Jmjd1c is dispensable for healthy adult hematopoiesis and Jak2V617F-driven myeloproliferative disease initiation in mice

Hans F. Staehle; Johannes Heinemann; Albert Gruender; Anne M. Omlor; Heike Luise Pahl; Jonas Samuel Jutzi

doi:10.1371/journal.pone.0228362

Peer Review History

Original SubmissionNovember 8, 2019
3 Dec 2019 Decision Letter - Kevin D Bunting, Editor PONE-D-19-29589 Jmjd1c is dispensable for healthy adult hematopoiesis and Jak2V617F-driven myeloproliferative disease initiation in mice PLOS ONE Dear Dr. Jutzi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ============================== Please add any additional details regarding the hematopoietic compartment of these knockout mice as suggested by the reviewer. ============================== We would appreciate receiving your revised manuscript by Jan 17 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 3. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In this manuscript, Staehle et al. investigated JMJD1C’s role in steady state hematopoiesis and JAK2V617F driven MPN. This study stems from their previous finding that JMJD1C is a target of NFE2, a transcription factor important for MPN, and acts in positive feedback loop to promote NFE overexpression in MPNs. The authors did a thorough validation of genetic Jmjd1c mouse model used. Their study showed that JMJD1C is dispensable for steady state hematopoiesis using a whole body knockout, largely similar to results from a previous published one. They further characterized the expression pattern of JMJD1C during embryogenesis. Moreover, they showed that JMJD1C loss is dispensable for JAK2V617F driven MPN. This study collaborated previous published hematopoietic phenotype of Jmjd1c knockout using a different Cre, expanded our understanding of the role of JMJD1C during early development and its role in MPN development. Overall, the results are supported by experiments that are well designed and performed with rigorous statistics. Major points 1. Have the authors examined whether there is any bone marrow cellularity changes in Jmjd1cd/d mice compare to Jmjd1c+/+ mice as shown by the previous publication using Vav1Cre? If so, are there any changes in absolute cell numbers of mature and HSPC compartments in the bone marrow? 2. As the authors pointed out in the Discussion section, it is possible that adaptation to JMJD1C loss may explain the lack of phenotype upon JAK2V617F induction in JMJD1C null background. It still remains possible that JMJD1C is required for the maintenance but not initiation of MPN. Along this line, no effect was observed on MLL-AF9 leukemia in a Jmjd1cf/f Vav1Cre background in the previous study. Have the authors tried to use conditional rather than null allele of Jmjd1c to address this possibility? 3. Both Jak2V617F and Jmjd1c deletion by themselves have been shown to reduce BM cellularity. Are there any changes in bone marrow cellularity in Jak2V617FJmjd1cd/d compare to Jak2V617F mice? If so, what are the changes in absolute cell numbers of mature and HSPC compartments? Minor points 1. Line 92-93, “shRNA-mediated JMJD1C depletion..”: reference cited used genetic model not shRNA. ****** 6. 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Revision 1
10 Jan 2020 Author Response To Prof. Dr. Kevin Bunting PLOS ONE Boston, January 9th, 2020 Dear Prof. Bunting, dear Kevin, Thank you for reviewing our manuscript entitled “Jmjd1c is dispensable for healthy adult hematopoiesis and Jak2V617F-driven myeloproliferative disease initiation in mice”. We addressed the reviewer’s comments point-by-point, see responses below. Moreover, in line with PLOS ONE’s journal requirements and guidelines, we included some additional information, including all original gel pictures into the revised manuscript. Reviewer’s comments: In this manuscript, Staehle et al. investigated JMJD1C’s role in steady state hematopoiesis and JAK2V617F driven MPN. This study stems from their previous finding that JMJD1C is a target of NFE2, a transcription factor important for MPN, and acts in positive feedback loop to promote NFE overexpression in MPNs. The authors did a thorough validation of genetic Jmjd1c mouse model used. Their study showed that JMJD1C is dispensable for steady state hematopoiesis using a whole body knockout, largely similar to results from a previous published one. They further characterized the expression pattern of JMJD1C during embryogenesis. Moreover, they showed that JMJD1C loss is dispensable for JAK2V617F driven MPN. This study collaborated previous published hematopoietic phenotype of Jmjd1c knockout using a different Cre, expanded our understanding of the role of JMJD1C during early development and its role in MPN development. Overall, the results are supported by experiments that are well designed and performed with rigorous statistics. Major points: 1. Have the authors examined whether there is any bone marrow cellularity changes in Jmjd1cd/d mice compare to Jmjd1c+/+ mice as shown by the previous publication using Vav1Cre? If so, are there any changes in absolute cell numbers of mature and HSPC compartments in the bone marrow? Response to 1.: Indeed, we examined the total bone marrow (BM) cellularity in the Jmjd1c d/d mice and compared it to Jmjd1c +/+ mice. We now include these data as supplemental figure 7A in the revised manuscript. There was no statistically significant difference in BM cellularity. We did not examine cellularity of sub-compartments of HSPCs. 