Author response:
The following is the authors’ response to the original reviews
Public Reviews:
Reviewer #1 (Public Review):
Summary:
The manuscript by Raices et al., provides novel insights into the role and interactions between SPO-11 accessory proteins in C. elegans. The authors propose a model of meiotic DSBs regulation, critical to our understanding of DSB formation and ultimately crossover regulation and accurate chromosome segregation. The work also emphasizes the commonalities and species-specific aspects of DSB regulation.
Strengths:
This study capitalizes on the strengths of the C. elegans system to uncover genetic interactions between a large number of SPO-11 accessory proteins. In combination with physical interactions, the authors synthesize their findings into a model, which will serve as the basis for future work, to determine mechanisms of DSB regulation.
Weaknesses:
The methodology, although standard, lacks quantification. This includes the mass spectrometry data , along with the cytology. The work would also benefit from clarifying the role of the DSB machinery on the X chromosome versus the autosomes.
• We have uploaded the MS data and added a summary table with the number of peptides and coverage.
• We have added statistics to the comparisons of DAPI body counts.
• We have provided additional images of the change in HIM-5 localization
• We have quantified the overlap (or lack thereof) between XND-1 and HIM-17 and the DNA axis
Reviewer #2 (Public Review):
Summary:
Meiotic recombination initiates with the formation of DNA double-strand break (DSB) formation, catalyzed by the conserved topoisomerase-like enzyme Spo11. Spo11 requires accessory factors that are poorly conserved across eukaryotes. Previous genetic studies have identified several proteins required for DSB formation in C. elegans to varying degrees; however, how these proteins interact with each other to recruit the DSB-forming machinery to chromosome axes remains unclear.
In this study, Raices et al. characterized the biochemical and genetic interactions among proteins that are known to promote DSB formation during C. elegans meiosis. The authors examined pairwise interactions using yeast two-hybrid (Y2H) and co-immunoprecipitation and revealed an interaction between a chromatin-associated protein HIM-17 and a transcription factor XND-1. They further confirmed the previously known interaction between DSB-1 and SPO-11 and showed that DSB-1 also interacts with a nematodespecific HIM-5, which is essential for DSB formation on the X chromosome. They also assessed genetic interactions among these proteins, categorizing them into four epistasis groups by comparing phenotypes in double vs. single mutants. Combining these results, the authors proposed a model of how these proteins interact with chromatin loops and are recruited to chromosome axes, offering insights into the process in C. elegans compared to other organisms.
Weaknesses:
This work relies heavily on Y2H, which is notorious for having high rates of false positives and false negatives. Although the interactions between HIM-17 and XND-1 and between DSB-1 and HIM-5 were validated by co-IP, the significance of these interactions was not tested, and cataloging Y2H interactions does not yield much more insight.
We appreciate that the reviewer recognized the value of our IP data, but we beg to differ that we rely too heavily on the Y2H. We also provide genetic analysis on bivalent formation to support the physical interaction data. We do acknowledge that there are caveats with Y2H, however, including that a subset of the interactions can only be examined with proteins in one orientation due to auto-activation. While we acknowledge that it would be nice to have IP data for all of the proteins using CRISPR-tagged, functional alleles, these strains are not all feasible (e.g. no functional rec-1 tag has been made) and are beyond the scope of the current work.
Moreover, most experiments lack rigor, which raises serious concerns about whether the data convincingly supports the conclusions of this paper. For instance, the XND-1 antibody appears to detect a band in the control IP; however, there was no mention of the specificity of this antibody.
We previously showed the specificity of this antibody in its original publication showing lack of staining in the xnd-1 mutant by IF (Wagner et al., 2010). To further address this, however, we have now included a new supplementary figure (Figure S1) demonstrating the specificity of the XND-1 antibody by Western blot. The antibody detects a distinct band in extracts from wild-type (N2) worms, but this band is absent in two independent xnd-1 mutant strains. This confirms that the antibody specifically recognizes XND-1, supporting the validity of the IP results shown in the main figures.
Additionally, epistasis analysis of various genetic mutants is based on the quantification of DAPI bodies in diakinesis oocytes, but the comparisons were made without statistical analyses.
We have added statistical analysis to all datasets where quantification was possible, strengthening the rigor and interpretation of our findings.
For cytological data, a single representative nucleus was shown without quantification and rigorous analysis. The rationale for some experiments is also questionable (e.g. the rescue by dsb-2 mutants by him-5 transgenes in Figure 2), making the interpretation of the data unclear. Overall, while this paper claims to present "the first comprehensive model of DSB regulation in a metazoan", cataloging Y2H and genetic interactions did not yield any new insights into DSB formation without rigorous testing of their significance in vivo. The model proposed in Figure 4 is also highly speculative.
Regarding the cytology, we provide new images and quantification of HIM-17 and XND-1 overlap with the DNA axes. We also added full germ line images showing HIM-5 localization in wild type and dsb-1 mutants, to provide a more complete and representative view of the observed phenotype. To further support our findings, we’ve also included images demonstrating that this phenotype is consistently observed with both in live worm with the the him-5::GFP transgene and in fixed worms with an endogenously tagged version of HIM-5.
Reviewer #3 (Public Review):
During meiosis in sexually reproducing organisms, double-strand breaks are induced by a topoisomerase-related enzyme, Spo11, which is essential for homologous recombination, which in turn is required for accurate chromosome segregation. Additional factors control the number and genome-wide distribution of breaks, but the mechanisms that determine both the frequency and preferred location of meiotic DSBs remain only partially understood in any organism.
The manuscript presents a variety of different analyses that include variable subsets of putative DSB factors. It would be much easier to follow if the analyses had been more systematically applied. It is perplexing that several factors known to be essential for DSB formation (e.g., cohesins, HORMA proteins) are excluded from this analysis, while it includes several others that probably do not directly contribute to DSB formation (XND-1, HIM-17, CEP-1, and PARG-1).
We respectfully disagree with the reviewer’s statement regarding the selection of factors included in our analysis. In this work, our focus was specifically on SPO-11 accessory factors — proteins that directly interact with or regulate SPO-11 activity during doublestrand break formation. Cohesins and chromosome axis proteins (such as the HORMA domain proteins) are essential for establishing the correct chromosome architecture that supports DSB formation, but there is no evidence that they are direct accessory factors of SPO-11. Therefore, they were intentionally excluded from this study to maintain a clear and focused scope on proteins that more directly modulate SPO-11 function.
Conversely, XND-1, HIM-17, CEP-1, and PARG-1 have all been implicated in regulating aspects of SPO-11-mediated DSB formation or its immediate environment. Although their contributions mayinvolve broader chromatin or DNA damage response regulation, prior literature supports their inclusion as relevant modulators of SPO-11 activity, justifying their analysis within the context of this work.
The strongest claims seem to be that "HIM-5 is the determinant of X-chromosome-specific crossovers" and "HIM-5 coordinates the actions of the different accessory factors subgroups." Prior work had already shown that mutations in him-5 preferentially reduce meiotic DSBs on the X chromosome. While it is possible that HIM-5 plays a direct role in DSB induction on the X chromosome, the evidence presented here does not strongly support this conclusion. It is also difficult to reconcile this idea with evidence from prior studies that him-5 mutations predominantly prevent DSB formation on the sex chromosomes, while the protein localizes to autosomes.
HIM-5 is not the only protein that is autosomally enriched but preferentially affects the X chromosome: MES-4 and MRG-1 are both autosomally-enriched but influence silencing of the X chromosome. While HIM-5 appears autosomally-enriched, it does not appear to be autosomal-exclusive. While we would ideally perform ChIP to determine its localization on chromatin, this method for assaying DSB sites is likely insufficient to identify DSB sites which differ in each nucleus and for which there are no known hotspots in the worm.
him-5 mutants confer an ~50% reduction in total number of breaks and a very profound change in break dynamics (seen by RAD-51 foci (Meneely et al., 2012)). Since the autosomes receives sufficient breaks in this context to attain a crossover in >98% of nuclei, this indicates that the autosomes are much less profoundly impacted by loss of DSB functions than is the X chromosome. Indeed, prior data from co-author, Monica Colaiacovo, showed that fewer breaks occur on the X (Gao, 2015) likely resulting from differences in the chromatin composition of the X and autosome resulting from X chromosome silencing.
The conclusion that HIM-5 must be required for breaks on the X comes from the examination of DSB levels and their localization in different mutants that impair but do not completely abrogate breaks. In any situation where HIM-5 protein expression is affected (xnd-1, him-17, and him-5 null alleles), breaks on the X are reduced/ eliminated. By contrast, in dsb-2 mutants, where HIM-5 expression is unaffected, both X and autosomal breaks are impacted equally. As discussed above, in the absence of HIM-5 function, there are ~15 breaks/ nucleus. The Ppie1::him-5 transgene is expressed to lower levels than Phim-5::him-5, but in the best case, the ectopic expression of this protein should give a maximum of ~15 breaks (the total # of breaks is thought to be ~30/nucleus). By these estimates, Ppie-1::him-5; him-17 and him-5 null mutants have the same number of breaks. Yet, in the former case, breaks occur on the X; whereas in the latter they do not. The best explanation for this discrepancy is that HIM-5 is sufficient to recruits the DSB machinery to the X chromosome.
The one experiment that seems to elicit the conclusion that HIM-5 expression is sufficient for breaks on the X chromosome is flawed (see below). The conclusion that HIM-5 "coordinates the activities of the different accessory sub-groups" is not supported by data presented here or elsewhere.
We have reorganized the discussion to more directly address the reviewers’ concerns. We raise the possibility that HIM-5 has an important role in bringing together the SPO-11 and its interacting components (DSB-1/2/3) with the other DSB inducing factors, including those factors that regulating DSB timing (XND-1), coordination with the cell cycle (REC-1), association with the chromosome axis (PARG-1, MRE-11), and coupling to downstream resection and repair (MRE-11, CEP-1).
This raises a natural question: if HIM-5 has such a central role, why are the phenotypes of HIM-5 so mild? We propose that while the loss of DSBs on the X appears mild, more profound effects are seen in the total number, timing, and placement of the DSBs across the genome- all of which are diminished or altered in the absence of HIM-5. The phenotypes of him-5 loss reminiscent of those observed in Prdm9-/- in mice where breaks are relocated to transcriptional start sites and show significant delay in formation. As with PRDM9, the comparatively subtle phenotypes of HIM-5 loss do not diminish its critical role in promoting proper DSB formation in most mammals.
Like most other studies that have examined DSB formation in C. elegans, this work relies on indirect assays, here limited to the cytological appearance of RAD-51 foci and bivalent chromosomes, as evidence of break formation or lack thereof. Unfortunately, neither of these assays has the power to reveal the genome-wide distribution or number of breaks. These assays have additional caveats, due to the fact that RAD-51 association with recombination intermediates and successful crossover formation both require multiple steps downstream of DSB induction, some of which are likely impaired in some of the mutants analyzed here. This severely limits the conclusions that can be drawn. Given that the goal of the work is to understand the effects of individual factors on DSB induction, direct physical assays for DSBs should be applied; many such assays have been developed and used successfully in other organisms.
We appreciate the reviewer’s thoughtful comments. We agree that RAD-51 foci are an indirect readout of DSB formation and that their dynamics can be influenced by defects in downstream repair processes. However, in C. elegans, the available methods for directly detecting DSBs are limited. Unlike other organisms, C. elegans lacks γH2AX, eliminating the possibility of using γH2AX as a DSB marker. TUNEL assays, while conceptually appealing, have proven unreliable and poorly reproducible in the germline context. Similarly, RPA foci do not consistently correlate with the number of DSBs and are influenced by additional processing steps.
Given these limitations, RAD-51 foci remain the most widely accepted surrogate for monitoring DSB formation in C. elegans. While we fully acknowledge the caveats associated with this approach — particularly the potential effects of downstream repair defects — RAD-51 analysis continues to provide valuable insight into DSB dynamics and regulation, especially when interpreted in combination with other phenotypic assessments.
Throughout the manuscript, the writing conflates the roles played by different factors that affect DSB formation in very different ways. XND-1 and HIM-17 have previously been shown to be transcription factors that promote the expression of many germline genes, including genes encoding proteins that directly promote DSBs. Mutations in either xnd-1 or him-17 result in dysregulation of germline gene expression and pleiotropic defects in meiosis and fertility, including changes in chromatin structure, dysregulation of meiotic progression, and (for xnd-1) progressive loss of germline immortality. It is thus misleading to refer to HIM-17 and XND-1 as DSB "accessory factors" or to lump their activities with those of other proteins that are likely to play more direct roles in DSB induction.
It is clear that we will not reach agreement about the direct vs indirect roles here of chromatin remodelers/transcription factors in break formation. In yeast, there is a precedent for SPP1 and in mouse for Prdm9, both of which could be described as transcription factors as well, as having roles in break formation by creating an open chromatin environment for the break machinery. We envision that these proteins function in the same fashion. The changes in histone acetylation in the xnd-1 mutants supports such a claim.
We do not know what the reviewer is referring to in statement that “XND-1 and HIM-17 have previously been shown to be transcription factors that promote the expression of many germline genes.” While the Carelli et al paper indeed shows a role for HIM-17 in expression of many germline genes, there is only one reference to XND-1 in this manuscript (Figure S3A) which shows that half of XND-1 binding sites overlap with the co-opted germline promoters. There is no transcriptional data at all on xnd-1 mutants, save our studies (referenced herein) that XND-1 regulates him-5 expression.
For example, statements such as the following sentence in the Introduction should be omitted or explained more clearly: "xnd-1 is also unique among the accessory factors in influencing the timing of DSBs; in the absence of xnd-1, there is precocious and rapid accumulation of DSBs as monitored by the accumulation of the HR strand-exchange protein RAD-51.
We are not sure what is confusing here. The distribution of RAD-51 foci is significantly altered in xnd-1 mutants and peak levels of breaks are achieved as nuclei leave the transition zone (Wagner et al., 2010; McClendon et al., 2016). There is no other mutation that causes this type of change in RAD-51 distribution.
"The evidence that HIM-17 promotes the expression of him-5 presented here corroborates data from other publications, notably the recent work of Carelli et al. (2022), but this conclusion should not be presented as novel here.
We have clarified this in the text. We note that this paper showed alterations in him-5 levels by RNA-Seq but they did not validate these results with quantitative RT-PCR. Thus, our studies do provide an important validation of their prior results.
The other factors also fall into several different functional classes, some of which are relatively well understood, based largely on studies in other organisms. The roles of RAD50 and MRE-11 in DSB induction have been investigated in yeast and other organisms as well as in several prior studies in C. elegans. DSB-1, DSB-2, and DSB-3 are homologs of relatively well-studied meiotic proteins in other organisms (Rec114 and Mei4) that directly promote the activity of Spo11, although the mechanism by which they do so is still unclear.
Whilst we agree that we understand some of the functions of the homologs, there are clearly examples in other processes of conserved proteins adopting unique regulatory function. We should not presume evolutionary conservation until proven. Indeed the comparison between the Mer2 proteins becomes particularly relevant here. For example, the RMM complex in plants does not contain PRD3, although this protein is thought to have function in DSB formation and repair (Lambing et al, 2022; Vrielynck et al., 2021; Thangavel et al., 2023). In Sordaria, as well, the Mer2 homolog has distinct functions (Tesse et al., 2017).
Mutations in PARG-1 (a Poly-ADP ribose glycohydrolase) likely affect the regulation of polyADP-ribose addition and removal at sites of DSBs, which in turn are thought to regulate chromatin structure and recruitment of repair factors; however, there is no convincing evidence that PARG-1 directly affects break formation.
Our prior collaborative studies on PARG-1 showed that is has a non-catalytic function that promote DSBs that is independent of accumulation of PAR (Janisiw et al., 2020; Trivedi et al., 2022)
CEP-1 is a homolog of p53 and is involved in the DNA damage response in the germline, but again is unlikely to directly contribute to DSB induction.
We respectfully disagree with the reviewer’s statement. While CEP-1 is indeed a homolog of p53 and plays a major role in the DNA damage response, prior work from Brent Derry’s lab and from our group (Mateo et al., 2016) demonstrated that specific cep-1 separationof-function alleles affect DSB induction and/or repair pathway choice independently of canonical DNA damage checkpoint activation. In particular, defects in DSB formation observed in certain cep-1 mutants can be rescued by exogenous irradiation, supporting a direct or closely linked role in promoting DSB formation rather than merely responding to damage. Thus, based on these functional data, we considered CEP-1 a relevant factor to include in our analysis. We have now clarified this rationale in the revised manuscript.
HIM-5 and REC-1 do not have apparent homologs in other organisms and play poorly understood roles in promoting DSB induction. A mechanistic understanding of their functions would be of value to the field, but the current work does not shed light on this. A previous paper (Chung et al. G&D 2015) concluded that HIM-5 and REC-1 are paralogs arising from a recent gene duplication, based on genetic evidence for a partially overlapping role in DSB induction, as well as an argument based on the genomic location of these genes in different species; however, these proteins lack any detectable sequence homology and their predicted structures are also dissimilar (both are largely unstructured but REC-1 contains a predicted helical bundle lacking in HIM-5). Moreover, the data presented here do not reveal overlapping sets of genetic or physical interactions for the two genes/proteins. Thus, this earlier conclusion was likely incorrect, and this idea should not be restated uncritically here or used as a basis to interpret phenotypes.
Actually, there is quite good bioinformatic analysis that the rec-1 and him-5 loci evolved from a gene duplication and that each share features of the ancestral protein (Chung et al., 2015). We are sorry if the reviewer casts aspersions on the prior literature and analyses. The homology between these genes with the ancestral protein is near the same degree as dsb-1, dsb-2, or dsb-3 to their ancestral homologs (<17%).
DSB-1 was previously reported to be strictly required for all DSB and CO formation in C. elegans. Here the authors test whether the expression of HIM-5 from the pie-1 promoter can rescue DSB formation in dsb-1 mutants, and claim to see some rescue, based on an increase in the number of nuclei with one apparent bivalent (Figure 2C). This result seems to be the basis for the claim that HIM-5 coordinates the activities of other DSB proteins. However, this assay is not informative, and the conclusion is almost certainly incorrect. Notably, a substantial number of nuclei in the dsb-1 mutant (without Ppie-1::him-5) are reported as displaying a single bivalent (11 DAPI staining bodies) despite prior evidence that DSBs are absent in dsb-1 mutants; this suggests that the way the assay was performed resulted in false positives (bivalents that are not actually bivalents), likely due to inclusion of nuclei in which univalents could not be unambiguously resolved in the microscope. A slightly higher level of nuclei with a single unresolved pair of chromosomes in the dsb-1; Ppie-1::him-5 strain is thus not convincing evidence for rescue of DSBs/CO formation, and no evidence is presented that these putative COs are X-specific. The authors should provide additional experimental evidence - e.g., detection of RAD-51 and/or COSA-1 foci or genetic evidence of recombination - or remove this claim. The evidence that expression of Ppie-1::him-5 may partially rescue DSB abundance in dsb-2 mutants is hard to interpret since it is currently unknown why C. elegans expresses 2 paralogs of Rec114 (DSB-1 and DSB-2), and the age-dependent reduction of DSBs in dsb-2 mutants is not understood.
We have removed this claim in part because we have been unable to create the triple mutants strains to analyze COSA-1 foci.
To the point about 11 vs 12 DAPI bodies: the literature is actually replete with examples of 11 DAPI bodies vs 12 in mutants with no breaks:
Hinman al., 2021: null allele of dsb-3 has an average of 11.6 +/- 0.6 breaks;
Stamper et al, 2013, show just over 60% of dsb-1 nuclei with 12 DAPI bodies and 5-10% with 10 DAPI bodies. (Figure 1);
In addition, we also previously showed (Machovina et al., 2016) that a subset of meiotic nuclei have a single RAD-51 focus and can achieve a crossover. RAD-51 foci in spo-11 were also reported in Colaiacovo et al., 2003.
Several of the factors analyzed here, including XND-1, HIM-17, HIM-5, DSB-1, DSB-2, and DSB-3, have been shown to localize broadly to chromatin in meiotic cells. Coimmunoprecipitation of pairs of these factors, even following benzonase digestion, is not strong evidence to support a direct physical interaction between proteins.
Similarly, the super-resolution analysis of XND-1 and HIM-17 (Figure 1EF) does not reveal whether these proteins physically interact with each other, and does not add to our understanding of these proteins functions, since they are already known to bind to many of the same promoters. Promoters are also likely to be located in chromatin loops away from the chromosome axis, so in this respect, the localization data are also confirmatory rather than novel.
While the binding to promoters would be expected to be on DNA loops, that has not been definitively shown in the worm germ line. The supplemental data of the Carelli paper suggests that there are ~250 binding sites for each protein at these coopted promoters. This could not account for crossover map seen in C. elegans.
The reviewer states correct that we do not reveal that these proteins interact, but we have shown that the two proteins co-IP and have a Y2H interaction. This interaction is supporedt by a recent publication (Blazickova et al., 2025) corroborating this conclusion and identifies XND-1 in HIM-17 co-IPs also in the presence of benzonase. We do now show, however, by immuno-localization that the two proteins appear to be adjacent, but nonoverlapping. As now described in the text, AlphaFold 3 modeling and structural analysis suggests that the two proteins do interact directly and that the tagged 5’ end of HIM-17 used in our studies is likely to be at least 200nm from the putative XND-1 binding interface, a distance that is consistent with our confocal images showing frequent juxtaposition of the two proteins.
The phenotypic analysis of double mutant combinations does not seem informative. A major problem is that these different strains were only assayed for bivalent formation, which (as mentioned above) requires several steps downstream of DSB induction. Additionally, the basis for many of the single mutant phenotypes is not well understood, making it particularly challenging to interpret the effects of double mutants. Further, some of the interactions described as "synergistic" appear to be additive, not synergistic. While additive effects can be used as evidence that two genes work in different pathways, this can also be very misleading, especially when the function of individual proteins is unknown. I find that the classification of genes into "epistastasis groups" based on this analysis does not shed light on their functions and indeed seems in some cases to contradict what is known about their functions. ‘
As described above, each of the proteins analyzed is thought to have a direct role in regulating meiotic DSB formation and single mutant phenotypes are consistent with this interpretation. In almost all-if not all- of these cases, IR induced breaks suppress univalent phenotypes (or uncover a downstream repair defect (e.g. in mre-11)) supporting this conclusion. We have changed the terminology from “epistasis groups” since this is not strict epistasis, but rather, “functional groups”.
The yeast two-hybrid (Y2H) data are only presented as a single colony. While it is understandable to use a 'representative' colony, it is ideal to include a dilution series for the various interactions, which is how Y2H data are typically shown.
The Y2H data are presented as spots on a plate and are from three to four individual transformants per interaction tested, and are not individual colonies. The experiment was repeated in triplicate from different transformations. We have now made this clearer in the materials and methods section. This approach has been successfully used to examine protein interactions in our prior manuscripts of yeast and human proteins [Gaines et al (2015) Nat. Comms 6:7834; Kondrashova et al (2017) Cancer Discovery 7:984; Garcin et al (2019) PLoS Genetics 15:e1008355; Bonilla et al (2021) eLife 1: e68080) Prakash et al (2022) PNAS 119: e2202727119, etc]
Additional (relatively minor) concerns about these data:
(1) Several interactions reported here seem to be detected in only one direction - e.g., MRE-11-AD/HIM-5-BD, REC-1-AD/XND-1-BD, and XND-1-AD/HIM-17-BD - while no interactions are seen with the reciprocal pairs of fusion proteins. I'm not sure if some of this is due to pasting "positive" colony images into the wrong position in the grid, but this should be addressed.
The asymmetry in the interactions observed is due to the well-known phenomenon in yeast two-hybrid (Y2H) assays where certain plasmids exhibit self-activation when fused in one orientation, making interpretation of reciprocal interactions challenging. In our experiment, some of the plasmids indeed showed self-activation in one direction, which likely accounts for the lack of interaction seen with the reciprocal pairs of fusion proteins. We have clarified this point in the Methods.
(2) DSB-3 was only assayed in pairwise combinations with a subset of other proteins; this should be explained; it is also unclear why the interaction grids are not symmetrical about the diagonal.
We have now completed the analysis by adding the interactions of DSB-3 with the remaining proteins that were missing from the initial set.
(3) I don't understand why the graphic summaries of Y2H data are split among 3 different figures (1, 2, and 3).
We chose to split the graphic summaries of the Y2H data across Figures 1, 2, and 3 because we felt this organization better aligns with the flow of the results presented in each figure. Each set of interactions is shown in the context of the specific experiments and findings discussed in those sections, which we believe helps provide a clearer and more logical presentation of the data.
Recommendations for the authors:
Reviewer #1 (Recommendations For The Authors):
Figure 1: B) The IP is difficult to interpret - there is a band of the corresponding size to XND-1 in the control lane calling into question the specificity of the IP/Western.
We added a supplemental figure with the specificity of the antibody showing that there is a background non-specific band.
C) More information about the mass spectrometry should be included. No indication of the number of times a peptide was identified, or the overall coverage of the identified proteins.
Done
This is important as in the results section (line 114) the authors indicate that there was "strong" interaction yet there is no way to assess this.
D) Why wasn't hatching measured in the him-5p::him-5; him-17(ok424) strain?
Great question. I guess we need to do this while back out for review. If anyone has suggestions of what to say here. Clearly we overlooked this point but do have the strain.
E) Quantification of the cytology should be included.
We have now quantified overlap between XND-1 and HIM-17
Figure 2: C) Statistics should be included.
Done
E) Quantification should be included for the cytology. I recommend changing the eals15 to HIM-5.
