Dear Dr. Brehelin,

Thank you very much for submitting your manuscript "Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ilya Ioshikhes

Associate Editor

PLOS Computational Biology

Erik van Nimwegen

Deputy Editor

PLOS Computational Biology

***********************

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Dear editor,

The author Menichelli et al has reported a method to identify long regulatory elements in Plasmodium falciparum and some other eukaryotes. The paper is of interest to the research community of apicomplexan research. I recommend the authors compile their findings, and give concrete numbers of LRE identified and make them easily accessible for other. Additionally, however, I have the following remarks and questions:

1.DexTER, the acronym for the method, was not defined until much later in the manuscript. A brief description of one or two sentences, of the method should be mentioned in the abstract.

2.“LERs appear to determine a very large part of gene expression variation”, page 2. This is not precise language for reporting science. Can the authors define “a very large part”

3.“For most specie, we show that these LRE are predictive of expression with an accuracy in between 50% and 60%, for P. falciparum, the accuracy even exceeds 70%”, page 5, these numbers 50%, 60% 70%, do not have a context. Please provide bench marking and rationale for the accuracy, because these number will determine the quality of the research

4.Related to 3, also page 7&8, please provide a benchmark comparison, how much can be recovered, which category, of LERs with at least one other species, e.g. human.

5.“We took 4kb centered either around the gene start, or around AUG ..”, page 6, please provide rationale for choosing 4000 bp.

6.Is there a supplemental file with all the LERs characterized? So that other researchers can perform independent validations. And the author’s work will be more accessible for the community.

7.Fig 5, why is P. falciparum’s result so different than that of P. berghei?

Reviewer #2: Summary comments:

Overall this is an interesting manuscript that explores the association of long repeat elements (LRE) with gene expression in a number of organisms. The main focus of the work is developing a systematic method to discover LREs adjacent to known or predicted genes and then to assess the association of those sequences with gene expression profiles. For most organisms, the association fluctuates around 60%, but in the case of Plasmodium falciparum is higher and this manuscript focuses on the implications for P. falciparum gene expression. In P. falciparum, whose genome is known to be strongly depleted of transcription factors, LREs appear to determine a very large part of gene expression variation, and their analyses show that they are involved in both transcriptomic and post-transcriptomic regulation mechanisms.

This work is novel and does provide an interesting set of correlative data for the potential role of long repeat elements in the regulation of P. falciparum gene expression. This is quite intriguing because there remain major questions as to the mechanism of gene regulation. There is a very structured program of gene regulation throughout the life cycle and a paucity of predicted transcriptional regulatory proteins. While the work presented here is of potential interest, a major question as to the relevance of these findings remains. The Plasmodium falciparum genome has an unbalanced base composition with 82% A+T on average across the genome. Most of the G+C pairs occur in genic regions and parts of the non-genic genome reach nearly 100% A+T with long stretches of homopolymer and low complexity (AT repeats) sequence surround genes. The comparisons to other organisms did not include organisms with similar high A+T content. This could influence the results. Of particular concern is the identified key LREs are TATAT, TTT, TATATA and AA all seem to show strong correlations.

On the positive side, the authors do show strong associations with particular sequences with certain biological features of gene expression in the parasite and the computational model may be teasing out relationships not previously recognized.

The authors extend their analysis to a functional test of the LREs by constructing two chimeric upstream regions, one with high ATA content and one with low ATA content attached to a GFP reporter and then inserting these in the Plasmodium pfs47 gene locus and measuring GFP expression. They observed a 10-fold difference in expression and while potentially interesting probably will require some additional characterization. Pfs47 is sexual stage specifically expressed in its native configuration so the CRISPR/Cas9 inserted construct has in some way changed the native expression profile – perhaps through the disruption of chromatin structure. Before definitive conclusions can be drawn, including additional work to determine if this difference in expression is context or sequence dependent. It would be particularly interesting of insertion of a specific LRE disrupted chromatin structure.

Overall, this work is of potential interest and may shed light on the mechanisms associated with the control of gene expression. The work is innovative and novel and has opened some new questions that merit further experimental exploration.

Reviewer #3: In the manuscript entitled “Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes” by Menichelli et al., authors have used a computational approach to identify the long regulatory elements (LREs) in the genomes of various eukaryotic systems. They have also used various conditions to train the program to eventually use it to identify the LREs and predict the gene expression from unknown sequences. Authors have used expression data as a readout for the direct association of the long regulatory elements with the regulation of the associated genes. Finally, they have validated the association between the LREs and gene expression in Plasmodium falciparum using two LREs of varying length in in vitro conditions. In this study, investigators have addressed an important long-standing question in the field and identified novel LREs and their association with gene expression. While the analysis is rigorous and potentially very interesting the manuscript needs clarity in presentation and explanation. Here are some of the concerns outlined.

1. One of the major concerns is that authors have used the association between LREs and gene expression as causation (i.e. regulation) interchangeably throughout the manuscript. Though causation is possible, the data provided in this manuscript is not sufficient to establish the causal relationship.

2. The correlation (which I believe is Pearson correlation) between LREs and expression is generally in the range of 0.2 to 0.5 in Figure 4. These are mostly weak or moderate associations.

