Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cells

Alyssa Kearly; Prontip Saelee; Jonathan Bard; Satrajit Sinha; Anne Satterthwaite; Lee Ann Garrett-Sinha

doi:10.1371/journal.pone.0308896

Peer Review History

Original SubmissionJuly 31, 2024
31 Jul 2024 Author Response https://doi.org/10.1371/journal.pone.0308896.r001
27 Sep 2024 Decision Letter - Roberto Mantovani, Editor PONE-D-24-32251Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cellsPLOS ONE Dear Dr. Garrett-Sinha, Thank you for submitting your manuscript to PLOS ONE. First, I have to apologize for the delay in the reviewing process. After very careful consideration, and revision by an expert in the field, 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 below. Please submit your revised manuscript by Nov 11 2024 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. 7. 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. [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. 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: In this paper, the authors set out to establish the regulatory regions that control Ets1 expression in B and T cells. Through combining chromatin state mapping and BAC transgenesis, they identify a region responsible for Ets-1 expression in B cells and establish that additional sequences outside this region are required for robust expression in T cells. The latter are not pursued. Functionally, the authors test various potential regulatory regions for enhancer activity but none are obviously active in that context. Furthermore, motif analysis identifies potential TF regulators but no experiments are provided to test their functionality in B cells. Overall, this study therefore contributes to our understanding of Ets1 expression control in immune cells but is rather preliminary in nature, stopping short of providing any detailed mechanistic insights rather than the current inferences. Generally, the data as presented are okay but there are a few places for improvement: (1) As indicated above, some functional testing of potential TF regulators would be a step forwards. (2) As the luciferase reporter assays are generally none informative, a better strategy would be to engineer changes in the regulatory regions either in the context of the BAC or via CRISPR in appropriate cells line models. (3) In Fig. 4B, adding the human data is okay but the rationale in the text is not really correct. Ie this does not allow “better definition of the regions” but instead provides evidence for evolutionary conservation. (4) In Fig. 5B, the second track should be labelled as Fo B cells. Also, what population of B cells does the bottom track come from? (5) In Fig. 6C, the percentage of regions containing each motif should be added. In the left table, the authors should comment on the second motif that resembles at ETS motif. On the right side, they cannot conclude this is Ets1 rather than another ETS TF, therefore the results text describing this needs rewording. (6) I was confused by Fig. 8C. It appears here that several regions seem to be repressive in nature (ie less than promoter alone), so not sure why the results text says that none of the regions show strong repressive activity. (7) Given that GWAS signals suggest the importance of the Ets1 locus, do any of the SNPs map to putative regulatory elements highlighted in this study? If so what is their predicted effect on activity (ideally would also be tested but as a minimum discussed). ****** 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.] 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.0308896.r002
Revision 1
12 Nov 2024 Author Response Response to Editor Comments: We have reformatted the manuscript to meet the PLoS One style and formatting. We included information about grant funding in the Cover Letter, as requested. Information about grant funding was removed from the Acknowledgements section. We removed reference to data not shown. We included captions for your Supporting Information files at the end of our manuscript, but also embedded them into the Supplemental Figures. Response to Reviewer Comments: Response to Reviewer Comments Reviewer #1: In this paper, the authors set out to establish the regulatory regions that control Ets1 expression in B and T cells. Through combining chromatin state mapping and BAC transgenesis, they identify a region responsible for Ets-1 expression in B cells and establish that additional sequences outside this region are required for robust expression in T cells. The latter are not pursued. Functionally, the authors test various potential regulatory regions for enhancer activity but none are obviously active in that context. Furthermore, motif analysis identifies potential TF regulators but no experiments are provided to test their functionality in B cells. Overall, this study therefore contributes to our understanding of Ets1 expression control in immune cells but is rather preliminary in nature, stopping short of providing any detailed mechanistic insights rather than the current inferences. Generally, the data as presented are okay but there are a few places for improvement: (1) As indicated above, some functional testing of potential TF regulators would be a step forwards. (2) As the luciferase reporter assays are generally none informative, a better strategy would be to engineer changes in the regulatory regions either in the context of the BAC or via CRISPR in appropriate cells line models. We agree with the reviewer that further studies are needed to identify relevant transcription factors and enhancer sequences needed for B cell expression. In this regard, Our lab has shown that signaling via the kinase IKK is required to downregulate Ets1 expression in response to BCR or TLR activation in B cells, although individually neither RelA nor cRel was required (see our published manuscript – PMID: 36445360). We also found that Foxo3a was not required for either the basal expression of Ets1 nor its downregulation in response to BCR ligation. We are continuing to perform follow-up experiments to examine roles for additional transcription factors (Pax5, Irf4, Ebf1, Bach2, PU.1 and Foxo1) that are expressed at high levels in resting B cells and that might be involved in regulating the basal levels of Ets1, but these studies have yet to yield conclusive results. We posit that combinatorial actions of transcription factors are likely to be at play, just as the case with the enhancers that we report in this study The reviewer brings up a valid point about the limitations of the transient transfection system with luciferase reporter genes. We are currently developing Crispr systems to mutate putative enhancers in a B cell line. However, these studies are at a very early stage and we are planning to include them in a future publication examining Ets1 regulation in more detail. Despite the fact that the studies reported in this manuscript have not yet identified a specific master transcription factor or enhancer segments crucial for regulating Ets1 expression, we feel that our studies still add valuable information to the knowledge of regulatory mechanisms that govern Ets1 expression. We have found that the Ets1 gene is regulated differently in B cells and T cells, something that was previously unknown. We have also found that sequences within and upstream of Ets1 are sufficient both for faithful expression in B cells, but also for stimulation induced decrease in Ets1 transcription as well as the decrease that occurs in plasma cells. We have also identified potential regulatory regions for Ets1 as marked by ATAC-seq peaks and histone marks. We have identified changes in chromatin accessibility that occur in B cells in response to BCR ligation – this experiment being a genome-wide study offers a valuable resource. While we have focused on describing such changes in the Ets1 locus, our ATAC-seq data have been submitted to the GEO databank and are available for other researchers to examine when assessing how BCR signaling alters chromatin in various gene loci. Finally, it is also important to note that journal information for PLoS One states that “We evaluate research on the basis of scientific validity, strong methodology, and high ethical standards—not perceived significance. Multidisciplinary and interdisciplinary research, replication studies, negative and null results are all in scope”. We feel that our manuscript currently meets this criterion for publication. (3) In Fig. 4B, adding the human data is okay but the rationale in the text is not really correct. i.e. this does not allow “better definition of the regions” but instead provides evidence for evolutionary conservation. We thank the reviewer for their thoughtful insights. We have modified this section of the text to better reflect the rationale. (4) In Fig. 5B, the second track should be labelled as Fo B cells. Also, what population of B cells does the bottom track come from? We have relabeled the figure. The bottom track in this figure is from data collected as part of this study (PMID: 25103404). It represents bulk CD3-, B220+, CD19+ B cells isolated from mouse spleen, which has been noted. (5) In Fig. 6C, the percentage of regions containing each motif should be added. In the left table, the authors should comment on the second motif that resembles at ETS motif. On the right side, they cannot conclude this is Ets1 rather than another ETS TF, therefore the results text describing this needs rewording. These motifs were discerned using the motif-finding algorithm HOMER (http://homer.ucsd.edu/homer/introduction/basics.html). HOMER scores motifs by looking for sequences with differential enrichment between two sets of sequences. In our case these two sets are DAR sequences from unstimulated versus stimulated B cells. HOMER calculates a p value for enrichment of known transcription motifs, but does not provide a value for the percentage of sequences containing a particular motif. Given the very small p values of the listed transcription factors, it is likely that they are strongly enriched. To identify potential transcription factors binding to the enriched sequences, HOMER uses the TRANSFAC database. TRANSFAC contains experimentally-determined motifs for many transcription factors including various members of the Ets gene family. While the core Ets binding motif is similar among the various Ets family members, the flanking sequences are somewhat different. Using the TRANSFAC database, HOMER found that the best match to the enriched sequence was the Ets1 binding motif. However, we do agree that other related transcription factors in the Ets gene family may also bind to this sequence. For that reason, we have revised the text to include this point. (6) I was confused by Fig. 8C. It appears here that several regions seem to be repressive in nature (ie less than promoter alone), so not sure why the results text says that none of the regions show strong repressive activity. The reviewers is correct and we have revised the text accordingly. (7) Given that GWAS signals suggest the importance of the Ets1 locus, do any of the SNPs map to putative regulatory elements highlighted in this study? If so what is their predicted effect on activity (ideally would also be tested but as a minimum discussed). Several disease-associated SNPs do map close to or within putative enhancer regions. We’ve included a new figure showing where the SNPs associated with lupus map (new Supplemental Figure S5). (8) Additional information for reviewer: It should also be noted that some changes have been made to format the revised manuscript according to guidelines for PLoS One. As part of this effort, we have moved the figure legends into the main body of the text just under the paragraph in which they are first mentioned. This is consistent with the recommended formatting style for this journal - https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf. The Supplemental Figure Legends are at the end of the manuscript and also included in the Supplemental figures themselves. Attachments Attachment Submitted filename: Response to Reviewer.pdf https://doi.org/10.1371/journal.pone.0308896.r003
