Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation

Petri Rummukainen; Kati Tarkkonen; Amel Dudakovic; Rana Al-Majidi; Vappu Nieminen-Pihala; Cristina Valensisi; R. David Hawkins; Andre J. van Wijnen; Riku Kiviranta

doi:10.1371/journal.pone.0265027

Peer Review History

Original SubmissionDecember 1, 2021
7 Jan 2022 Decision Letter - Dengshun Miao, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-21-38069Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiationPLOS ONE Dear Dr. Rummukainen, 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 submit your revised manuscript by Feb 21 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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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. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 study, they examined roles of LSD1 in the osteoblast phenotypes in vitro studies using MC3T3-E1 cells and in vivo mouse study. They showed that LSD1 inhibition suppresses the expression of osteogenic genes, ALP statin and mineralization in MC3T3-E1 cells. Moreover, they confirmed the roles of LSD1 using Prrx1-Cre LSD1 deleted mice. Moreover, they presented some evidence of the epigenetic regulation of LSD1 in osteoblast differentiation. They presented enough detailed data supporting their conclusion, which will result in the addition and extension of previously reported studies. The manuscript and figures are generally well prepared. There are a few minor points, which might be addressed. 1. In some figures, number of samples and significant asterisks are not adequately shown, which should be added carefully. 2. The origin of MC3T3-E1 cells should be written clearly. 3. Re: Line 359, Data should be shown in supplemental figure. 4. BMD data could be added in Figures 3 and 4. 5. Some syntactic errors of English should be carefully corrected. For example, the second sentence of Abstract. Reviewer #2: Petri Rummukainen et al. investigate the function of lysine-specific demethylase 1 (LSD1) in osteoblast differentiation. Mechanistically, they find Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 that has an essential role for bone formation. The experiments are decently performed, and the conclusions are solid. I only have a few comments below. 1. Fig 1: Western blot assay will be needed to show the protein levels of LSD1 during osteoblast differentiation. 2. Fig 1 and Fig 2: The bar figures should be correctly labeled if they have statistically significance. 3. Fig 3F: a representative image should be provided, as calcein double-labelling assay is used to be measured the distance between the two dyes of the indicated time. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. 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Revision 1
17 Feb 2022 Author Response PONE-D-21-38069 Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation PLOS ONE We would like to thank the editor and reviewers for the peer-review of our manuscript as well as excellent comments and suggestions. We have carefully reviewed the editorial as well as reviewers’ comments on our manuscript, performed additional analysis and modified the manuscript accordingly to address these important points raised. Please find our detailed, point to point responses below. Journal Requirements: 1. 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 https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Thank you for this point. We have thoroughly gone through the style requirements and edited the manuscript accordingly. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This study was funded by the Academy of Finland (298625, 268535, and 139165 to RK), as well as foundation grants (Emil Aaltonen Foundation, Sigrid Juselius Foundation and Finnish Cultural Foundation). Support was also provided by the National Institutes of Health (R01-AR09069 to AJvW). " We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "RK: 298625, Academy of Finland, https://www.aka.fi/ RK: 268535, Academy of Finland, https://www.aka.fi/ RK: 139165, Academy of Finland, https://www.aka.fi/ AvW: R01-AR09069, National Institutes of Health, https://www.nih.gov/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Thank you for this valuable correction. We have removed the funding information from the manuscript and would like to edit the Funding Statement to following: "RK: 298625, Academy of Finland, https://www.aka.fi/ RK: 268535, Academy of Finland, https://www.aka.fi/ RK: 139165, Academy of Finland, https://www.aka.fi/ KT: 275001, Academy of Finland, https://www.aka.fi/ AvW: R01-AR09069, National Institutes of Health, https://www.nih.gov/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation 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. We have edited the manuscript accordingly, complementing this manuscript with one new supplemental figure (now S1 Figure) to bring forward this additional data as well as removing one reference to unshown data. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Captions for Supporting Information files have been moved to the end of the manuscript from their previous locations. 5. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Thank you for this important note. Reference list has been reviewed and analyzed by scite.ai tool, which determines whether or not the references listed have received any editorial notices. Of the 95 references in this manuscript, 3 articles referred had been corrected or amended post the original publication: 40. Ye L, Fan Z, Yu B, Chang J, Al Hezaimi K, Zhou X, et al. Histone demethylases KDM4B and KDM6B promotes osteogenic differentiation of human MSCs. Cell Stem Cell. 2012 Jul;11(1):50–61: A duplicated reference photo had been replaced with the correct images, not impacting the interpretation of the results. 