We included better images showing whole gonads instead of one or two nuclei. We were not sure what the reviewers want us to quantify here since the relocalization of the protein to the cytoplasm is very clear.
I have a general issue with the use of the term epistasis - this is used to order gene function based on different mutant phenotypes, usually with null alleles. While I think the authors have valid points with how they group the different SPO-11 accessory proteins, I do not think they should use the word epistasis, but rather genetic interactions.
We appreciate the reviewers thoughts on this matter and have removed the term epistasis and use functional groups or genetic interactions throughout the text.
Figure 4 and the nature of the X chromosome: First, I think it would help the non-C. elegans reader to include a little more information on the X chromosome with respect to its differences compared to the autosomes. I also think that, if possible, it would be beneficial to include a model of the X in Figure 4.
We have added more about X/autosome differences in the intro and during the discussion of HIM-5 function and have added a figure showing difference in the behavior of the X/autosomes during DSB/crossover formation.
Minor points:
Abstract: Given the findings of Silva and Smolikove on SPO-11 breaks, I recommend removing "early" from line 28 in the Abstract.
Done
Introduction (line 93): I think "biochemical studies" is a stretch here - I recommend "interaction studies".
Done
Results: (lines 160-161): mutations are not required for breaks. Line 172, there is a problem with the sentence.
Corrected
Reviewer #2 (Recommendations For The Authors):
Major comments:
(1) Figure 1B- The signal for XND-1 seems to appear both in the control and him-17::HA IP. Do the authors have tested the specificity of the XND-1 antibody?
We included a supplementary figure demonstrating the specificity of the XND-1 antibody by Western blot. This was also previously published (Wagner et al., 2010)
(2) Figure 1D - can the authors provide an explanation why the him-5p::him-5 transgene that drives a higher expression than pie-1p::him-5 fails to suppress the Him phenotype seen in him-17? What are the HIM-5 levels like in these two strains compared to N2 and him-17 null mutants? Can this information provide explanation for the differential effect of the him-5 transgenes?
We previously reported that him-5p::him-5 drives higher expression than pie-1p::him-5 (McClendon et al, 2016).
The reason that him-5p::him-5 does not rescue, despite higher wild type expression is that HIM-17 directly regulates expression of him-5. Since HIM-17 does not regulate the pie-1 promoter, the pie-1p::him-5 construct can at least partially suppress the him-17 mutation.
We have (hopefully) explained this better in the text.
(3) Line 102- the subheading "HIM-5 is the essential factor for meiotic breaks in the Xchromosome" may not be appropriate for this section. This is what has previously been known. However, the results in Figure 1 demonstrate that a him-5 transgene can partially rescue the him-17 and ¬xnd-1 phenotype, but not that it is essential for meiotic DSB formation on X chromosomes.
We think some of the concern here is sematic and have changed the phraseology to say that HIM-5 is SUFFICIENT for DSBs on the X… which had not previously been shown.
Vis-à-vis the X chromosome, in all genetic backgrounds examined, the absence of HIM-5 consistently results in a complete lack of DSBs on the X. For instance, in dsb-2 mutants— where HIM-5 is still expressed—DSBs are reduced genome-wide, but the X chromosome occasionally retains breaks. In contrast, even a weak allele of him-17 results specifically in the loss of X chromosome breaks, underscoring a unique requirement for HIM-5 in promoting DSBs on the X. While Figure 1 shows that a him-5 transgene can partially rescue him-17 and xnd-1 phenotypes, the consistent observation that X breaks are absent without HIM-5 supports its classification as sufficient for DSB formation on the X chromosome.
(4) Figure 1E - please consider enlarging the images and showing multiple examples.
Done.
I also suggest that the authors perform a more rigorous analysis to support the conclusion that XND-1 and HIM-17 localize away from the axis by quantifying multiple images and doing line-scan analysis.
Provided. New images are provided in both, the main and supplemental figures, and quantification is included. There is no detectable overlap of the two protein with one another or the DNA axes (see quantification of overlap in Fig. 1).
(5) Line 162 - This is the first mention of DSB-1, DSB-2, and DSB-3 in the paper. DSB-1 and DSB-2 are Rec114 homologs in C. elegans (Tesse et al., 2017), while DSB-3 is a homolog of Mei4 (Hinman et al., 2021). These proteins should be properly introduced in the introduction with appropriate citations.
Done. We appreciate the reviewer pointing out that this was the first reference to these genes.
(6) Line 169 - the rationale for this experiment is unclear. Why did the Y2H interaction between HIM-5 and DSB-1 prompt the authors to test the rescue of dsb-1 or dsb-2 phenotypes by the ectopic expression of him-5? Do the authors have evidence that HIM-5 level is reduced in dsb-1 or dsb-2 mutants?
We have reorganized this section to better explain the motivation for looking at these interactions. We did see a difference in the localization in HIM-5 in the dsb-1 mutant animals and we did have a sense that HIM-5 was critical for breaks on the X. We reasoned that it could have independent functions in promoting breaks that were not yet appreciated so wanted to do this experiment.
(7) Line 172 - "very slightly reduced". This claim requires statistical analysis.
We added statistical analysis, but we also removed this claim.
(8) Figures 2C and 2D - Can the authors provide an explanation why the pie-1p::him-5 transgene fails to suppress the phenotypes in dsb-1, while the him-5p::him-5 trasgene can? Again, the rationale for these experiments is unclear. Because of this, the interpretation is also unclear.
The difference in rescue between the pie-1p::him-5 and him-5p::him-5 transgenes likely reflects differences in expression levels. As previously shown (McClendon et al., 2016), the him-5p::him-5 construct results in significantly higher expression of HIM-5 protein compared to pie-1p::him-5. This elevated expression likely explains its ability to partially rescue the dsb-1 phenotype. In contrast, the lower expression driven by the pie-1 promoter is insufficient to compensate for the absence of dsb-1 function. We have clarified the rationale and interpretation of these experiments in the revised manuscript to better reflect this point.
(9) Lines 184-185 - the data for endogenously tagged HIM-5::3xHA are not shown anywhere in the paper. This must be shown.
We have added this in the supplemental figures.
(10) Figure 2D and 2E - what does the localization of pie-1p::him-5::GFP (eaIs15) and him5p::him-5::GFP (eaIs4) look like in wild-type and dsb-1 mutants? Are the cytoplasmic aggregates caused by increased levels of HIM-5 expression? Can the differential behavior of him-5 transgenes provide explanation for differential rescues?
We now show both live and fixed images of Phim-5::him-5::gfp transgenes, as well as the localization of the endogenously HA-tagged HIM-5 locus (Figure 2 and S3). In all cases, the protein is initially nuclear and then absent from meiotic nuclei with similar timing. The Ppie1::him-5 transgene was very difficult to image due to low expression (even in wild type) so it not shown here. We presume it is the slightly elevated level of expression of the Phim5::him-5::gfp that can explain the differential rescue.
(11) Lines 221-222, where are the results shown? Please refer to Figure S3.
Done
(12) Figure S3 - these need statistical analyses.
Done
(13) Lines 230-231 - what about the rec-1; parg-1; cep-1 triple mutant?
This is an excellent suggestion and not one we have not yet pursued. Given the lack of strong phenotypes in all combination of double mutants, we prioritized other experiments . However, we agree that examining the rec-1; parg-1; cep-1 triple mutant would provide a valuable test of whether these factors act in the same pathway, and we appreciate the reviewer highlighting this potential future direction.
(14) Line 298 - I suggest the authors take a look at the Alphafold prediction of DSB-1/DSB-2/DSB-3 and the comparison to human and budding yeast Rec114/Mei4 complex in Guo et al., 2022 eLife, which could provide insights into the Y2H results.
We thank the reviewer for these comments and have indeed used these interactions and predicted homologies to zero in a region of interaction between these proteins that resembles what is seen in humans and yeast with a dimer of REC114 like proteins wraps stabilizing a central Mei4 helix . This is now shown in Figure 3H, I. Satisfyingly, this modeling predicts that a trimer comprised of 2 DSB-1 proteins with DSB-3 is more stable than a DSB1-DSB-2-DSB-3 trimer. This might explain why DSB-2 is not required in young adults and only becomes essential as DSB-1 levels drop in older animals (Rosu et al., 2013)
(15) Can the authors introduce mutations within the DSB-1 interfaces that disrupt the interaction to either SPO-11 or DSB-2?
We have begun to address this question by introducing targeted mutations within DSB-1. As shown in Figure 3E and 3F, mutations in the C-terminal region of DSB-1—which includes a core of four α-helices—disrupt its interaction with DSB-2 and DSB-3, but not with SPO-11. These findings suggest that the C-terminus mediates interactions specifically with DSB2 and DSB-3
(16) Line 323 - The him-5 phenotypes are too weak to support the idea that it serves as the linchpin for the whole DSB complex. Do the authors have an explanation for why him-5 mutants exhibit X-chromosome-specific DSB defects?
In response to the reviewer, above, and in the text, we have included a more detailed explanation of why we think HIM-5 has a key role in coordinating meiotic break formation. Although, identified for its role on the X, the phenotypes associated with DSB formation in the mutant are really quite pleiotropic and severe.
(17) Line 436 - C. elegans lacks DSB hotspots.
Removed
Minor comments:
(1) Figure 1A - please show the raw data for the yeast two-hybrid.
We show representative yeast colonies in Figure S3.
(2) It looks like the labeling for Figure 1B and 1C are switched.
Fixed.
(3) Figure 1B - what does the red box indicate? Please explain it in the legend.
It indicates the XND-1 band. We added that information in the legend.
(4) Figure 1C - in the legend, it was noted that the results are from GFP pulldowns of HIM17::GFP. However, the method for Figure 1B and the method section noted that HIM-17 was tagged with 3xHA, and the pull-down was performed using anti-HA affinity matrix. Please reconcile this discrepancy.
That’s because they were done in two different sets of experiments. For the IPs we used a HIM-17::HA strain and for the MS, a HIM-17::GFP strain.
(5) Also in Figure 1C - please call Table S2 in the main text when discussing the mass spec results. Also, it is not clear what HIM-17 and GFP indicate in the table. What makes CKU80 different from the other proteins listed under GFP? Please explain more clearly in the legend.
We have move the table to supplemental data where we have included all of the peptide counts and gene coverage. We have included in the revised method rationale for inclusion in this table which explains why CKU-80 differs.
(6) Line 527 - it is unclear what experiment was done for HIM-17. Please revise it to indicate that this is for "HIM-17 immunoprecipitation". Also please indicate the strain used for HIM17 pull-down (AV280?).
(7) Line 113- please be specific about how the HIM-17 IP was performed. Which epitope and strains are used for pull-downs?
This indeed was AV280. This has been added to the text and methods.
(8) Figure 1D- What does ND mean? In the text, it was stated that there was only a minor suppression of hatching rates. The hatching rate for him-5p::him-5; him-17 must have been measured, and the data must be presented.
ND does mean not determined. We have removed the statement about “minor suppression”. We only tested the overall population dynamics in the Phim-5::him-5;him17(ok424) and the DAPI body counts. The failure to suppress the latter suggests there would be no enect on hatching rates, although we did not test this directly. Since we had done this for the Ppie-1::him-5;him-17 strain, we provided this information to further support the claims of genetic rescue by ectopic expression.
(9) Line 151 - please specify that STED was used.
We have removed the STED images, and just show the confocal images with Lightning Processing.
(10) Figure 1E- the authors suggested that HIM-17 and XND-1 mainly localize to autosomes but not the X chromosome. However, there is not enough evidence that the chromosome excluded from HIM-17 staining is indeed an X chromosome.
(11) Figure 1E (Line 154) - what are the active chromatin markers examined? Where are the data?
We have previously shown that the chromosome lacking XND-1 staining is the X (Wagner et al., 2010). The X is heterochromatic and chromatin marks associated with active transcription, including H3K4me3 and HTZ-1 (a variant H2A), preferentially localize to autosomes, effectively anti-marking the X chromosome. As shown in the new Figure 1E, a single chromosome has very little XND-1 and HIM-17 associated proteins. This is the X chromosome.
(12) Line 172 - It should be a comma instead of the period after "In dsb-1 mutants".
Fixed
(13) Figure S3H-K - I suggest the authors indicate the alleles of mre-11 (null vs. iow1) on the graph, similarly to him-5(e1490) to avoid confusion.
Done
(14) Lines 294 and 600 - Guo et al. 2022 is now published in eLife. The authors must cite the published paper, not the preprint.
Fixed
(15) Line 407 - the reference Carelli et al., 2022 is missing.
Added
(16) Line 766 - please remove "is" before nuclear.
Done
Reviewer #3 (Recommendations For The Authors):
Major issues:
In my view, the most interesting mechanistic finding in the paper is the evidence that HIM-5 may not bind to chromatin in the absence of DSB-1. If validated, this would suggest that HIM-5 is likely to be directly involved in a process that promotes break formation, in contrast to factors such as HIM-17 and XND-1. It does not, however, support the idea that HIM-5 is at the top of a hierarchy of DSB factors, as it is interpreted here. More importantly, the data supporting this claim are unconvincing; only a single image of an unfixed gonad from an animal expressing HIM-5::GFP is shown. Immunofluorescence should be performed and the results must be quantified.
We have provided additional images of the HIM-5 relocalization to show that we observed this in both fixed and live worms with two different tagged strains. The exclusion from the nucleus is seen in all scenarios. Whether the protein now accumulates exclusively in the cytoplasm/ is destabilized is challenging to address with the fixed images due to the arbitrariness of defining “background” staining.
More generally, this type of analysis, looking at the interdependence of different factors for their association with chromosomes, is much more informative than the genetic interaction data presented in the paper, which does not seem to provide any mechanistic insights into the functions of the factors analyzed. The paper could potentially be greatly improved through a more extensive, systematic analysis of the interdependence of DSBpromoting factors for their localization to chromosomes.
We have at least added this for XND-1 and HIM-17 and show they are not interdependent for chromosome association. We also provide for the first time data on the localization of HIM-5 in the dsb-1 mutant. Many of the other interactions have already been shown in the literature and/or were not warranted base on the lack of genetic interaction we present here.
Minor issues:
The title is vague and inconclusive. A more concrete title summarizing the major findings would help readers to assess whether the work is of interest.
We have discussed the title extensively with all authors and all would like to keep the current title.
The authors claim that the expression of HIM-5 from a different promoter (Ppie-1::him-5) but not its endogenous promoter (Phim-5::him-5) can partially rescue the DSB defect in him-17 mutants. To support this claim, they should really quantify the germline expression of HIM-5 in wild-type, him-17, him-17; Ppie-1::him-5, and Phim-5::him-5; him-17.
We had previously reported the expression in the N2 background of both transgenes (McClendon et al., 2016)
Panel O appears to be missing from Figure S3.
Fixed
The evidence for chromosome fusions in cep-1; mre-11 mutants shown in S4D is not convincing and the claim should be removed unless stronger evidence can be obtained.
A clearer image has been added
The basis of the following statement is unclear: "Furthermore, rec-1;him-5 double mutants give an age-dependent severe loss of DSBs (like dsb-2 mutants) suggesting that the ancestral function of the protein may have a more profound effect on break formation." The manuscript does not seem to include data regarding age-dependent loss of DSBs and no other publication is cited to support this claim. The interpretation is also perplexing; I think that it may be predicated on the idea that REC-1 and HIM-5 are paralogs, but as stated above, this claim is not well supported and is likely specious.
We have added the reference. This was shown in Chung et al., 2013 – the paper that presented the cloning of the rec-1 locus.
