Regulation of DNA Methylation Through EBP1 Interaction with NLRP2 and NLRP7
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsHuman NLRP2 and NLRP7 are known maternal effect genes that regulate early embryonic development. Both genes have been implicated in the regulation of DNA methylation; however, the mechanisms involved remain poorly understood. In this manuscript, Son et al. report that NLRP2 and NLRP7 interact with EBP1 (also known as PA2G4), and that EBP1 expression is modulated by NLRP2/7. The authors further propose that global DNA methylation levels decrease upon co-expression of NLRP2/7 and EBP1, suggesting that DNA methylation may be regulated through the aforementioned interactions. While the study addresses an interesting biological question, several key claims are not fully supported by the presented data. Detailed comments follow below.
Text and Fig. 3:
The authors state in the abstract that “global DNA methylation decreased when NLRP2 and NLRP7 were expressed with EBP1,” and further claim in the discussion that “our methylation analysis revealed a significant co-functional effect of NLRP2/7 and EBP1 on DNA methylation in HEK293T cells.” However, the data presented in Figure 3 do not adequately support these conclusions. While a further reduction in DNA methylation upon co-expression is suggested, no statistical comparison is provided between the single transfections (NLRP2 or NLRP7 alone) and the co-transfection conditions. As currently presented, the effects of NLRP2 and NLRP7 appear significant and may be epistatic to EBP1, rather than synergistic.
Additionally, a more informative approach may involve analyzing DNA methylation at specific genomic loci known to be regulated during early development. It would also be beneficial to validate these findings in other cell lines beyond HEK293T to assess their generalizability.
Finally, the authors are encouraged to mutate EBP1 to test whether its interaction with NLRP2/7 is necessary for the observed methylation effects. Demonstrating that mutation of EBP1 can rescue or diminish the methylation phenotype would provide more direct evidence of a mechanistic link.
Figure 1A:
Example images of the 2Y hybrid plates should be provided, perhaps as supplementary material. Furthermore, the authors must demonstrate at least the reproducibility of the interaction results using a plasmid expressing EBP1 and negative controls.
Figure 2A:
The term “IP cont.” is not defined in the figure legend. It is presumed to refer to a GFP antibody control, but this must be explicitly stated. Moreover, a more appropriate control would be immunoprecipitation with an HA antibody in cells transfected only with myc-tagged NLRP2 or NLRP7, to control for potential non-specific binding.
Minor issues:
Abstract:
Define NLPR2 and NLRP7
Figure 1B-C:
The nature of the PCR reactions in these panels is unclear. The oligonucleotides used should be described or depicted, and corresponding negative controls must be also shown.
Figure 3A, B.D:
Clarify in the figure legend whether these panels represent global DNA methylation levels. Additionally, indicate that “CO” stands for co-transfection, to avoid ambiguity.
Figure 3C:
The rationale for presenting the expression level of EBP1 under co-transfection conditions is unclear. If the goal is to demonstrate that EBP1 is more expressed upon co-transfection, the authors should compare EBP1 levels in single versus co-transfection conditions.
Author Response
Reviewer Comment: “The authors state in the abstract that ‘global DNA methylation decreased when NLRP2 and NLRP7 were expressed with EBP1,’ and further claim in the discussion that ‘our methylation analysis revealed a significant co-functional effect of NLRP2/7 and EBP1 on DNA methylation in HEK293T cells.’ However, the data presented in Figure 3 do not adequately support these conclusions. While a further reduction in DNA methylation upon co-expression is suggested, no statistical comparison is provided between the single transfections (NLRP2 or NLRP7 alone) and the co-transfection conditions. As currently presented, the effects of NLRP2 and NLRP7 appear significant and may be epistatic to EBP1, rather than synergistic.”
Response: We acknowledge that our conclusion on co-functionality was speculative and based on observed trends rather than direct statistical comparisons between the single and co-expression groups. We have revised the text in the abstract and discussion to more accurately reflect the current data, now stating that “expression of NLRP2/7 with EBP1 appeared to further reduce global DNA methylation,” and avoiding definitive claims of synergy or co-functionality. We believe this clarification addresses the concern and aligns the interpretation more closely with the presented results.
Reviewer Comment: “Additionally, a more informative approach may involve analyzing DNA methylation at specific genomic loci known to be regulated during early development.”
Response: We agree that analysis of locus-specific methylation, particularly at developmentally regulated genes, would provide additional mechanistic insight. However, the current study was designed to assess global methylation trends, and we were unable to perform additional targeted analyses. We have added this as a limitation in the discussion and noted it as an important direction for future research.
Reviewer Comment: “It would also be beneficial to validate these findings in other cell lines beyond HEK293T to assess their generalizability.”
Response: HEK293T cells were chosen for their high transfection efficiency and ease of manipulation in initial functional screening. We acknowledge that the generalizability of our findings remains to be established in other cellular contexts, and we have now included this as a study limitation in the revised manuscript.
Reviewer Comment: “Finally, the authors are encouraged to mutate EBP1 to test whether its interaction with NLRP2/7 is necessary for the observed methylation effects.”
Response: We agree that mutational analysis of EBP1 would provide mechanistic insight into its interaction with NLRP2/7. We have just mutated Ebp1’s nuclear localization sequence within the last week, but we have yet to perform any experiments with this mutant. It would take several months to verify expression of this mutant and then perform the experiments to determine the effects on NLRP2/7 interaction and DNA methylation. If the editor and reviewers feel this is important for the current manuscript and are willing to wait until October for the data and revised manuscript, then we would be happy to provide these data. Currently, we have updated the manuscript to acknowledge this point in the discussion and highlighted it as an important follow-up direction.
Reviewer Comment: “Example images of the Y2H plates should be provided, perhaps as supplementary material. Furthermore, the authors must demonstrate at least the reproducibility of the interaction results using a plasmid expressing EBP1 and negative controls.”
Response: We have now provided the Y2H results as Supplementary Figure S1. Regarding the reproducibility of the EBP1 interaction, while we are unable to perform additional yeast-based assays at this time, we would like to highlight that the interaction between NLRP2/7 and EBP1 was independently validated through multiple orthogonal methods in mammalian HEK293T cells. Specifically:
- Co-immunoprecipitation confirmed the interaction of Myc-tagged NLRP2/7 with HA-tagged EBP1 (Figure 2A)
- Confocal immunofluorescence microscopy demonstrated cytoplasmic co-localization (Figure 2B)
- Three-channel corrected FRET analysis further confirmed a specific molecular interaction between NLRP2/7 and EBP1, but not NLRP12 and EBP1, which served as a functional negative control (Figure 2C–D)
Together, these complementary experiments provide strong evidence supporting the specificity and reproducibility of the NLRP2/7–EBP1 interaction in human cells where the interaction is more relevant as opposed to yeast, that do not naturally express these proteins.
Reviewer Comment: “The term ‘IP cont.’ is not defined in the figure legend. It is presumed to refer to a GFP antibody control, but this must be explicitly stated. Moreover, a more appropriate control would be immunoprecipitation with an HA antibody in cells transfected only with myc-tagged NLRP2 or NLRP7, to control for potential non-specific binding.”
Response: We have revised the legend for Figure 2A to clarify that “IP cont.” refers to immunoprecipitation with anti-HA antibody in cells transfected with a GFP control plasmid, serving as a non-specific binding control. Regarding the recommended control, immunoprecipitation using HA antibody in cells transfected only with Myc-tagged NLRP2 or NLRP7, we agree that this would provide a more stringent assessment of non-specific interactions. However, we emphasize that specificity of the interaction was further supported through independent confocal co-localization and FRET analyses, which included NLRP12 as a negative control. This multi-method validation strengthens the confidence in the observed interaction between NLRP2/7 and EBP1.
Minor comments such as grammatical or wording changes have been addressed and are indicated in the revised manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn the present work, Nayeon et al. try to determine the regulation of DNA methylation through ErbB3-53 Binding Protein 1 (EBP1) interaction with NLRP2 and NLRP7. This article indicates that NLRP2 and NLRP7 regulate EBP1 gene expression and global DNA methylation decreased when NLRP2 and NLRP7 were expressed with EBP1. However, there are some questions that should be explained.
Major concerns
- There are many NLR family pyrin domains, including NLRP1, NLRP2, NLRP3 and NLRP7 et al. Why only NLRP2 and NLRP7 are selected. Only NLRP12 was selected as a negative control.
- Latest references in this manuscript is 2021. More latest articles should be introduced. For example,
Kim Y, Ko HR, Hwang I, Ahn JY. ErbB3 binding protein 1 contributes to adult hippocampal neurogenesis by modulating Bmp4 and Ascl1 signaling. BMB Rep. 2024;57(4):182-187.
- The percent match is 40%, which is too higher, and should be reduced.
- English grammar and writing style should be checked and revised throughout the manuscript.
Minor concerns
- There are so many ‘we’. As a scientific paper, it is necessary to adopt the third person.
- Keywords, delete ‘NOD-Like Receptors’.
- Introduction section, the aim and hypothesis should be added in the end of introduction section. However, the results of this manuscript should not present in the introduction section.
