Y' elements act in cis to suppress axis protein association at chromosome ends. While this is an attractive model, the current analyses do not yet provide sufficient support for it.
Thank you for this comment. Our data indeed appeared contradictory, because XY’ ends showed overall lower axis protein enrichment, yet our analysis of chromosome fusions, which also eliminated Y’ elements at some the fused ends provided no evidence for an effect of Y’ elements at those ends. As noted above, we now compared axis protein enrichment between S288c and SK1, which differ substantially in their number and distribution of Y’ elements (Supplementary Fig. 5). We found that axis protein distribution and enrichment was very consistent between SK1 and S288c when correcting for the displacement caused by the presence of Y' elements and other subtelomeric sequences. These data support the conclusion that axis protein enrichment levels are primarily encoded in cis and indicate that the presence of Y’ elements does not affect axis protein levels beyond displacing the axis-recruiting sequences further into the chromosome interior (giving rise to the apparently lower axis protein enrichment on XY’ ends).
(1a) In Figure S4c, the authors masked Y' elements to rule out the possibility that reduced binding within Y' elements themselves accounts for the overall underrepresentation in subtelomeric regions. However, since the authors propose that Y' elements suppress axis protein binding in surrounding regions in cis, it is appropriate to perform this analysis specifically on chromosome ends containing XY'.
Thank you for this suggestion. We agree that this would specifically affect the XY’ ends. However, given that we did not see a change even with all the ends, we do not expect a change with just the XY’ ends.
(1b) In Figure 2b-c, the authors conclude that removal of Y' elements by chromosome fusion does not reveal a long-range suppressive effect. However, the spatial extent of Y'-mediated suppression is not defined, making it unclear whether this experiment can test the proposed model. Perhaps plotting the averaged axis protein profile as a function of distance from Y' elements could help define the effective range of suppression and clarify whether the fusion experiment is informative in this context.
Thank you. As noted above we now compared SK1 and S288c ends, which provided further evidence that Y’ elements do not affect axis protein enrichment beyond displacing binding sites further into the chromosome interior. In addition, we substantially expanded the description of the chromosome fusion experiment to more clearly outline the setup and the limitations of this experiment.
(2) L402: "one of the first pieces of direct evidence that nucleosomes block meiotic DSB formation in vivo" sounds overstated, considering past publications (e.g., ref 45 and S. pombe ade6-M26 papers).
We toned this down and added the references.
(3) Figure 2e and other scatter plots: Correlation coefficients are reported without p-values. If the authors prefer to use confidence intervals from linear regression instead, they should justify this approach.
We added p-values to all scatter plots.
(4) Figure 7f. Explain blue dots.
We apologize for this oversight (also applies to Supplementary Fig. 10). The blue dots are measurements within 5 kb of an X element. The red dots are the rest of the genome. We now included a legend in the panel to clarify this notation.
(5) Figure 8d. To assess whether the conclusion can be generalized, the authors could plot the MNase and TrAEL-seq signal fold changes (sir3Δ/SIR3) for hotspots within 5 kb of X elements.
We attempted various analyses in this direction. However, the range of the MNase-seq effect in sir3 mutants is much greater than the effect on DSBs, making it difficult to make any correlative statements. There are clearly additional layers of DSB suppression in the telomeric regions, and loss/gain of nucleosomes is not sufficient to switch hotspots on/off at most hotspots. We now included a statement to this end in the abstract and also further discuss this notion in the discussion.
(6) Figure S1c. The apparent difference in X-element distribution may be influenced by bin size. This could be tested by repeating the analysis using smaller bins, comparable in size to the X elements, for all regions.
We thank the reviewer for this thoughtful suggestion. To address this concern, we repeated the analysis using smaller bins comparable in size to X elements (450 bp) across all region types. Specifically, X elements were analyzed per annotated element, while Y′ elements, subtelomeric 20 kb regions, and internal regions were subdivided into fixed 450 bp windows, and mean input coverage was calculated for each window using the same width-weighted approach.
This reanalysis did not materially alter the overall distribution patterns observed in Figure S1c. We observed only minor shifts in absolute values, which are expected when changing bin granularity.