43. Whyte WA, Bilodeau S, Orlando DA, Hoke HA, Frampton GM, Foster CT, et al. Enhancer decommissioning by LSD1 during embryonic stem cell differentiation. Nature. 2012 Feb;482(7384):221–5: A duplicated western blot image was corrected to represent the actual images, not altering the analyzed tubulin level. Also black lines were added for extra clarity on lane recognition. 81. Yu Y, Schleich K, Yue B, Ji S, Lohneis P, Kemper K, et al. Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma. Cancer Cell [Internet]. 2018 Feb 12;33(2):322-336.e8: The name of author Mark R. Silvis’s name was spelled incorrectly as Mark S. Silvis. Other references did not flag any editorial concerns in the scite.ai analysis and the corrections in these mentioned articles do not impact the value of their science for this manuscript. Reviewer #1: In this study, they examined roles of LSD1 in the osteoblast phenotypes in vitro studies using MC3T3-E1 cells and in vivo mouse study. They showed that LSD1 inhibition suppresses the expression of osteogenic genes, ALP statin and mineralization in MC3T3-E1 cells. Moreover, they confirmed the roles of LSD1 using Prrx1-Cre LSD1 deleted mice. Moreover, they presented some evidence of the epigenetic regulation of LSD1 in osteoblast differentiation. They presented enough detailed data supporting their conclusion, which will result in the addition and extension of previously reported studies. The manuscript and figures are generally well prepared. There are a few minor points, which might be addressed. 1. In some figures, number of samples and significant asterisks are not adequately shown, which should be added carefully. We warmly thank the reviewer for these comments. The number of samples and significances have been added where missing and applicable. 2. The origin of MC3T3-E1 cells should be written clearly. Thank you for this important point. The origin of MC3T3-E1 cells has been added to material and methods: Mouse MC3T3-E1 cells were purchased from ATCC (MC3T3-E1 Subclone 4, CRL-2593) and maintained in �MEM supplemented with 100 U/ml penicillin-streptomycin, 10% fetal calf serum and 2 mM L-glutamine (maintenance medium). 3. Re: Line 359, Data should be shown in supplemental figure. We thank the reviewer for this comment and this information has been added to the new S1 figure. 4. BMD data could be added in Figures 3 and 4. Cortical BMD data is available in Figures 3 and 4 labeled “uCT cort BMD”. BMD of trabecular bone, often reported as tissue mineral density or TMD is not reported here as it is not a part of our regular analysis pattern due to the difficulties of defining a ROI in such a multifaceted surface and higher structural variability. 5. Some syntactic errors of English should be carefully corrected. For example, the second sentence of Abstract. Attention has been directed to syntactic errors in this article. Fixed second sentence of abstract to “Histone methylation is controlled by multiple lysine demethylases and is an important step in controlling local chromatin structure and gene expression.” Reviewer #2: Petri Rummukainen et al. investigate the function of lysine-specific demethylase 1 (LSD1) in osteoblast differentiation. Mechanistically, they find Lsd1 occupies Runx2-binding cites at H3K4me2 and H3K4me3 that has an essential role for bone formation. The experiments are decently performed, and the conclusions are solid. I only have a few comments below. 1. Fig 1: Western blot assay will be needed to show the protein levels of LSD1 during osteoblast differentiation. We thank the reviewer for their important comments on this paper. In the previous version of this manuscript the LSD1 expression was shown only on mRNA level using both RNA-seq and qPCR level due to the abundant RNA expression levels as well as difficulties of extracting proteins from differentiated osteoblast cultures where extracellular matrix formation is extensive. Data has now been added to the new S1 figure. 2. Fig 1 and Fig 2: The bar figures should be correctly labeled if they have statistically significance. We thank the reviewer for this important point. Labels for statistical significance has been added to figures 1 and 2. 3. Fig 3F: a representative image should be provided, as calcein double-labelling assay is used to be measured the distance between the two dyes of the indicated time. Thank you for this good point. The images chosen to Figure 3F represent accurately the label presence in these samples. In all of our samples the presence of double labeling is confirmed from the cortical bone before analysis. Our current microscope setup does not accurately visualize the different colors of labels where double labels are present, where calcein label is dominantly green but demeclocycline also appears as a milder green label. To accurately measure both single- and double labeling in these samples, we used an alternative filter to visualize only demeclocycline and then combine this information to the calcein label data to allow for an accurate, precise analysis. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0265027.r002
21 Feb 2022 Decision Letter - Dengshun Miao, Editor Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation PONE-D-21-38069R1 Dear Dr. Rummukainen, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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. Kind regards, Dengshun Miao Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0265027.r003
Formally Accepted
24 Feb 2022 Acceptance Letter - Dengshun Miao, Editor PONE-D-21-38069R1 Lysine-Specific Demethylase 1 (LSD1) epigenetically controls osteoblast differentiation Dear Dr. Rummukainen: I'm 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 let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Dengshun Miao Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265027.r004

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