22 Nov 2024 Decision Letter - Roberto Mantovani, Editor PONE-D-24-32251R1Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cellsPLOS ONE Dear Dr. Garrett-Sinha, Thank you for re-submitting your manuscript to PLOS ONE. After further consideration, we feel that it has merit but it still 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 Jan 06 2025 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 . When you're 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. 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). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Roberto Mantovani Academic Editor PLOS ONE Journal Requirements: 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. 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: 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: Most things have been addressed. I appreciate this is PLOSONE and there are criteria for publication, and my initial comments were based on making the paper more conclusive rather than suggestive. I do take the arguments they make but it would be useful to add caveats about the limitations of the approach (ie surrounding previous points 1 and 2 about more appropriate definitive assays). One this not addressed was "In Fig. 6C, the percentage of regions containing each motif should be added. In the left table, the authors should comment on the second motif that resembles at ETS motif. " The authors have not commented on the ETS-like motif. For the percentage of regions with motifs issue, then this is still useful to include as it provides a feel for what proportion of the effect is driven by a particular TF. You can get highly enriched regions in a low percentage of regions if the genomic back ground is also low for example. Again, this is not essential but just helps the reader to gauge the influence of ETS TFs. new Supplemental Figure S5 was not included or referred to in the revised manuscript so was impossible to evaluate. ****** 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.] 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.0308896.r004
Revision 2
24 Nov 2024 Author Response Response to Reviewer Comments Reviewer #1: Most things have been addressed. I appreciate this is PLOSONE and there are criteria for publication, and my initial comments were based on making the paper more conclusive rather than suggestive. I do take the arguments they make but it would be useful to add caveats about the limitations of the approach (ie surrounding previous points 1 and 2 about more appropriate definitive assays). We have added some sentences to address this on pages 22-23 of the revised paper. One this not addressed was "In Fig. 6C, the percentage of regions containing each motif should be added. While we would like to address this point, unfortunately the software used to identify motifs (HOMER software - http://homer.ucsd.edu/homer/) does not provide the percentage of sequences that contain a motif. It simply identifies motifs that are over-represented in one dataset (e.g., stimulated B cells) as compared to the other dataset (e.g., unstimulated B cells) and gives a p value for the significance of the over-representation. The p values for the motifs we list are all very small, indicating that these sequences are highly enriched in one dataset or the other. But we cannot provide a percentage of sites that contain those particular motifs. In the left table, the authors should comment on the second motif that resembles at ETS motif. " The authors have not commented on the ETS-like motif. For the percentage of regions with motifs issue, then this is still useful to include as it provides a feel for what proportion of the effect is driven by a particular TF. You can get highly enriched regions in a low percentage of regions if the genomic back ground is also low for example. Again, this is not essential but just helps the reader to gauge the influence of ETS TFs. We agree with the reviewer’s point, but unfortunately as described above, we have no information on the percentages of sequences with a particular motif. new Supplemental Figure S5 was not included or referred to in the revised manuscript so was impossible to evaluate. Sorry, this was a mistake on our part. The extra figure is Supplemental Figure 4, which was included in the figures along with a figure legend, but was not referenced in the text. We have added sentences on page 14 of the manuscript to refer to the new figure. Attachments Attachment Submitted filename: Response to Reviewer.docx https://doi.org/10.1371/journal.pone.0308896.r005
12 Dec 2024 Decision Letter - Roberto Mantovani, Editor Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cells PONE-D-24-32251R2 Dear Dr. Garrett-Sinha, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Roberto Mantovani Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0308896.r006
Formally Accepted
Acceptance Letter - Roberto Mantovani, Editor PONE-D-24-32251R2 PLOS ONE Dear Dr. Garrett-Sinha, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Prof. Roberto Mantovani Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0308896.r007

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