Any residual differences likely reflect underlying copy number of X elements at chromosome ends. Importantly, all ChIP signals in the manuscript are normalized to their corresponding input (ChIP/Input), which mitigates potential biases arising from local copy number variation.
(7) Figure S2. X elements are difficult to find (e.g., chrVII-L).
We now included arrowheads at locations with full-length X elements. Partial X elements are marked with stars.
(8) Figure S7. Please indicate the endpoints of spreading.
As apparent in this figure and also indicated in the quantification in Supplementary Fig. 9a, spreading of the Sir complex is in most cases quite limited. The example in Supplementary Fig. 9b is one of the largest spreads we observed. The scale of the spreading is hard to meaningfully visualize in Supplementary Fig. 8 given the relatively large genomic distances shown in these profiles. We therefore refer the reader to the analyses shown in Supplementary Fig. 9a, which shows chromosome-resolved extent of spreading.
Reviewer #2 (Recommendations for the authors):
To go beyond the correlation between the presence of Y' elements and low levels of protein axis binding, subtelomeres could be easily truncated. Analyzing strains with different distributions of Y' elements would also be informative. The correlative analysis could also be expanded to compare how far the influence of Y' elements goes and whether the number of Y' impacts the extent of protein axis depletion.
We respectfully disagree with the assertion that subtelomeres could easily be truncated. The high repetitiveness of these sequences makes targeted manipulations of the extreme ends where the Y’ elements are located essentially impossible and is the main reasons for the limitations associated with the analysis of the chromosome fusions as outlined in the response to reviewer 1.
However, we would like to thank the reviewer for their suggestion to analyze different strain backgrounds. We now compared axis protein enrichment between S288c and SK1. For this analysis, we took advantage of a dataset we had produced previously that measures Red1 enrichment in SK1/S288c hybrid strains, which provide a powerful internally controlled setup that eliminates effects caused by differential timing and synchrony between samples. As now shown in Supplementary Fig. 5, SK1 and S288c differ substantially in their subtelomeric architecture at many ends, including extensive differences in the number and distribution of Y’ elements. Importantly, axis protein distribution was very consistent between SK1 and S288c when correcting for the differences in length of individual chromosome ends, supporting the conclusion that axis protein enrichment levels are primarily encoded in cis. This analysis is now shown in Fig. 2c. These data also indicate that the presence of Y’ elements does not affect axis protein levels beyond displacing the axis-recruiting sequences further into the chromosome interior.
Given the separation between protein axis loading and DSB induction, it would be interesting to test whether the presence of Y' elements influences the frequency and position of DSB induction.
We agree that this experiment would be very interesting. However, given the experimental challenges associated with targeted manipulation of Y’ elements as outlined above, we believe that this experiment lies outside the scope of this study. Our observations that Y’ elements do not grossly influence axis protein enrichment in their vicinity may also make an effect on DSB formation less likely.
The effect of Dot1 on Red1 loading is intriguing because it is at least partially independent of its main known target H3K79, yet fully dependent on Sir3. However, this effect extends far beyond Sir3 binding as detected by ChIP. This is surprising because Dot1 has a limited effect on Sir3 binding as detected by ChIP, and SIR3 deletion has no impact on Red1 binding. However, Dot1 was shown to limit Sir3 spreading to 20 kb on average when overexpressed (Katan-Khaykovich and Struhl 2005; Hocher et al, 2018). It would be interesting to test whether the regions affected by DOT1 deletion coincide with the zone covered by Sir3 upon overexpression (Extended Silent Domains: ESDs, Hocher et al., 2018).
We agree that this would be an interesting analysis. Unfortunately, the available data on the extended silent domains were not obtained in SK1 and, as noted above, the chromosome end structure differs substantially between the strains, preventing direct comparisons without repeating all the relevant analyses in S288c. In addition, the available data was collected in vegetative cells, although this may be less of an issue given that our analyses show similar spreading in vegetative and meiotic cells. However, short of repeating SIR3 overexpression in meiosis (which also would require a different overexpression regimen as galactose interferes with meiosis), we are not in a position to do this analysis.
As mentioned in the manuscript, the interplay between the Sir complex and Dot1 has been shown to affect checkpoint regulation during meiotic recombination. However, a discussion on how this relates to the observations reported here is missing.
Thank you. We included a discussion of this role and its relation to our observations.
Also, it is unclear why the authors did not investigate the impact of Dot1 and Sir3 impact on the binding of Hop1 rather than Red1, given that Hop1 is currently « the most upstream regulator of recombination known to be depleted about 20 kb from chromosome ends. »
We changed this statement in the introduction to avoid confusion and also included a model figure that specifically highlights the Rec8-dependent recruitment as a regulatory target.
Our data show that most of the telomere-proximal effects seem to act through the Rec8-dependent recruitment pathway for which Red1 is the most upstream regulator known. So, although the most upstream factor known before this study was Hop1, our data now identify the interaction between Red1 and Rec8 as the most upstream regulatory node.
Sir3's impact on DSB induction is compelling, yet it only accounts for a small proportion of DSB depletion in subtelomeric regions. Thus, the main mechanisms suppressing crossover close to the ends of chromosomes remain to be deciphered. This should be acknowledged and discussed.
In addition to the explicit statement of this conclusion in the results, we now added another statement in the abstract and also expanded the discussion of the fact that there are clearly additional levels of regulation that remain to be discovered.
Reviewer #3 (Recommendations for the authors):
Major points:
It would be nice to show a schematic summary of the authors' main conclusion.
Thank you, we now included a model schematic as Fig. 9.
Minor points:
(1) Supplemental Figure 2: A small box for the X element is marked with the same color as the Y' element, and so it is very hard to find the X element. Please use the clearer color, and it would be nice to show the chromosome ends without the X element (lines 129-130).
We now included arrowheads at locations with full-length X elements. Partial X elements are marked with stars. This notation also makes it obvious which ends lack annotated X elements.
(2) Line 156-163, Figure 2b: In the main text, "chromosome fusion between chromosome IV right arm and chromosome I left arm" should be mentioned. Moreover, it isn't very clear to have the data in the S288C background. The fusion points are different between S288C and SK1 (the structures of these ends are quite different). Please explain the authors' logic in the text.
To improve clarity, we have included a schematic to better highlight which chromosomal sequences were removed. We have also substantially expanded the description of this experiment and explicitly state the limitations of this assay.
(3) Supplemental Figure 6: Since the sir3 mutation affects the binding of Red1 EARs (and centromeres). It would be nice to show the similar sets for the HML, MAT, and HMR loci (and intergenic regions as a control).
We are unfortunately statistically underpowered to perform a meta-analysis of just HML, HMR and MAT. However, we now indicated the positions of HML and HMR in Supplementary Fig. 2 and 8, so the binding of the axis proteins and Sir3 can be inspected directly. MAT is not within 50 kb of a chromosome end and thus was not captured in these analyses.
(4) Line 322-, the section: From here, the authors switched their story from the sir3 to the sir2. It would be nice to provide the logic with a small introduction on the relationship between Sir2 and Sir3.
We apologize for this confusion. We are not switching our story to Sir2 but rather are taking advantage of an available dataset that analyzed DSBs in sir2 mutants. We then return to Sir3 to also analyze DSBs in the sir3 mutant and analyze its interaction with a dot1 mutation. To better support the logic, we now briefly reiterate that Sir2 and Sir3 are part of the same complex at the beginning of this section.
(5) Line 330-331, Figure 8a (and also Supplemental Fig. 8c): Would you explain a bit more about matched strain in the text or figure legend? Each dot represents a strain. If so, please show the strains used here.
Each dot refers to an individual X or Y’ element that is shown matched in WT and mutant to highlight the trends at the level of individual elements. This is noted in the figure legend.
(6) Supplemental Figure 7 (and 2): It would be nice to show the position of the HML, MAT, and HMR loci as well as the centromeres in the Figure.
We now indicated the positions of HML and HMR in Supplementary Fig. 2 and 8. MAT and the centromeres are not located within 50 kb from chromosome ends.
