PONE-D-19-26238

Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced

PLOS ONE

Dear Banerjee,

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.

The reviewers have identified a number of major and minor concerns. It is important to fully address them in your revised manuscript. These include, but are not limited to, providing evidence about the functional significance of the discovered PTMs, providing more details and further clarifying the results related to SLiMs, among other issues raised by the reviewers.

We would appreciate receiving your revised manuscript by Nov 29 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,

Jinsong Zhang

Academic Editor

PLOS ONE

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements.

Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

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.

[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: I Don't Know

Reviewer #2: N/A

**********

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: 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

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: The MS by Banerjee et al. reports on the extensive characterization of postranslational modifications (PTMs) targeting the Drosophila Hox protein Sex combs reduced (Scr). This is to my knowledge the first extensive description of Hox associated PTMs, providing qualitative and quantitative insights of how PTMs can impact Hox proteins. Consistent with role of PTMs affecting Hox protein function, a large proportion of the identified PTMs targets regions of the Scr protein previously known to be functionally important. The MS however do not experimentally address the functional relevance of any of the PTMs identified.

The MS is overall well written, the material and methods are extensive, and Fig. 7 and 8 nicely summarizes the content of the MS. Some Figures with low info content may be combined (Fig.1 to 3).

Specific comments:

- The authors address whether phosphorylation sites preferentially map to SLiM motifs. It is not clear why this specific question is raised, and also why it is addressed only for phosphorylation.

- Fig. 1 highlights FUNCTIONAL Scr protein domains. The bases for that claim for each motif should be provided. Also it may be useful to complement that figure to show to what extent these domains are conserved over short and/or long evolutionary distances.

- Fig. 2 shows a single Scr band, raising the issue of whether/how much of these PTMs likely “coexist”. May be a more resolutive gel and less exposed picture would help appreciating the point.

- Quality of the Figure may be improved for a few panels (in Fig. 3, larvae are apparently not at equivalent stages, and one panel of Fig. 4B is strongly over exposed).

- Discussion, p32, the statement “that the modifications map to functionally important region” is not supported. This point is accurately described in the abstract.

- The authors do not discuss one important limitation that relates the “ectopic” approach. Scr is only expressed very locally in the Drosophila embryo. Following heat shock it is expressed at high levels in all embryonic cells, which may reveal PTMs that do not occur in cells “normally” expressing Scr.

Reviewer #2: This manuscript reports results of a study of the post-translational modifications of the Drosophila Hox protein SCR. The study design was to overexpress a tagged version of Scr in embryos, extract it and analyze modifications by Tandem Mass Spectrometry. The study identified multiple protein modifications including phosphorylation, acetylation, formylation, carboxylation and hydroxulation for a total of 44 modifications. Many of these map to regions of the protein previously shown to be important for function. The study then did bioinformatics analysis to identify SLiMs and found that 66% of SCR is comprised of these, of which >30% are evolutionarily conserved.

The report of PTMs for SCR is of interest and worthy of publication. This analysis appears to have been carefully done and provides worthwhile information for the study of Hox protein function in general. I have two major reservations:

1. The PTMs were analyzed on SCR protein that was expressed throughout the embryo , at high levels and over a very long time period. While this study design is understandable, in that large amounts of protein are needed, it raises the problem that expression outside of the native SCR domain may result in PTMs that do not occur in the cells in which SCR is endogenously expressed. Therefore, the functional significance of the different modifications is unclear. I would like to see at least some functional testing of residues – perhaps at least the phosphorylated residues predicted to be in regions important for function – in order to be able to evaluate the significance of the PTMs identified.

