Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production

Naoko Hirahara; Hiroko Misawa Nakamura; Shigekazu Sasaki; Akio Matsushita; Kenji Ohba; Go Kuroda; Yuki Sakai; Shinsuke Shinkai; Hiroshi Haeno; Takuhiro Nishio; Shuichi Yoshida; Yutaka Oki; Takafumi Suda

doi:10.1371/journal.pone.0227646

Peer Review History

Original SubmissionAugust 14, 2019
3 Oct 2019 Decision Letter - Hiroyoshi Ariga, Editor PONE-D-19-22945 Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for the thyrotropin production PLOS ONE Dear Sasaki, 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. We would appreciate receiving your revised manuscript by Nov 17 2019 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Hiroyoshi Ariga Academic Editor PLOS ONE Journal requierments 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 2. 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. 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. 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. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. 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: Hirahara et al studied GATA2 promoter function as well as expression of endogenous GATA2 in cultured cells. They demonstrated that GATA2 promoter is down regulated by T3 in the presence of T3Rß2. They also showed endogenous GATA2 is down regulated in LßT2 cells in a manner which is not inhibited by MG132. In general the experiments are well designed and quality of the data is good. This reviewer have one major comment and several minor. Major. Fig.2 T3 is added only in GATA2+ T3Rß2 cells. Effect of T3 should be analyzed (although the authors may have shown similar data in previous papers), in cells with vector only and GATA2 only. Minor. p. 3 L83, “has not performed” should be “has not been performed” p.6 L190, p.7 L198, 201, period is not required for “hr” or they should be replaced to “hrs”. L 222, same comment. L225-227, one closing bracket is lacking. p.9 284, “theoretically”?, shouldn’t it be “experimentally”? Please rephrase the sentence. Also this reviewer would like to confirm the authors whether “no study showed that liganded TR functioned as a transcriptional repressor”, including the authors’ preceding studies. Or, these statements should be moved to after the sentence in L290-295, to make sense. p.11 L344 “ as log scale” should be “ in a log scale”. As it appears that articles are lacking or inappropriate here and there throughout the manuscript, further English editing is recommended. p.14, l430 to later, “Several mechanisms” should be explained more clearly. The sentences following “ first, seconde,…” should simply state the mechanism, but not the reasoning. For an example, “T3 may destabilize TSH beta mRNA” should follow immediately after “Third”…something like that. Employing LßT2 cells cannot be one of the additional mechanisms. ****** 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. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227646.r001
Revision 1
23 Nov 2019 Author Response REFERENCE: PONE-D-19-22945 Title: "Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production” AUTHORS: Naoko Hirahara and Hiroko Misawa Nakamura et al. PLOS ONE Academic Editor: Hiroyoshi Ariga Please re-evaluate our revised manuscript. Although our previous manuscript was screened once by a native English speaker before first submission, the referee recommended us to edit it again regarding in particular to the usage of articles. So we asked another (more experienced) person to re-check it. Based on his recommendation, we deleted “the” from “the thyrotropin production” in the title. If it can be now acceptable in your journal, we would be very much pleased. Thank you for your kind arrangement. Shigekazu Sasaki. MD. Senior Assistant Professor Second Division of Internal Medicine Hamamatsu University School of Medicine 1-20-1 Handayama Higashi-ku Hamamatsu 431-3192, Japan E-mail: sasakis@hama-med.ac.jp RESPONSE TO THE COMMENTS FROM REVIEWER Major. Regarding to the effect of T3 on the CAT reporter genes co-transfected with vector only and GATA2 only (Fig.2), To evaluate the effect of T3 (100 nM) on the activities of CAT reporter genes co-transfected with vector only and GATA2 only, we conducted additional CAT assays for five times independently. Using the value of the activity of the CMV-based CAT reporter gene (inter assay control) as 100, we combined these data with the original data of Fig. 2A and created new Fig. 2A. We did not find any statistical significance in CAT activities between absence or presence of T3 when we co-transfected vector only or GATA2 only. Minor. Regarding to “has not performed” of p. 3 L83 in the previous manuscript, we added “been”. Regarding to period for “hr” in p.6 L190, p.7 L198, p.7 L201 and p.7 L222 of the previous manuscript, we deleted all the periods for “hr”. Regarding to lack of a closing bracket in L225-227 of the previous manuscript, we added a closing bracket. Regarding to “theoretically” in p.9 284 of the previous manuscript, we rephrased the sentence as follows. Using the experimental system in Fig. 2B, we wanted to know whether the liganded TR functions as a transcriptional repressor that was defined by the theory of multi-dimensional in put function proposed by Uri Alon (Fig. 3 in set) [49]. We