3. Most of the housekeeping genes in higher eukaryotic systems (human and mouse) have high CpG content at promoters. Thus sequence itself is governing the gene expression in this case. How do the authors explain the sinusoidal pattern of gene expression in Plasmodium with LREs?

4. I was wondering if DExTER has also identified CpG elements in the human/mouse genome? Does it correlate with gene expression?

5. The design for in vitro experimental validation is not clear. In this experiment, the authors have used two different sequences and compared the GFP expression. As these are two different sequences they cannot be directly compared. Authors should take varying repetitions of the ATA sequences (eg. 3, 2, and 1) to see the effect of this LTR on expression. Moreover, if they want to demonstrate this association for causation, they need to mutate the LRE of the same length.

6. It would be important that authors provide information pertinent to the important LREs identified in a tabulated form. LRE-sequence/associated with number of genes/LRE count/gene ontology/significance value.

7. Which version of the Plasmodium genome is used for the analysis?

8. Authors have also looked at conservation of the identified LREs along evolution. Why do authors believe that there will be conservation in LREs when genomes are so different? Please include an explanation.

9. The text in figure 1 is very hard to read due to the very small font size.

10. Axis label is missing in figure 2. Also, tissue type/stages of development are not explained properly in the figure or the text.

11. Figure 4: The ‘most important variables’ indicated on the graph are hard to read due to the poor contrast and small font size.

12. In general, the font sizes of the axis labels are too small for many supplementary figures. Figure caption is missing for figure S3. Axis label is missing in Figure 7a, S5 and S9.

13. Authors should specify moderate/ average/higher gene expression correlations.

14. The assumption that LREs are involved in post-transcriptional regulation is not supported in this study. Plasmodium also produces a lot of anti-sense RNAs and these LREs might have a role in transcriptional regulation of anti-sense RNAs.

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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 Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure 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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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, please see http://journals.plos.org/compbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Brehelin,

Thank you very much for submitting your manuscript "Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes" for consideration at PLOS Computational Biology.

As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the comments of reviewer 2.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ilya Ioshikhes

Deputy Editor

PLOS Computational Biology

Erik van Nimwegen

Deputy Editor

PLOS Computational Biology

***********************

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #1: Most of my questions have been answered or addressed.

Reviewer #2: The authors have responded with new data and a modified text to the comments by the referees. The addition of the analysis of other high A+T content organisms provides additional new data and strengthens the manuscript. There remains one issue that was not adequately addressed. The functional validation remains a weak point in the manuscript. The authors have not addressed issues of genomic context - in particular, the authors have used a gene that is normally only expressed in the mosquito stages and yet they measure the transcription in the asexual stages. There are no controls presented to demonstrate that transcriptional levels of other genes have similar transcriptional levels in the two genetically modified parasites. There is not confirmation that the two lines are assayed in parasites at the same stage of the life cycle. Without additional experiments including key control experiments, the data presented cannot be used as proof of validation. This is the beginning of a validation process, but far from the acceptable standard.

Reviewer #3: In th manuscript entitled “Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes” by Menichelli et al., Authors have satisfactorily answered all my concerns.

**********

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 Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: None

Reviewer #2: Yes

Reviewer #3: None

**********

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

Reviewer #3: No

Figure 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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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, please see http://journals.plos.org/compbiol/s/submission-guidelines#loc-materials-and-methods

Dear Dr. Brehelin,

Thank you very much for submitting your manuscript "Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes" for consideration at PLOS Computational Biology. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by the Reviewer #2 who recommended in the confident comments to the editor several changes that must be implemented in order for the manuscript be processed further. The reviewer appreciated the attention to an important topic. Based on the confidential comments, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the reviewers recommendations.

Please have the additional data presented only for the review be included in supplemental material.  We are also asking you to qualify the conclusions you draw from the validation experiments, citing the many caveats to this work, which were previously mentioned.  Specifically lines 539-541 are a broad overstatement.  Lines 478-479 should be deleted - at best these results indicate that there should be further investigation of these LREs for a potential role in transcriptional regulation.  We recommend to delete the last line of the Abstract.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ilya Ioshikhes

Deputy Editor

PLOS Computational Biology

Erik van Nimwegen

Deputy Editor

PLOS Computational Biology

***********************

A link appears below if there are any accompanying review attachments. If you believe any reviews to be missing, please contact ploscompbiol@plos.org immediately:

[LINK]

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #2: none

**********

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 Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #2: Yes

**********

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 #2: No

Figure 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. 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example in PLOS Biology see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit 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/ploscompbiol/s/submission-guidelines#loc-materials-and-methods

References:

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.

Dear Dr. Brehelin,

We are pleased to inform you that your manuscript 'Identification of long regulatory elements in the genome of Plasmodium falciparum and other eukaryotes' has been provisionally accepted for publication in PLOS Computational Biology.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Computational Biology. 

Best regards,

Ilya Ioshikhes

Deputy Editor

PLOS Computational Biology

Erik van Nimwegen

Deputy Editor

PLOS Computational Biology

***********************************************************

Reviewer's Responses to Questions

Comments to the Authors:

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

Reviewer #2: none

**********

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 Computational Biology data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #2: Yes

**********

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 #2: No