2. The finding that most of SCR is covered by SLiMs is very surprising to me and I am, quite frankly, skeptical about this result and its potential importance. At a minimum, this needs a lot more explanation. How do you define a SLiM? What are your cut-offs? What are you comparing? Same questions for the conservation. What is compared? How broadly conserved are these? Across drosophilid SCRs? Other species? I would add main text figures on this, and show the sequence alignments with sequences (not just blocks). The Tables in the Supplement are fine but are basically just lists. The main text needs to summarize this information in more than just the schematic shown in the main Figure. I really wonder what the significance of all these is? Would you find this many SLiMs in any protein analyzed? This needs to be tested in order to understand whether this finding is significant.

Other specific comments:

Line 257: Indicate C-terminal or N-terminal fusion; replace “behind” with “downstream of”

Line 262: This is an enormous difference. It might be worth stating here which GAL4 was used (even though it's in the methods) and perhaps commenting on whether that is considered a "strong" GAL4 line. Just to point to how generalizable this is - that it is so much stronger (which is something many of us assume but I have not noticed it directly compared like this before to a ubiquitous GAL4 driver)

Line 287: It would be helpful if the WT control were imaged in a way that shows the T1 beard. Also, it is now pretty standard to use a UAS-gfp as control rather than a WT animal

Line 291: Perhaps worth a mention here or in the methods how you managed the heat shock with 3 grams of embryos - size of tools used to collect, size of mesh - just a few words to point out that this is a larger mass of embryos than your standard collection. Also, please include stage and length of heat shock, here even though it is in the methods

Line 300: This is confusing if you are saying that the purified band had less than you need for the MS. You just need more sentences here to explain. It is also notable that a lot of background bands came through the whole purification. Obviously you got the right product in the end but I don't know if you want to comment on that point when discussing Fig. 4. You really cannot see the SCR band above background levels in the Coomassie stained gel.

Fig. 5: I would make this a supplementary figure. It's kind of distracting to the main story line to see a figure with sequence and then that sequence isn't SCR.

**********

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: Yes: Leslie Pick

[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.

PONE-D-19-26238R1

Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced

PLOS ONE

Dear Banerjee,

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.

Although Reviewer 1 has indicated that your revised manuscript has addressed the previous concerns, Reviewer 2 has remaining concerns that need to be further addressed, which include clarification of the species related to the "conserved" SLiMs, and some validation of the significance of the predicted SLiMs from the database used in the manuscript.

We would appreciate receiving your revised manuscript by Jan 24 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,

Jinsong Zhang

Academic Editor

PLOS ONE

[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: All comments have been addressed

Reviewer #2: (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

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: I Don't Know

**********

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

Reviewer #2: 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

Reviewer #2: 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: The authors have properly addressed the issue raised in my first round of review and modified accordingly the manuscript.

Reviewer #2: This revised manuscript is much improved and many of my suggestions have been addressed. I can agree with the argument that this study is worthy of publication before functional testing because of the complications that those experiments present, as discussed by the authors both in the rebuttal and in the text. The one additional major point I would like to see addressed though is the finding that so much of the SCR protein is comprised of SLiMs. First, the authors should make clear in the text and figure that “conserved” in this case refers to Drosophila species. This is quite different from an earlier figure where conserved included all arthropods - hundreds of millions of years of difference in divergence. Second, I ask that the authors experimentally (bioinformatically) address the question I raised previously: Would you find this many SLiMs in any protein analyzed? This needs to be tested in order to understand whether this finding is significant, especially since experimental testing of individual sequences is not realistic in this system. Are there examples of proteins analyzed in this way for which the function of the predicted SLiMs has been experimentally tested or verified? Basically, the conclusion now is based on analysis in one database. I personally am not familiar with the stringency of this database but I am still very skeptical about the finding. I would be happy to be convinced of its validity – it would help our own research enormously – but I need more evidence. Most of the other points were addressed adequately although I did not some unusual grammar and punctuation in some of the changes – it needs a quick proofreading before resubmission.

**********

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

Reviewer #2: Yes: Leslie Pick

[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.

Post-translational modifications of Drosophila melanogaster HOX protein, Sex combs reduced

PONE-D-19-26238R2

Dear Dr. Banerjee,

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,

Jinsong Zhang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: