Dear Dr. Cockerill,

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.

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The data presented in this manuscript are compelling and constitute a meaningful contribution to the existing body of work in this area. Overall, the study is well executed and clearly written. With a small number of minor revisions and the addition of one clarifying experimental component, the manuscript would be strengthened further.

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Purnima Singh, PhD

Academic Editor

PLOS One

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Biotechnology and Biological Sciences Research Council grant BB/E023002/1 awarded to P.N.C.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: N/A

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: CTCF binding at DNA sequences not only insulates enhancer-promoter interactions but also plays essential role in chromatin boundary formation. It is not known whether or how those functions are distinguished at the molecular level and if any DNA sequence can specify TAD function over insulator function. Drs. Bowers and Cockerill revisit the IL3 insulator they characterized earlier. A secondary DNA sequence upstream of the CTCF core motif binding site was recently reported to be part of the strongest enhancer–promoter blocker CTCF binding sites. Using EMSA the authors find that this sequence in the IL3 insulator is, indeed, important for CTCF binding. Based on similarity they predict that CTCF ZF9-11 are in contact with this secondary motif (in addition to ZF4-7 at the core motif). In luciferase assays they find that the secondary motif is important for insulating enhancer-promoter interaction in a linear transfected DNA template. They also provide genome browser views from a published ChIP-seq dataset suggesting that such composite CTCF sites are not only present in known insulators but are also located at several known chromatin boundaries, suggesting a function at TAD formation. The authors then provide an interesting discussion based on their findings and observations. This Discussion should motivate follow-up studies that may include a more in-depth bioinformatics analysis of the two types of CTCF binding sites, whether the composite site always occurs at boundaries, whether it co-occurs with cohesin, and whether the simple site (without secondary motif) forms boundaries. In addition, future studies may test the role of the secondary sequence in boundary function by genetics.

Reviewer #2: Summary

In this study, the authors present data on the molecular role of an upstream secondary DNA binding motif for the CTCF insulator protein in vertebrates. Using a combination of bioinformatics, in vitro binding assays and an in vivo cell-based reporter system, they demonstrate a clear role for the secondary binding motif in mediating high affinity CTCF binding and functional activity as an enhancer-blocking insulator at IL3. The methods and results are scientifically sound, detailed, clearly presented and will be of interest to the transcriptional regulation, genome organization and developmental biology communities. Minor comments and one experimental suggestion are provided below to enhance the paper.

Minor comments

1. Adding a more graphical representation to summarize the CTCF protein domain organization and previously characterized binding modes (of the ZFs) would enhance Figure 1.

2. The sub-section “The upstream CTCF motif is a common feature of enhancer insulators and chromatin barriers” should be moved to the beginning of the Results section.

3. A control should be added to the luciferase assay experiments described in lines 182-188. The CTCF site should also be tested in the forward orientation to complement the reverse orientation results. Based on the hypothesis presented, this should reduce the insulator activity and importantly would shed light on the functional role of the molecular organization of the protein-DNA complex.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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An upstream secondary DNA motif within the IL3 insulator CTCF binding site is required for enhancer-blocking insulator activity

PONE-D-25-67228R1

Dear Dr. Cockerill,

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.

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Kind regards,

Purnima Singh, PhD

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #2: Yes

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Reviewer #2: The authors have addressed all prior (minor) concerns satisfactorily. I still believe the opposite orientation control suggested would provide additional mechanistic insight, but acknowledge that it may lie beyond the scope of the current manuscript.

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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

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