Hierarchical regulation of autophagy during adipocyte differentiation

Mahmoud Ahmed; Trang Huyen Lai; Trang Minh Pham; Sahib Zada; Omar Elashkar; Jin Seok Hwang; Deok Ryong Kim

doi:10.1371/journal.pone.0250865

Peer Review History

Original SubmissionApril 21, 2021
21 Apr 2021 Author Response Attachments Attachment Submitted filename: Responsestoreviewers_1.docx https://doi.org/10.1371/journal.pone.0250865.r001
8 Jul 2021 Decision Letter - Naresh Doni Jayavelu, Editor PONE-D-21-12883 Hierarchical Regulation of Autophagy During Adipocyte Differentiation PLOS ONE Dear Dr. Deok Ryong Kim, 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. Based on the two reviewers comments, it seems that this manuscript has utilized datasets from your previous publications. Therefore it is important to address how this study is different from the previously published ones. Please submit your revised manuscript by October 5th 2021. 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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We will update your Data Availability statement to reflect the information you provide in your cover letter. [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: Partly Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 Reviewer #2: No ****** 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 Reviewer #2: No ****** 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 their manuscript, “Hierarchical Regulation of Autophagy During Adipocyte Differentiation,” Ahmed and colleagues has attempted to identify the order of autophagic event during adipocyte regulation. To do this, they made use of the datasets that are previously available. The manuscript reports a series of temporal and spatial organization between adipogenic transcription factors (TFs) and co-factors in the regulation of autophagy-related gene expression by integrating multiple high-throughput sequencing data including RNA-seq, histone ChIP-seq and TF ChIP-seq data. On the one, the efforts and steps taken to identify the series of events is highly appreciated - is extremely important and interesting. On the other hand, the paper is rather disappointing, because the authors did not attempt to validate the prediction. However, I think if the premise of the paper could be strengthened by either 1) assessing functional outcome of the prediction, for example, studies to correlate autophagy with gene expression. (or) 2) I understand these studies could be time consuming, in that case I suggest providing the separate limitation section and alter the language throughout by not making any strong claims. As one of the reviewer pointed out early, Fig2A, B and 6A are hard to understand. I suggest authors to come up with different way of representing the data. Minor corrections: All the figure legends repeat the same first sentence again and again. Legends could benefit from detailed info. Also, the legends give a feel that the actual screens were performed in this paper. Legends need serious attention. Too many figures in the main text. Consider moving some of them as supplementary. Finally, the manuscript could benefit from rigorous language editing. Reviewer #2: As have been reported and analyzed in prior work by the same group (https://doi.org/10.3390/cells8111321, https://doi.org/10.1080/21623945.2019.1697563, https://doi.org/10.1080/21623945.2020.1829852), all the gene expression and the DNA-binding events have already been displayed using the same datasets. The present study does not provide anything new and exciting. Also I agree with the following comments that the autophagy gene regulation might not be as specific as the authors stated as thousands of genes will be indcued during adipogenesis by adipogenic transcription factors. I doubt why the authors only choose Cebpb and Pparg, not Cebpa and Fabp4, which are also important. Most importantly, the different autophagy stages are not clear as the authors have no experimental data for support. In conclusion, the idea maybe interesting, but the data is too old and the model is not supportive. ****** 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 Reviewer #2: 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.] 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0250865.r002
Revision 1
10 Sep 2021 Author Response Reviewer #1: In their manuscript, “Hierarchical Regulation of Autophagy During Adipocyte Differentiation,” Ahmed and colleagues has attempted to identify the order of autophagic event during adipocyte regulation. To do this, they made use of the datasets that are previously available. The manuscript reports a series of temporal and spatial organization between adipogenic transcription factors (TFs) and co-factors in the regulation of autophagy-related gene expression by integrating multiple high-throughput sequencing data including RNA-seq, histone ChIP-seq and TF ChIP-seq data. On the one, the efforts and steps taken to identify the series of events is highly appreciated - is extremely important and interesting. On the other hand, the paper is rather disappointing, because the authors did not attempt to validate the prediction. However, I think if the premise of the paper could be strengthened by either 1. assessing functional outcome of the prediction, for example, studies to correlate autophagy with gene expression. (or) I understand these studies could be time consuming, in that case I suggest providing the separate limitation section and alter the language throughout by not making any strong claims. As one of the reviewer pointed out early, Fig2A, B and 6A are hard to understand. I suggest authors to come up with different way of representing the data. We expanded the limitations part of the discussion to address the reviewer's concerns. In addition, we edited the figures and the legends to better describe the data presented and how it can be interpreted. Minor corrections: • All the figure legends repeat the same first sentence again and again. We edited the figure legends to better reflect the data shown in them. • Legends could benefit from detailed info. Also, the legends give a feel that the actual screens were performed in this