- Line 41, move ‘nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs)’ to Line 38.
- Table 1, complete ‘For’ and ‘Rev’.
- Lines 88-92, ‘1%NP-40’, ‘4ËšC’, ‘300-500μl’, and ‘2μg’. A space should be added. Please check these throughout the manuscript.
- Line 150, ‘p<0.05’. ‘p’ should be italic. Please check this throughout the manuscript.
- Lines 168-175, different word size with others.
- Figure 1, the DNA ladder should be added the production size.
- Figure 2, the molecular weight should be added in Figure 2A. Bars should be added in Figure 2B.
- Reference section, some references are only present one author, but others are three authors. Some references are no page number. For example, Ref. 4. Please check these throughout the reference section.
The English could be improved to more clearly express the research.
Author Response
Reviewer #2:
Reviewer Comment: There are many NLR family pyrin domains, including NLRP1, NLRP2, NLRP3 and NLRP7 et al. Why only NLRP2 and NLRP7 are selected. Only NLRP12 was selected as a negative control.
Repones: NLRP2 and NLRP7 were specifically selected for this study based on strong prior evidence linking these genes to reproductive disorders and early embryonic development. NLRP2 and NLRP7 have been classified as maternal effect genes, and mutations in both genes are associated with abnormal DNA methylation patterns in oocytes and early embryos. In contrast, other NLRP family members such as NLRP1 and NLRP3 are more predominantly involved in inflammasome formation and immune responses, with no known roles in embryogenesis or imprinting regulation. To ensure specificity, we included NLRP12 as a negative control in both FRET and DNA methylation assays. NLRP12 was selected based on its immune-specific function and lack of reproductive phenotype, making it a biologically appropriate comparator.
Reviewer Comment:
Latest references in this manuscript is 2021. More latest articles should be introduced. For example, Kim Y, Ko HR, Hwang I, Ahn JY. ErbB3 binding protein 1 contributes to adult hippocampal neurogenesis by modulating Bmp4 and Ascl1 signaling. BMB Rep. 2024;57(4):182-187.
Response: we have incorporated the 2024 study by Kim et al. into the Discussion section, noting that EBP1 continues to be implicated in developmentally important pathways beyond its role in early embryogenesis. Additionally, we have included two more recent studies: Chen et al. (2025), which demonstrates a role for NLRP7 in maintaining genomic stability during early embryogenesis through alternative splicing, and Zhang et al. (2024), which highlights emerging epigenetic functions of NLRP2. Together, these updates strengthen the discussion by reinforcing the broader relevance of NLRP2, NLRP7, and EBP1 in developmental and epigenetic regulation.
Reviewer Comment:
The percent match is 40%, which is too higher, and should be reduced.
Response: It is not clear what the reviewer is referencing with the “40% match.” If this is referring to how closely this manuscript matches previously published research, it is not clear what it is matching, as this article has never been published in any peer-reviewed journal previously. It is possible that it matches Nayeon Son’s Master’s Thesis, which is held in the Missouri State University library archives, but there is no other legitimate match. Please advise us if our interpretation is incorrect.
Minor comments such as grammatical or wording changes have been addressed and are indicated in the revised manuscript.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsI am satisfied with the authors' response.
Author Response
There are no additional comments.
Reviewer 2 Report
Comments and Suggestions for AuthorsThanks for author’s responses. However, there are some questions that should be explained.
- The reasons that only NLRP2 and NLRP7 are selected should be added.
- The percent match (iThenticate report) is 40%, which is higher, and should be reduced.
- Reference section, some references are only present one author, but others are three authors. Please revise the references one by one according to MDPI style.
Author 1, A.B.; Author 2, C.D. Title of the article. Abbreviated Journal Name Year, Volume, page range.
Comments on the Quality of English LanguageThe English could be improved to more clearly express the research.
Author Response
We thank the reviewer for their helpful comments.
1. The reasons that only NLRP2 and NLRP7 are selected should be added.
Response: We have now added a paragraph to the introduction outlining why NLRP2 and NLRP7 were selected for this research. In summary, both NLRP2 and NLRP7 mutations are associated with reproductive wastage. Therefore, studying them together was a logical approach.
2. The percent match (iThenticate report) is 40%, which is higher, and should be reduced.
Response: We appreciate the reviewer's concern about any similarities and potential plagiarism. The only significant similarity between this manuscript and other works is with the student's master's thesis. The Editor has confirmed that this is not an issue for publication as similarities with a thesis are acceptable. No changes are needed per the editor.
3. Reference section, some references are only present one author, but others are three authors. Please revise the references one by one according to MDPI style.
Response: We have updated the references to match the MDPI style.
