The conserved ATPase PCH-2 controls the number and distribution of crossovers by antagonizing crossover formation in C. elegans

Reviewer #1 (Public review):

The conserved AAA-ATPase PCH-2 has been shown in several organisms including C. elegans to remodel classes of HORMAD proteins that act in meiotic pairing and recombination. In some organisms the impact of PCH-2 mutations is subtle but becomes more apparent when other aspects of recombination are perturbed. Patel et al. performed a set of elegant experiments in C. elegans aimed at identifying conserved functions of PCH-2. Their work provides such an opportunity because in C. elegans meiotically expressed HORMADs localize to meiotic chromosomes independently of PCH-2. Work in C. elegans also allows the authors to focus on nuclear PCH-2 functions as opposed to cytoplasmic functions also seen for PCH-2 in other organisms.

The authors performed the following experiments:

(1) They constructed C. elegans animals with SNPs that enabled them to measure crossing over in intervals that cover most of four of the six chromosomes. They then showed that double-crossovers, which were common on most of the four chromosomes in wild-type, were absent in pch-2. They also noted shifts in crossover distribution in the four chromosomes.

(2) Based on the crossover analysis and previous studies they hypothesized that PCH-2 plays a role at an early stage in meiotic prophase to regulate how SPO-11 induced double-strand breaks are utilized to form crossovers. They tested their hypothesis by performing ionizing irradiation and depleting SPO-11 at different stages in meiotic prophase in wild-type and pch-2 mutant animals. The authors observed that irradiation of meiotic nuclei in zygotene resulted in pch-2 nuclei having a larger number of nuclei with 6 or greater crossovers (as measured by COSA-1 foci) compared to wildtype. Consistent with this observation, SPO11 depletion, starting roughly in zygotene, also resulted in pch-2 nuclei having an increase in 6 or more COSA-1 foci compared to wild type. The increased number at this time point appeared beneficial because a significant decrease in univalents was observed.

(3) They then asked if the above phenotypes correlated with the localization of MSH-5, a factor that stabilizes crossover-specific DNA recombination intermediates. They observed that pch-2 mutants displayed an increase in MSH-5 foci at early times in meiotic prophase and an unexpectedly higher number at later times. They conclude based on the differences in early MSH-5 localization and the SPO-11 and irradiation studies that PCH-2 prevents early DSBs from becoming crossovers and early loading of MSH-5. By analyzing different HORMAD proteins that are defective in forming the closed conformation acted upon by PCH-2, they present evidence that MSH-5 loading was regulated by the HIM-3 HORMAD.

(4) They performed a crossover homeostasis experiment in which DSB levels were reduced. The goal of this experiment was to test if PCH-2 acts in crossover assurance. Interestingly, in this background PCH-2 negative nuclei displayed higher levels of COSA-1 foci compared to PCH-2 positive nuclei. This observation and a further test of the model suggested that "PCH-2's presence on the SC prevents crossover designation."

(5) Based on their observations indicating that early DSBS are prevented from becoming crossovers by PCH-2, the authors hypothesized that the DNA damage kinase CHK-2 and PCH-2 act to control how DSBs enter the crossover pathway. This hypothesis was developed based on their finding that PCH-2 prevents early DSBs from becoming crossovers and previous work showing that CHK-2 activity is modulated during meiotic recombination progression. They tested their hypothesis using a mutant synaptonemal complex component that maintains high CHK-2 activity that cannot be turned off to enable crossover designation. Their finding that the pch-2 mutation suppressed the crossover defect (as measured by COSA-1 foci) supports their hypothesis.

Based on these studies the authors provide convincing evidence that PCH-2 prevents early DSBs from becoming crossovers and controls the number and distribution of crossovers to promote a regulated mechanism that ensures the formation of obligate crossovers and crossover homeostasis. As the authors note, such a mechanism is consistent with earlier studies suggesting that early DSBs could serve as "scouts" to facilitate homolog pairing or to coordinate the DNA damage response with repair events that lead to crossing over. The detailed mechanistic insights provided in this work will certainly be used to better understand functions for PCH-2 in meiosis in other organisms. My comments below are aimed at improving the clarity of the manuscript.

Comments

(1) It appears from reading the Materials and Methods that the SNPs used to measure crossing over were obtained by mating Hawaiian and Bristol strains. It is not clear to this reviewer how the SNPs were introduced into the animals. Was crossing over measured in a single animal line? Were the wild-type and pch-2 mutations made in backgrounds that were isogenic with respect to each other? This is a concern because it is not clear, at least to this reviewer, how much of an impact crossing different ecotypes will have on the frequency and distribution of recombination events (and possibly the recombination intermediates that were studied).

(2) The authors state that in pch-2 mutants there was a striking shift of crossovers (line 135) to the PC end for all of the four chromosomes that were tested. I looked at Figure 1 for some time and felt that the results were more ambiguous. Map distances seemed similar at the PC end for wildtype and pch-2 on Chrom. I. While the decrease in crossing over in pch-2 appeared significant for Chrom. I and III, the results for Chrom. IV, and Chrom. X. seemed less clear. Were map distances compared statistically? At least for this reviewer the effects on specific intervals appear less clear and without a bit more detail on how the animals were constructed it's hard for me to follow these conclusions.

(3) Figure 2. I'm curious why non-irradiated controls were not tested side-by-side for COSA-1 staining. It just seems like a nice control that would strengthen the authors' arguments.

(4) Figure 3. It took me a while to follow the connection between the COSA-1 staining and DAPI staining panels (12 hrs later). Perhaps an arrow that connects each set of time points between the panels or just a single title on the X-axis that links the two would make things clearer.

Reviewer #2 (Public review):

Summary:

This paper has some intriguing data regarding the different potential roles of Pch-2 in ensuring crossing over. In particular, the alterations in crossover distribution and Msh-5 foci are compelling. My main issue is that some of the models are confusingly presented and would benefit from some reframing. The role of Pch-2 across organisms has been difficult to determine, the ability to separate pairing and synapsis roles in worms provides a great advantage for this paper.

Strengths:

Beautiful genetic data, clearly made figures. Great system for studying the role of Pch-2 in crossing over.

Weaknesses:

(1) For a general audience, definitions of crossover assurance, crossover eligible intermediates, and crossover designation would be helpful. This applies to both the proposed molecular model and the cytological manifestation that is being scored specifically in C. Elegans.

(2) Line 62: Is there evidence that DSBs are introduced gradually throughout the early prophase? Please provide references.

(3) Do double crossovers show strong interference in worms? Given that the PC is at the ends of chromosomes don't you expect double crossovers to be near the chromosome ends and thus the PC?

(4) Line 155 - if the previous data in Deshong et al is helpful it would be useful to briefly describe it and how the experimental caveats led to misinterpretation (or state that further investigation suggests a different model etc.). Many readers are unlikely to look up the paper to find out what this means.

(5) Line 248: I am confused by the meaning of crossover assurance here - you see no difference in the average number of COSA-1 foci in Pch-2 vs. wt at any time point. Is it the increase in cells with >6 COSA-1 foci that shows a loss of crossover assurance? That is the only thing that shows a significant difference (at the one time point) in COSA-1 foci. The number of dapi bodies shows the loss of Pch-2 increases crossover assurance (fewer cells with unattached homologs). So this part is confusing to me. How does reliably detecting foci vs. DAPI bodies explain this?

(6) Line 384: I am confused. I understand that in the dsb-2/pch2 mutant there are fewer COSA-1 foci. So fewer crossovers are designated when DSBs are reduced in the absence of PCH-2. How then does this suggest that PCH-2's presence on the SC prevents crossover designation? Its absence is preventing crossover designation at least in the dsb-2 mutant.

(7) Discussion Line 535: How do you know that the crossovers that form near the PCs are Class II and not the other way around? Perhaps early forming Class I crossovers give time for a second Class II crossover to form. In budding yeast, it is thought that synapsis initiation sites are likely sites of crossover designation and class I crossing over. Also, the precursors that form class I and II crossovers may be the same or highly similar to each other, such that Pch-2's actions could equally affect both pathways.

Reviewer #3 (Public review):

Summary:

This manuscript describes an in-depth analysis of the effect of the AAA+ ATPase PCH-2 on meiotic crossover formation in C. elegant. The authors reach several conclusions, and attempt to synthesize a 'universal' framework for the role of this factor in eukaryotic meiosis.

Strengths:

The manuscript makes use of the advantages of the 'conveyor' belt system within the c.elegans reproductive tract, to enable a series of elegant genetic experiments.

Weaknesses:

A weakness of this manuscript is that it heavily relies on certain genetic/cell biological assays that can report on distinct crossover outcomes, without clear and directed control over other aspects and variables that might also impact the final repair outcome. Such assays are currently out of reach in this model system.

In general, this manuscript could be more generally accessible to non-C.elegans readers. Currently, the manuscript is hard to digest for non-experts (even if meiosis researchers). In addition, the authors should be careful to consider alternative explanations for certain results. At several steps in the manuscript, results could ostensibly be caused by underlying defects that are currently unknown (for example, can we know for sure that pch-2 mutants do not suffer from altered DSB patterning, and how can we know what the exact functional and genetic interactions between pch-2 and HORMAD mutants tell us?). Alternative explanations are possible and it would serve the reader well to explicitly name and explain these options throughout the manuscript.

Author response:

Public Reviews:

Reviewer #1 (Public review):

The conserved AAA-ATPase PCH-2 has been shown in several organisms including C. elegans to remodel classes of HORMAD proteins that act in meiotic pairing and recombination. In some organisms the impact of PCH-2 mutations is subtle but becomes more apparent when other aspects of recombination are perturbed. Patel et al. performed a set of elegant experiments in C. elegans aimed at identifying conserved functions of PCH-2. Their work provides such an opportunity because in C. elegans meiotically expressed HORMADs localize to meiotic chromosomes independently of PCH-2. Work in C. elegans also allows the authors to focus on nuclear PCH-2 functions as opposed to cytoplasmic functions also seen for PCH-2 in other organisms.

The authors performed the following experiments:

(1) They constructed C. elegans animals with SNPs that enabled them to measure crossing over in intervals that cover most of four of the six chromosomes. They then showed that doublecrossovers, which were common on most of the four chromosomes in wild-type, were absent in pch-2. They also noted shifts in crossover distribution in the four chromosomes.

(2) Based on the crossover analysis and previous studies they hypothesized that PCH-2 plays a role at an early stage in meiotic prophase to regulate how SPO-11 induced double-strand breaks are utilized to form crossovers. They tested their hypothesis by performing ionizing irradiation and depleting SPO-11 at different stages in meiotic prophase in wild-type and pch-2 mutant animals. The authors observed that irradiation of meiotic nuclei in zygotene resulted in pch-2 nuclei having a larger number of nuclei with 6 or greater crossovers (as measured by COSA-1 foci) compared to wildtype. Consistent with this observation, SPO11 depletion, starting roughly in zygotene, also resulted in pch-2 nuclei having an increase in 6 or more COSA-1 foci compared to wild type. The increased number at this time point appeared beneficial because a significant decrease in univalents was observed.

(3) They then asked if the above phenotypes correlated with the localization of MSH-5, a factor that stabilizes crossover-specific DNA recombination intermediates. They observed that pch-2

mutants displayed an increase in MSH-5 foci at early times in meiotic prophase and an unexpectedly higher number at later times. They conclude based on the differences in early MSH-5 localization and the SPO-11 and irradiation studies that PCH-2 prevents early DSBs from becoming crossovers and early loading of MSH-5. By analyzing different HORMAD proteins that are defective in forming the closed conformation acted upon by PCH-2, they present evidence that MSH-5 loading was regulated by the HIM-3 HORMAD.

(4) They performed a crossover homeostasis experiment in which DSB levels were reduced. The goal of this experiment was to test if PCH-2 acts in crossover assurance. Interestingly, in this background PCH-2 negative nuclei displayed higher levels of COSA-1 foci compared to PCH-2 positive nuclei. This observation and a further test of the model suggested that "PCH-2's presence on the SC prevents crossover designation."

(5) Based on their observations indicating that early DSBS are prevented from becoming crossovers by PCH-2, the authors hypothesized that the DNA damage kinase CHK-2 and PCH2 act to control how DSBs enter the crossover pathway. This hypothesis was developed based on their finding that PCH-2 prevents early DSBs from becoming crossovers and previous work showing that CHK-2 activity is modulated during meiotic recombination progression. They tested their hypothesis using a mutant synaptonemal complex component that maintains high CHK-2 activity that cannot be turned off to enable crossover designation. Their finding that the pch-2 mutation suppressed the crossover defect (as measured by COSA-1 foci) supports their hypothesis.

Based on these studies the authors provide convincing evidence that PCH-2 prevents early DSBs from becoming crossovers and controls the number and distribution of crossovers to promote a regulated mechanism that ensures the formation of obligate crossovers and crossover homeostasis. As the authors note, such a mechanism is consistent with earlier studies suggesting that early DSBs could serve as "scouts" to facilitate homolog pairing or to coordinate the DNA damage response with repair events that lead to crossing over. The detailed mechanistic insights provided in this work will certainly be used to better understand functions for PCH-2 in meiosis in other organisms. My comments below are aimed at improving the clarity of the manuscript.

We thank the reviewer for their concise summary of our manuscript and their assessment of our work as “convincing” and providing “detailed mechanistic insight.”

Comments

(1) It appears from reading the Materials and Methods that the SNPs used to measure crossing over were obtained by mating Hawaiian and Bristol strains. It is not clear to this reviewer how the SNPs were introduced into the animals. Was crossing over measured in a single animal line? Were the wild-type and pch-2 mutations made in backgrounds that were isogenic with respect to each other? This is a concern because it is not clear, at least to this reviewer, how much of an impact crossing different ecotypes will have on the frequency and distribution of recombination events (and possibly the recombination intermediates that were studied).

We will clarify these issues in the Materials and Methods of an updated preprint. The control and pch-2 mutants were isogenic in either the Bristol or Hawaiian backgrounds. Control lines were the original Bristol and Hawaiian lines and pch-2 mutants were originally made in the Bristol line and backcrossed at least 3 times before analysis. Hawaiian pch-2 mutants were made by backcrossing pch-2 mutants at least 7 times to the Hawaiian background and verifying the presence of Hawaiian SNPs on all chromosomes tested in the recombination assay. To perform the recombination assays, these isogenic lines were crossed to generate the relevant F1s.

(2) The authors state that in pch-2 mutants there was a striking shift of crossovers (line 135) to the PC end for all of the four chromosomes that were tested. I looked at Figure 1 for some time and felt that the results were more ambiguous. Map distances seemed similar at the PC end for wildtype and pch-2 on Chrom. I. While the decrease in crossing over in pch-2 appeared significant for Chrom. I and III, the results for Chrom. IV, and Chrom. X. seemed less clear. Were map distances compared statistically? At least for this reviewer the effects on specific intervals appear less clear and without a bit more detail on how the animals were constructed it's hard for me to follow these conclusions.

We hope that the added details above makes the results of these assays more clear. Map distances were compared and did not satisfy statistical significance, except where indicated. While we agree that the comparisons between control animals and pch-2 mutants may seem less clear with individual chromosomes, we argue that more general patterns become clear when analyzing multiple chromosomes. Indeed, this is why we expanded our recombination analysis beyond Chromosome III and the X Chromosomes, as reported in Deshong, 2014.

(3) Figure 2. I'm curious why non-irradiated controls were not tested side-by-side for COSA-1 staining. It just seems like a nice control that would strengthen the authors' arguments.

We will add these controls in the updated preprint.

(4) Figure 3. It took me a while to follow the connection between the COSA-1 staining and DAPI staining panels (12 hrs later). Perhaps an arrow that connects each set of time points between the panels or just a single title on the X-axis that links the two would make things clearer.

We will make changes in the updated preprint to make this figure more clear.

Reviewer #2 (Public review):

Summary:

This paper has some intriguing data regarding the different potential roles of Pch-2 in ensuring crossing over. In particular, the alterations in crossover distribution and Msh-5 foci are compelling. My main issue is that some of the models are confusingly presented and would benefit from some reframing. The role of Pch-2 across organisms has been difficult to determine, the ability to separate pairing and synapsis roles in worms provides a great advantage for this paper.

Strengths:

Beautiful genetic data, clearly made figures. Great system for studying the role of Pch-2 in crossing over.

We thank the reviewers for their constructive and useful summary of our manuscript and the analysis of its strengths.

Weaknesses:

(1) For a general audience, definitions of crossover assurance, crossover eligible intermediates, and crossover designation would be helpful. This applies to both the proposed molecular model and the cytological manifestation that is being scored specifically in C. elegans.

We will make these changes in an updated preprint.

(2) Line 62: Is there evidence that DSBs are introduced gradually throughout the early prophase? Please provide references.

We will reference Woglar and Villeneuve 2018 and Joshi et. al. 2015 to support this statement in the updated preprint.

(3) Do double crossovers show strong interference in worms? Given that the PC is at the ends of chromosomes don't you expect double crossovers to be near the chromosome ends and thus the PC?

Despite their rarity, double crossovers do show interference in worms. However, the PC is limited to one end of the chromosome. Therefore, even if interference ensures the spacing of these double crossovers, the preponderance of one of these crossovers toward one end (and not both ends) suggest something functionally unique about the PC end.

(4) Line 155 - if the previous data in Deshong et al is helpful it would be useful to briefly describe it and how the experimental caveats led to misinterpretation (or state that further investigation suggests a different model etc.). Many readers are unlikely to look up the paper to find out what this means.

We will add this to the updated preprint.