co-transfected various amounts of---. Regarding to “ as log scale” in the previous manuscript, we corrected “ as log scale” to “ in a log scale” Regarding to lacking or inappropriate usage of articles, Thank you for your recommendation. Although a native English speaker screened our previous manuscript once before submission, we asked another (more experienced) person to re-check English including usage of articles. He pointed a lot of mistakes in the previous manuscript. We corrected all of them. In addition, based on his recommendation, we deleted “the” from “the thyrotropin production” in the title (Please see “Track Changes”). We appreciate your recommendation very much. Regarding to “Several mechanisms” in p.14, l430 of the previous manuscript, We altered the sentences as follows. Third, T3 destabilizes the TSHβ mRNA but not CGA mRNA [78]. Fourth, T3 may inhibit the expression of Pit1 [79], which is a critical transcription factor for the TSHβ gene (Fig.7), but not the CGA gene [2]. Finally, the cell cycle of thyrotrophs [80, 81] may also be involved because sustained hypothyroidism often causes thyrotroph hyperplasia, resulting in the massive enlargement of the anterior pituitary [82]. In the course of this revision, we found two mistakes in p.18 L554 and L556: the phrase “the CAT activity” in these sentences was corrected to “the GATA2/GAPDH ratio”. Attachments Attachment Submitted filename: PLoSOneRebuttal.docx https://doi.org/10.1371/journal.pone.0227646.r002
29 Nov 2019 Decision Letter - Hiroyoshi Ariga, Editor PONE-D-19-22945R1 Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production PLOS ONE Dear Dr. Sasaki, 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. We would appreciate receiving your revised manuscript by Jan 13 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Hiroyoshi Ariga Academic Editor PLOS ONE ------------------------------- Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No ****** 6. 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: The authors performed additional experiments according to the query raised by this reviewer. In addition, the manuscript has been improved considerably by further editing. This manuscript is almost acceptable, however, I still found some points that should be corrected. Line 43, “a determinants” should be “ a determinant” or “one of the determinants” Line 288, “ wanted to know” in an original scientific article sounds a bit unusual to me. Maybe, “aimed to clarify” or “tried to evaluate” should be suitable. ****** 7. 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. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227646.r003
Revision 2
20 Dec 2019 Author Response REFERENCE: PONE-D-19-22945 Title: "Liganded T3 receptor _2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production” AUTHORS: Naoko Hirahara and Hiroko Misawa Nakamura et al. PLOS ONE Academic Editor: Hiroyoshi Ariga Please re-evaluate our revised manuscript. We corrected the sentences that the referee pointed. In addition, we transferred the original figure file to seven separate tif-style files using PACE home page (https://pacev2.apexcovantage.com/). If it can be now acceptable in your journal, we would be very much pleased. Thank you for your kind arrangement. Shigekazu Sasaki. MD. Senior Assistant Professor Second Division of Internal Medicine Hamamatsu University School of Medicine 1-20-1 Handayama Higashi-ku Hamamatsu 431-3192, Japan E-mail: sasakis@hama-med.ac.jp RESPONSE TO THE COMMENTS FROM REVIEWER We corrected the sentences that the referee pointed as follows. Line 43, “a determinants” We corrected as “ a determinant” Line 288, “ wanted to know” We corrected as “aimed to clarify”. Attachments Attachment Submitted filename: PLoSOneRebuttalR011218.docx https://doi.org/10.1371/journal.pone.0227646.r004
26 Dec 2019 Decision Letter - Hiroyoshi Ariga, Editor Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production PONE-D-19-22945R2 Dear Dr. Sasaki, 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, Hiroyoshi Ariga Academic Editor PLOS ONE ------------------------------ Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. 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: Hirahara et al studied GATA2 promoter function as well as expression of endogenous GATA2 in cultured cells. They demonstrated that GATA2 promoter is down regulated by T3 in the presence of T3Rß2. They also showed endogenous GATA2 is down regulated in LßT2 cells in a manner which is not inhibited by MG132. In general the experiments are well designed and quality of the data is good. As the authors adequately addressed to the comments raised by this reviewer, this reviewer feels that the manuscript is now acceptable. ****** 7. 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. Reviewer #1: No https://doi.org/10.1371/journal.pone.0227646.r005
Formally Accepted
30 Dec 2019 Acceptance Letter - Hiroyoshi Ariga, Editor PONE-D-19-22945R2 Liganded T3 receptor β2 inhibits the positive feedback autoregulation of the gene for GATA2, a transcription factor critical for thyrotropin production Dear Dr. Sasaki: 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. Hiroyoshi Ariga Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227646.r006

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .