Phosphoproteomics of ATR signaling in mouse testes
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Evaluation Summary:
This study describes a phosphoproteomic analysis of the ATR kinase signaling pathway in mouse testis. The study is well designed and performed, the manuscript is properly constructed and written, and the conclusions are supported by the data. The phosphoproteomic data obtained will be very useful resource for the DNA repair, meiosis, and reproductive biology communities studying the roles of the ATR-dependent DNA damage response pathway.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
The phosphatidylinositol 3′ kinase (PI3K)‐related kinase ATR is crucial for mammalian meiosis. ATR promotes meiotic progression by coordinating key events in DNA repair, meiotic sex chromosome inactivation (MSCI), and checkpoint-dependent quality control during meiotic prophase I. Despite its central roles in meiosis, the ATR-dependent meiotic signaling network remains largely unknown. Here, we used phosphoproteomics to define ATR signaling events in testes from mice following chemical and genetic ablation of ATR signaling. Quantitative analysis of phosphoproteomes obtained after germ cell-specific genetic ablation of the ATR activating 9-1-1 complex or treatment with ATR inhibitor identified over 14,000 phosphorylation sites from testes samples, of which 401 phosphorylation sites were found to be dependent on both the 9-1-1 complex and ATR. Our analyses identified ATR-dependent phosphorylation events in crucial DNA damage signaling and DNA repair proteins including TOPBP1, SMC3, MDC1, RAD50, and SLX4. Importantly, we identified ATR and RAD1-dependent phosphorylation events in proteins involved in mRNA regulatory processes, including SETX and RANBP3, whose localization to the sex body was lost upon ATR inhibition. In addition to identifying the expected ATR-targeted S/T-Q motif, we identified enrichment of an S/T-P-X-K motif in the set of ATR-dependent events, suggesting that ATR promotes signaling via proline-directed kinase(s) during meiosis. Indeed, we found that ATR signaling is important for the proper localization of CDK2 in spermatocytes. Overall, our analysis establishes a map of ATR signaling in mouse testes and highlights potential meiotic-specific actions of ATR during prophase I progression.
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Author Response:
Reviewer #2 (Public Review):
The authors try to identify ATR-mediated phosphorylation sites in male meiosis of mice and performed phosphoproteomics using two distinct mouse models. The paper focuses on important topics in the field. Since ATR has key functions in meiosis, successful identification of ATR-mediated phosphorylation sites would have a profound impact.
The study has certain technical issues in experimental design and data interpretations.
The rationale as to why they used Rad1-cKO was not well described. According to the co-submitted manuscript, Rad1-cKO spermatocytes experience meiotic arrest, and the cellular composition is totally different between controls and Rad1-cKO testes. The "RAD1-dependent" phenotype may simply reflect the difference in cellular composition in testis. With this criterion, …
Author Response:
Reviewer #2 (Public Review):
The authors try to identify ATR-mediated phosphorylation sites in male meiosis of mice and performed phosphoproteomics using two distinct mouse models. The paper focuses on important topics in the field. Since ATR has key functions in meiosis, successful identification of ATR-mediated phosphorylation sites would have a profound impact.
The study has certain technical issues in experimental design and data interpretations.
The rationale as to why they used Rad1-cKO was not well described. According to the co-submitted manuscript, Rad1-cKO spermatocytes experience meiotic arrest, and the cellular composition is totally different between controls and Rad1-cKO testes. The "RAD1-dependent" phenotype may simply reflect the difference in cellular composition in testis. With this criterion, any phosphorylation sites present after the mid-pachytene stage in normal spermatogenesis can be categorized as "RAD1-dependent".
We have altered the figure and text in the manuscript to more clearly explain the rationale for using Rad1-cKO and combining the generated data with the data from the rapid 4 hour ATRi treatment. Importantly, we now consider the phosphorylation sites impaired after a quick 4 hour treatment with ATRi (New Supplementary File 1), which is expected to be too quick to induce an appreciable pachytene arrest. Therefore, the final ATR-dependent and RAD1-dependent dataset is unlikely to include phosphorylation sites that are only shown as being depleted due to a persistent mid-pachytene arrest (these sites should appear as RAD1-dependent and ATR-independent).
There are two different experiments for ATR inhibitor (ATRi)-treated mice (2 pairs after 2.5-3 days of treatment, and 2 pairs 4 hours after a single dose). However, these results are not distinguished in the analysis, and there is no evaluation of testicular morphology after ATRi treatment.
We addressed the point of separating the data from 4 hour and 2-3 days of treatment. We also have now also addressed testicular morphology after 4 hour ATRi treatment and did not observe any defect (new Figure 5-figure supplement 3A-B).
Finally, the authors showed ATR-dependent localization of SETX and RANBP3 and discussed interesting data. However, it has not been determined whether these localization changes were due to the functions of identified phosphorylation sites or some other mechanisms.
We agree with the reviewer that it would be very interesting to address the role of specific phosphorylation sites in SETX and RANBP3. However, we feel this would require significantly additional effort and time, which would not be realistic in the current manuscript, and is beyond the scope of this resource paper.
Reviewer #3 (Public Review):
In this study, Sims et al. perform a phosphoproteomic analysis of the ATR signaling pathway in mouse testis. By studying the different phosphorylated peptides found in testis samples from ATR inhibited mice and from mutant mice for the member of the ATR-activating 9-1-1 complex, RAD1, authors defined a comprehensive map of the ATR signaling pathway in the mouse testis. In general, the methodological approach performed is appropriate to accomplish the desired goal and the results obtained are well explained and properly discussed. The conclusions raised by the authors are supported by the results obtained and the manuscript reads easily. Thus, overall the manuscript is of high quality. Furthermore, the information provided in this study is novel since to my knowledge this is the first attempt to characterize the ATR signaling pathway in the testis. In my opinion, these data will be very relevant to better understand the role of the ATR in mouse spermatogenesis, and in meiosis in particular, in the future.
Thank you, we appreciate the positive remarks.
Nonetheless, I have a few major concerns about this manuscript. Firstly, I think an important part of the description of the results is placed in a related preprint by the authors (Pereira et al. https://www.biorxiv.org/content/10.1101/2021.04.09.439198v1). In my opinion, this manuscript lacks a more detailed analysis of the ATR signaling on DNA repair and chromosome axis structure, which are fundamental to understand the meiotic prophase. Secondly, the manuscript falls short of providing novel insights about ATR roles during the meiotic prophase. As ATR function on the meiotic prophase has been extensively studied, the ATR phosphoproteome should provide either some clues about possible novel functions ATR may do during the meiotic prophase or spermatogenesis, or provide a mechanistic explanation of how ATR performs its meiotic functions (e.g., meiotic sex chromosome inactivation or meiotic recombination). The final section of the results is an attempt at doing sol, but to me, the data provided only suppose a small incremental advance in our knowledge of how ATR promotes MSCI. I would have liked the authors to expand this section to prove the utility of the data.
We agree with the reviewer that it would be very interesting to address more details of the roles of ATR in meiosis and the underlying molecular mechanisms. However, we feel this would require significantly additional effort and time, which would not be realistic in the current manuscript, and is beyond the scope of this resource paper. We note that the revised version of the manuscript now reports the exciting finding that ATR is important for the proper localization of CDK2 in meiotic spreads. While the details and mechanisms remain unknown, we believe this finding, together with other reported findings in this resource paper, open new directions to study meiotic ATR signaling.
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Evaluation Summary:
This study describes a phosphoproteomic analysis of the ATR kinase signaling pathway in mouse testis. The study is well designed and performed, the manuscript is properly constructed and written, and the conclusions are supported by the data. The phosphoproteomic data obtained will be very useful resource for the DNA repair, meiosis, and reproductive biology communities studying the roles of the ATR-dependent DNA damage response pathway.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The paper by Sim et al describes phospho-proteomic analysis of ATR kinase-dependent pathway in mouse spermatocytes. By administrating an ATR inhibitor, AZ20, to mice and using Rad1 (a component of 911 DNA damage clamp) conditional knockout mouse (cKO), the authors isolated testis from these mice, isolated phospho-peptides and analyzed with Tandem Mass Tag (TMT). The analyses identified 37,180 phosphorylation sites and created the data base for them. Importantly, in-depth analysis of the phosphorylation sites revealed an unique consensus site of the ATR-dependent phosphorylation; S/TPXK, whose kinase has not been identified yet. In addition, the authors showed new ATR-dependent phosphorylation sites in proteins in RNA metabolisms including piRNA biogenesis for transposon silencing and showed ATR-dependent …
Reviewer #1 (Public Review):
The paper by Sim et al describes phospho-proteomic analysis of ATR kinase-dependent pathway in mouse spermatocytes. By administrating an ATR inhibitor, AZ20, to mice and using Rad1 (a component of 911 DNA damage clamp) conditional knockout mouse (cKO), the authors isolated testis from these mice, isolated phospho-peptides and analyzed with Tandem Mass Tag (TMT). The analyses identified 37,180 phosphorylation sites and created the data base for them. Importantly, in-depth analysis of the phosphorylation sites revealed an unique consensus site of the ATR-dependent phosphorylation; S/TPXK, whose kinase has not been identified yet. In addition, the authors showed new ATR-dependent phosphorylation sites in proteins in RNA metabolisms including piRNA biogenesis for transposon silencing and showed ATR-dependent localization of two RNA processing enzymes, SETX helicase, and RANBP3 in meiosis sex chromosome inactivation (MSCI). This is an important body of work as a good resource for phosphorylation in mouse germ cells. The study had been done in a great care with proper controls. The data set in the paper is very much useful and of great interest to researchers in meiosis and DNA damage response (DDR) field.
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Reviewer #2 (Public Review):
The authors try to identify ATR-mediated phosphorylation sites in male meiosis of mice and performed phosphoproteomics using two distinct mouse models. The paper focuses on important topics in the field. Since ATR has key functions in meiosis, successful identification of ATR-mediated phosphorylation sites would have a profound impact.
The study has certain technical issues in experimental design and data interpretations.
The rationale as to why they used Rad1-cKO was not well described. According to the co-submitted manuscript, Rad1-cKO spermatocytes experience meiotic arrest, and the cellular composition is totally different between controls and Rad1-cKO testes. The "RAD1-dependent" phenotype may simply reflect the difference in cellular composition in testis. With this criterion, any phosphorylation sites …
Reviewer #2 (Public Review):
The authors try to identify ATR-mediated phosphorylation sites in male meiosis of mice and performed phosphoproteomics using two distinct mouse models. The paper focuses on important topics in the field. Since ATR has key functions in meiosis, successful identification of ATR-mediated phosphorylation sites would have a profound impact.
The study has certain technical issues in experimental design and data interpretations.
The rationale as to why they used Rad1-cKO was not well described. According to the co-submitted manuscript, Rad1-cKO spermatocytes experience meiotic arrest, and the cellular composition is totally different between controls and Rad1-cKO testes. The "RAD1-dependent" phenotype may simply reflect the difference in cellular composition in testis. With this criterion, any phosphorylation sites present after the mid-pachytene stage in normal spermatogenesis can be categorized as "RAD1-dependent".
There are two different experiments for ATR inhibitor (ATRi)-treated mice (2 pairs after 2.5-3 days of treatment, and 2 pairs 4 hours after a single dose). However, these results are not distinguished in the analysis, and there is no evaluation of testicular morphology after ATRi treatment.
Finally, the authors showed ATR-dependent localization of SETX and RANBP3 and discussed interesting data. However, it has not been determined whether these localization changes were due to the functions of identified phosphorylation sites or some other mechanisms.
-
Reviewer #3 (Public Review):
In this study, Sims et al. perform a phosphoproteomic analysis of the ATR signaling pathway in mouse testis. By studying the different phosphorylated peptides found in testis samples from ATR inhibited mice and from mutant mice for the member of the ATR-activating 9-1-1 complex, RAD1, authors defined a comprehensive map of the ATR signaling pathway in the mouse testis. In general, the methodological approach performed is appropriate to accomplish the desired goal and the results obtained are well explained and properly discussed. The conclusions raised by the authors are supported by the results obtained and the manuscript reads easily. Thus, overall the manuscript is of high quality. Furthermore, the information provided in this study is novel since to my knowledge this is the first attempt to characterize …
Reviewer #3 (Public Review):
In this study, Sims et al. perform a phosphoproteomic analysis of the ATR signaling pathway in mouse testis. By studying the different phosphorylated peptides found in testis samples from ATR inhibited mice and from mutant mice for the member of the ATR-activating 9-1-1 complex, RAD1, authors defined a comprehensive map of the ATR signaling pathway in the mouse testis. In general, the methodological approach performed is appropriate to accomplish the desired goal and the results obtained are well explained and properly discussed. The conclusions raised by the authors are supported by the results obtained and the manuscript reads easily. Thus, overall the manuscript is of high quality. Furthermore, the information provided in this study is novel since to my knowledge this is the first attempt to characterize the ATR signaling pathway in the testis. In my opinion, these data will be very relevant to better understand the role of the ATR in mouse spermatogenesis, and in meiosis in particular, in the future.
Nonetheless, I have a few major concerns about this manuscript. Firstly, I think an important part of the description of the results is placed in a related preprint by the authors (Pereira et al. https://www.biorxiv.org/content/10.1101/2021.04.09.439198v1). In my opinion, this manuscript lacks a more detailed analysis of the ATR signaling on DNA repair and chromosome axis structure, which are fundamental to understand the meiotic prophase. Secondly, the manuscript falls short of providing novel insights about ATR roles during the meiotic prophase. As ATR function on the meiotic prophase has been extensively studied, the ATR phosphoproteome should provide either some clues about possible novel functions ATR may do during the meiotic prophase or spermatogenesis, or provide a mechanistic explanation of how ATR performs its meiotic functions (e.g., meiotic sex chromosome inactivation or meiotic recombination). The final section of the results is an attempt at doing sol, but to me, the data provided only suppose a small incremental advance in our knowledge of how ATR promotes MSCI. I would have liked the authors to expand this section to prove the utility of the data.
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