paper. Legends need serious attention. We edited the figure legends to include detailed descriptions of the data and analysis presented in each. • Too many figures in the main text. Consider moving some of them as supplementary. We reduced the number of figures by using more compact representations and moving two figures to supplementary materials • Finally, the manuscript could benefit from rigorous language editing. We revised the language in the manuscript to correct grammatical mistakes and typos and to improve clarity. We also plan to use professional editing service to revise the manuscript before the publication. Reviewer #2: As have been reported and analyzed in prior work by the same group (https://doi.org/10.3390/cells8111321, https://doi.org/10.1080/21623945.2019.1697563, https://doi.org/10.1080/21623945.2020.1829852), all the gene expression and the DNA-binding events have already been displayed using the same datasets. The present study does not provide anything new and exciting. In a previous study, we showed that two important adipogenic transcription factors (PPARG and CEBPB) regulate the expression of autophagy-related genes either directly or indirectly through other specific transcription factors. In this study, we investigated the spatial and temporal arrangement of those two factors and their cofactors. We believe that these findings elaborate on the mechanisms of the transcription regulation of autophagy by these transcription factors and co-factors. In addition, we showed that these regulatory links are potentially involved in the induction of specific forms of autophagy, have a strong temporal dimension, and work through specific effectors (other factors or kinases) compared to the regulation of lipogenesis. In response to the reviewers' comments, we revised the Introduction & Discussion section to stress the value added by the study to previous work. Also I agree with the following comments that the autophagy gene regulation might not be as specific as the authors stated as thousands of genes will be indcued during adipogenesis by adipogenic transcription factors. The reason we believe autophagy genes are regulated by adipogenic transcription factors is the following 1. Previous studies have shown that knocking out ATG5/7 in preadipocytes resulted in failure of differentiation. 2. The expression of autophagy genes changed over the course of differentiation 3. We found binding sites of PPARG and CEBPB on autophagy genes 4. The knockdown of PPARG or CEBPB in preadipocytes resulted in an autophagy gene expression signature that is different during the course of differentiation compared to control cells. Our initial goal was to study the regulation of autophagy during adipogenesis mainly through gene expression data. In previous work, we found that autophagy genes are regulated as part of the transcriptional program and adipogenic transcription factors and co-factors were the obvious candidates as drives of the gene expression. As a result, we used ChIP-seq data to identify binding patterns and correlate them with changes in gene expression. The role of these factors is well described in the case of the processes that are directly related to adipogenesis and lipogenesis. Therefore, we believe it is useful to study these patterns of regulation in the case of autophagy. We also found key differences between the way autophagy and lipogenesis genes are regulated by these factors. Namely, the autophagic response to the adipogenic induction is biphasic and the adipogenic factors target other factors and kinases more specifically. We clarified the motivation for the study and the focus on autophagy genes in the Introduction section. I doubt why the authors only choose Cebpb and Pparg, not Cebpa and Fabp4, which are also important. Our analysis doesn't rule out the involvement of other transcription factors in regulating autophagy genes. We observed significant binding and gene expression changes during adipogenesis that seem to be correlated with those two factors. In addition, the two factors and their co-factors explain a significant portion of the variance over time. We discussed this issue in the limitations of the study in the Discussion section Most importantly, the different autophagy stages are not clear as the authors have no experimental data for support. In conclusion, the idea maybe interesting, but the data is too old and the model is not supportive. Our analysis is based on two data types (RNA and ChIP-seq). We reported observations on the changes in gene expression, DNA-binding, and the correlation of the two. We don't make any explicit claims on the function, since our analysis doesn't include any functional data. We recognize that this kind of observation is only valid insofar as phenotypes reflect the underlying gene expression. We used the gene set enrichment and over-representation analysis to statistically quantify these observations at the gene set level, with is more likely to correspond to biological functions than claims based on changes in the expression of individual genes. We explained this view with the other limitations of the study in the Discussion section. Attachments Attachment Submitted filename: rebuttal_PONE-D-21-12883.pdf https://doi.org/10.1371/journal.pone.0250865.r003
6 Dec 2021 Decision Letter - Naresh Doni Jayavelu, Editor Hierarchical Regulation of Autophagy During Adipocyte Differentiation PONE-D-21-12883R1 Dear Dr. Deok Ryong Kim, 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, Naresh Doni Jayavelu, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Authors sufficiently addressed both reviewers comments and added limitation of the current study. I think it is ready for publication now. Reviewers' comments: https://doi.org/10.1371/journal.pone.0250865.r004
Formally Accepted
31 Dec 2021 Acceptance Letter - Naresh Doni Jayavelu, Editor PONE-D-21-12883R1 Hierarchical Regulation of Autophagy During Adipocyte Differentiation Dear Dr. Kim: 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. Naresh Doni Jayavelu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0250865.r005

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