(5) Line 248: I am confused by the meaning of crossover assurance here - you see no difference in the average number of COSA-1 foci in Pch-2 vs. wt at any time point. Is it the increase in cells with >6 COSA-1 foci that shows a loss of crossover assurance? That is the only thing that shows a significant difference (at the one time point) in COSA-1 foci. The number of dapi bodies shows the loss of Pch-2 increases crossover assurance (fewer cells with unattached homologs). So this part is confusing to me. How does reliably detecting foci vs. DAPI bodies explain this?

We apologize for the confusion and will make this more clear in an updated perprint. The reviewer is correct that we do not see a difference in the average number of GFP::COSA1 foci at all time points in this experiment, even though we do see a difference in the number of DAPI stained bodies (an increase in crossover assurance in pch-2 mutants). What we meant to convey is that because of PCH-2’s dual role in regulating crossover formation (inhibiting it in early prophase, guaranteeing assurance later), the average number of GFP::COSA-1 foci at all time points also reflects this later role, resulting in this average being lower than if PCH-2 only inhibited crossovers early in meiotic prophase. We have shown that this later role does not significantly affect the average number of DAPI stained bodies, allowing us to see the role of PCH-2 in early meiotic prophase on crossover formation more clearly.

(6) Line 384: I am confused. I understand that in the dsb-2/pch2 mutant there are fewer COSA-1 foci. So fewer crossovers are designated when DSBs are reduced in the absence of PCH-2.

How then does this suggest that PCH-2's presence on the SC prevents crossover designation? Its absence is preventing crossover designation at least in the dsb-2 mutant.

We will also make this more clear in an updated preprint, as well as provide additional evidence to support this claim. In this experiment, we had identified three possible explanations for why PCH-2 persists on some nuclei that do not have GFP::COSA-1 foci: 1) PCH-2 removal is coincident with crossover designation; 2) PCH-2 removal depends on crossover designation; and 3) PCH-2 removal facilitates crossover designation. The decrease in the number of GFP::COSA-1 foci in dsb-2::AID;pch-2 mutants argues against the first two possibilities, suggesting that the third might be correct. We have additional evidence that we will include in an updated preprint that should provide stronger support and make this more clear.

(7) Discussion Line 535: How do you know that the crossovers that form near the PCs are Class II and not the other way around? Perhaps early forming Class I crossovers give time for a second Class II crossover to form. In budding yeast, it is thought that synapsis initiation sites are likely sites of crossover designation and class I crossing over. Also, the precursors that form class I and II crossovers may be the same or highly similar to each other, such that Pch-2's actions could equally affect both pathways.

We do not know that the crossovers that form near the PC are Class II but hypothesize that they are based on the close, functional relationship that exists between Class I crossovers and synapsis and the apparent antagonistic relationship that exists between Class II crossovers and synapsis. We agree that Class I and Class II crossover precursors are likely to be the same or highly similar, exhibit extensive crosstalk that may complicate straightforward analysis and PCH-2 is likely to affect both, as strongly suggested by our GFP::MSH-5 analysis. We present this hypothesis based on the apparent relationship between PCH-2 and synapsis in several systems but agree that it needs to be formally tested. We will make this argument more clear in an updated preprint.

Reviewer #3 (Public review):

Summary:

This manuscript describes an in-depth analysis of the effect of the AAA+ ATPase PCH-2 on meiotic crossover formation in C. elegant. The authors reach several conclusions, and attempt to synthesize a 'universal' framework for the role of this factor in eukaryotic meiosis.

Strengths:

The manuscript makes use of the advantages of the 'conveyor' belt system within the c.elegans reproductive tract, to enable a series of elegant genetic experiments.

We thank this reviewer for the useful assessment of our manuscript and the articulation of its strengths.

Weaknesses:

A weakness of this manuscript is that it heavily relies on certain genetic/cell biological assays that can report on distinct crossover outcomes, without clear and directed control over other aspects and variables that might also impact the final repair outcome. Such assays are currently out of reach in this model system.

In general, this manuscript could be more generally accessible to non-C.elegans readers. Currently, the manuscript is hard to digest for non-experts (even if meiosis researchers). In addition, the authors should be careful to consider alternative explanations for certain results. At several steps in the manuscript, results could ostensibly be caused by underlying defects that are currently unknown (for example, can we know for sure that pch-2 mutants do not suffer from altered DSB patterning, and how can we know what the exact functional and genetic interactions between pch-2 and HORMAD mutants tell us?). Alternative explanations are possible and it would serve the reader well to explicitly name and explain these options throughout the manuscript.

We will make the manuscript more accessible to non-C. elegans readers and discuss alternate explanations for specific results in an updated preprint.