Dear Dr Imhof,

Thank you very much for submitting your Research Article entitled 'The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila' to PLOS Genetics.

The manuscript was fully evaluated by independent peer reviewers at Review Commons. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on your proposed responses to the reviewer comments, we would be willing to review a much-revised version. We cannot, of course, promise publication at that time as the number of changes will require a re-review from at least one of the original reviewers.

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To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.

Yours sincerely,

Harmit S. Malik

Associate Editor

PLOS Genetics

Kirsten Bomblies

Section Editor: Evolution

PLOS Genetics

Dear Dr Imhof,

Thank you very much for submitting your Research Article entitled 'The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers who previously evaluated your paper. All reviewers are now largely satisfied but each identified some concerns that we ask you address in a revised manuscript- in particular you to be clear when referring to 'hybrid' phenotypes that are inferred indirectly and to make the distinction between transgenes and endogenously expressed genes.

We therefore ask you to modify the manuscript according to the review recommendations. Your revisions should address the specific points made by each reviewer.

In addition we ask that you:

1) Provide a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

2) Upload a Striking Image with a corresponding caption to accompany your manuscript if one is available (either a new image or an existing one from within your manuscript). If this image is judged to be suitable, it may be featured on our website. Images should ideally be high resolution, eye-catching, single panel square images. For examples, please browse our archive. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License. Note: we cannot publish copyrighted images.

We hope to receive your revised manuscript within the next 30 days. If you anticipate any delay in its return, we would ask you to let us know the expected resubmission date by email to plosgenetics@plos.org.

If present, accompanying reviewer attachments should be included with this email; please notify the journal office if any appear to be missing. They will also be available for download from the link below. You can use this link to log into the system when you are ready to submit a revised version, having first consulted our Submission Checklist.

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. 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 be aware that our data availability policy requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as "data not shown" or "unpublished results" in manuscripts. All points should be backed up by data provided with the submission.

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

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.

PLOS has incorporated Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.

To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.

[LINK]

Please let us know if you have any questions while making these revisions.

Yours sincerely,

Harmit S. Malik

Associate Editor

PLOS Genetics

Kirsten Bomblies

Section Editor: Evolution

PLOS Genetics

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript “The integrity of the speciation core complex is necessary for centromeric binding and reproductive isolation in Drosophila” by Lukacs and colleagues was previously reviewed via Review Commons by these reviewers. Therefore, we focused on how the concerns raised were addressed, whether the manuscript has improved and whether any outstanding issues still remain that would preclude publication in PLoS Genetics.

In our view, the concerns raised by all three reviewers have been adequately addressed.

The following specific points have been addressed to our satisfaction:

1) The overall flow of the manuscript reads much better and is less speculative.

2) The addition of the IF study on how HMR localizes with CENP-C or HP1a in ovaries are an important addition, which help clarify the dynamics of HMR binding in development and explain how these dynamics correlate with TE suppression.

3)The finding that the CTD of HMR is important for HMR localization to pericentromeric HP1a provides an important mechanistic insight.

4) The observation that HRM binds two distinct chromatin domains, both of which are necessary for proper development, are insightful from the dynamic perspective of chromatin-binding factors.

Overall, these data imply that both spatiotemporal regulation of chromatin factors and interaction with other chromatin-associated factors are critical for establishing and maintaining chromatin function and subsequent downstream effects. It will be interesting to learn how future studies will tease apart how the binding of HMR to these two chromatin domains are regulated. The updated discussion succinctly captures this point.

A few minor points could benefit from editing:

+ On page 16, the sentence “To have a more comprehensive of HMR’s localization in flies …" does not flow well.

+ On page 17, the use of the word “flies” in “… can also be observed in flies, we wanted …” is a little broad. Maybe write out more specifically which fly they are referring to?

Reviewer #2: Review is uploaded as an attachment.

Reviewer #3: I’ve looked through previous reviews and responses to them. It is somewhat unclear exactly at what stage I was brought in to review this at PLoS Genetics.

In previous version(s?) of this manuscript, the reviewers generally agreed that the data quality is good but the interpretations are not well-supported. In this revised version, the authors have improved the writing considerably, and the overall conclusions are reasonably supported. Specific comments below cover several additional issues along this line, but once they are addressed, this manuscript will be of considerable interest for the field.

-Page 13-14: They infer that the pulldown experiment under Hmr overexpression condition mimics hybrid condition. However, it should be noted that this is still in pure species condition (absence of any simulans proteins). Of course, they don’t need to do such experiments, but this difference needs to be clearly mentioned.

-Figure2, referring Hmr ectopic expression to as ‘Hmr+’ is very misleading. I understand this means ‘wild type transgene’, but in the context of differentiating Hmr-endo vs. Hmr+, it’s hard to understand ‘Hmr+’ indicates ectopic expression.

-Please label Figure 5A.

-Fig7 title ‘Hmr C-ter is required for hybrid lethality’ is misleading. Although this statement is not wrong, the results might simply indicate that Hmr-deltaC is just non-functional, as is shown in Figure 6. But the figure title/subheading indicate a little more than that (as if it is specifically required for hybrid lethality, without affecting its endogenous function in pure species context). I recommend a slightly more cautious description.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes: Yamini Dalal and Daniel Melters

Reviewer #2: No

Reviewer #3: Yes: Yukiko Yamashita

Attachments

Attachment

Submitted filename: Review_PLoS Genetics.docx

Dear Dr Imhof,

We are pleased to inform you that your manuscript entitled "The Integrity of the HMR complex is necessary for centromeric binding and reproductive isolation in Drosophila" has been editorially accepted for publication in PLOS Genetics. Congratulations!

Before your submission can be formally accepted and sent to production you will need to complete our formatting changes, which you will receive in a follow up email. Please be aware that it may take several days for you to receive this email; during this time no action is required by you. Please note: the accept date on your published article will reflect the date of this provisional acceptance, but your manuscript will not be scheduled for publication until the required changes have been made.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!

Yours sincerely,

Harmit S. Malik

Associate Editor

PLOS Genetics

Kirsten Bomblies

Section Editor: Evolution

PLOS Genetics

www.plosgenetics.org

Twitter: @PLOSGenetics

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Comments from the reviewers (if applicable):

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