2. As the authors pointed out in the Discussion section, it is possible that adaptation to JMJD1C loss may explain the lack of phenotype upon JAK2V617F induction in JMJD1C null background. It still remains possible that JMJD1C is required for the maintenance but not initiation of MPN. Along this line, no effect was observed on MLL-AF9 leukemia in a Jmjd1cf/f Vav1Cre background in the previous study. Have the authors tried to use conditional rather than null allele of Jmjd1c to address this possibility? Response to 2.: Thank you for this question. Indeed, while we examined the diseases initiation process by Jak2V617F induction in the Jmjd1c d/d background, we have not knocked out Jmjd1c in an already established Jak2V617F-driven disease background. We established a constitutive Jak2V617F model which we planned to use for crossing with the inducible Jmjd1c ko mouse model. While we obtained viable Jak2V617F offspring, it was almost impossible to maintain the line itself due to fertility problems of the females. Crossing them to Jmjd1c fl/fl mice proved unfeasible. It is known in literature and we have shown as well (Jutzi et al., HemaSphere, 2018) that floxed Jak2V617F mice in an Mx1Cre background already show a full-blown phenotype without previous induction, probably due to a pro-inflammatory feed-forward activation of the Mx promoter, followed by cre recombinase expression. This knowledge thwarted our alternative approach of using Jmjd1 fl/fl BM in a retroviral Jak2V617F model followed by pi:pC induction as it would not be a clean model to study disease maintenance. The leakiness would cause Jmjd1c excision by the cre recombinase already prior to retroviral infection with the Jak2 mutation. We therefore focused on disease initiation entirely. 3. Both Jak2V617F and Jmjd1c deletion by themselves have been shown to reduce BM cellularity. Are there any changes in bone marrow cellularity in Jak2V617FJmjd1cd/d compare to Jak2V617F mice? If so, what are the changes in absolute cell numbers of mature and HSPC compartments? Response to 3.: We have determined the total BM cellularity of both genotypes and have not found a statistically significant difference between Jak2V617F and Jak2 V617F Jmjd1c d/d mice. We have not further subdivided the examined cellularity into the HSPC sub-compartments. We included the data in the revised manuscript as supplemental figure 7B. Minor points: 1. Line 92-93, “shRNA-mediated JMJD1C depletion...”: reference cited used genetic model not shRNA. Thank you for pointing out this error. Indeed, it is of course a genetic deletion of Jmjd1c with a comparable approach as ours. We have corrected the paragraph in the introduction in the revised manuscript and apologize for the mistake made. Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response to 1.: We have now addressed all the PLOS ONE style requirements, in the cases where we hadn’t addressed it before (e.g. title page). 2. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. Response to 2.: In line with PLOS ONE guidelines, we expanded the statistical section in the methods part of the manuscript. 3. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. Response to 3.: We now included the data on beta-galactosidase staining previously referred to as “data not shown” in the revised manuscript: We included a “negative control” for the staining in supplemental figure 6E to show that there is background activity in the intestine at time point E17.5. Missing data points: The whole study took us more than 12 months after establishing the mouse lines. This is due to the size of the cohort and expected Mendelian ratios as well as the long follow-up of up to 40 weeks. In light of that, we were not able to obtain all data points from all mice at all time points, mostly due to technical errors or premature deaths of single mice. In the revised manuscript, we now include a supplemental table 2 that lists all lost data points and the specific reasons for it. 4. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Response to 4.: Following the guidelines, we included original gel pictures for all gels included in the main figures, into a new supplemental figure 4 (S4 Fig.). Because of this, there is a shift in the numbering of the originally submitted supplemental figures. All changes are highlighted in red. All figures meet the journal’s requirement now, including visualization of individual data points (figure 4 C-E, G and figure 5A-J). Important to note: The western blot membrane used for experiments depicted in figure 3B and revised supplemental figure 4 D+E has been previously used for unrelated experiments, in which it was also probed with the JMJD1C antibody. The lanes labeled “unrelated experiments” show JMJD1C and actin bands of samples used for a different publication from our lab: Peeken et al., Blood, 2018. Additional changes and remarks by the authors: Copy and paste error in figure 5I: During the process of harmonizing our data to the journal’s requirement, meaning showing individual data points, we realized an error in the uploaded figure 5. While the content of the results section and the figure legend was correct, a copy and paste error occurred and figure 5H was shown twice. We have now replaced figure 5I with the originally intended data showing erythropoietic cells defined by flow in the spleen of Jak2V617F and Jak2V617F Jmjd1cd/d mice. We would like to apologize for this mistake! Spleen weight of Jmjd1c d/d: Using the Mann-Whitney U test, the spleen weights of Jmjd1c d/d are indeed statistically significantly lower when compared to WT littermate controls. However, we failed to mark the statistical significance in the initially submitted manuscript. We sincerely apologize for this mistake. The revised figure 4G contains the asterisk to indicate a p value of <.05. We inserted a corrected statement into the results section, highlighted in red. However, this does not change the conclusion that we find only minor differences with questionable biological implications. We hope that with these changes made and additional data included, you consider our manuscript worthy of publication in PLOS ONE. Thank you for your time and effort in reviewing the manuscript. Sincerely, Jonas S. Jutzi, MD, PhD Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0228362.r002
14 Jan 2020 Decision Letter - Kevin D Bunting, Editor Jmjd1c is dispensable for healthy adult hematopoiesis and Jak2V617F-driven myeloproliferative disease initiation in mice PONE-D-19-29589R1 Dear Dr. Jutzi, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Kevin D Bunting Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0228362.r003
Formally Accepted
15 Jan 2020 Acceptance Letter - Kevin D Bunting, Editor PONE-D-19-29589R1 Jmjd1c is dispensable for healthy adult hematopoiesis and Jak2^V617F-driven myeloproliferative disease initiation in mice Dear Dr. Jutzi: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Kevin D Bunting Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228362.r004

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