Unraveling the Role of JMJD1B in Genome Stability and the Malignancy of Melanomas

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting paper which relies on the role of JMJD1B/KDM3B in preserving genome integrity of melanoma cells through the maintenance of histone supply to the nucleus, extending its role beyond the regulation of gene expression.

I have only few considerations to be addressed.

Perhaps the authors could explore more deeply the transcriptomic data on DEGs. Enrichment analysis and correlation of these genes using TCGA (melanoma datasets) focusing on mutational burden and patients’ prognostic may add significant value to the study.  

Title is too generic and should be presented in the context of melanoma.

In figure 1 A and B, indicate the statistical differences direct in the graphics based on proper time of observation.

Author Response

We thank the reviewer for all the very constructive comments. We believe that those comments have improved the quality of our work. Here is our response to the commentaries:

Comment 1: Perhaps the authors could explore more deeply the transcriptomic data on DEGs. Enrichment analysis and correlation of these genes using TCGA (melanoma datasets) focusing on mutational burden and patients’ prognostic may add significant value to the study.  

Answer 1: We have performed initial transcriptomic analyses on DEGs. However, it is not possible to demonstrate a direct significant prognostic effect on survival, even though this may underlie the expression of other co-expressed genes. To further interpret the differential expression and enrichment analyses, it would be necessary to significantly expand the manuscript and consider other types of cancer. This would require additional time and may exceed our initial scope.

Comment 2: The title is too generic and should be presented in the context of melanoma.

Answer 2: We have modified the title to as “Unraveling the Role of JMJD1B in Genome Stability and Malignancy of Melanoma”, as suggested by the reviewer.

Comment 3: In figure 1 A and B, indicate the statistical differences direct in the graphics based on proper time of observation.

Answer 3: We have revised Figures 2A and 2D as per the reviewer´s suggestion, adding the statistical differences within the graphs. It is worth noting that the reviewer mentioned Figures 1A and B, however, there are no statistic data on those figures. We assumed the reviewer referred to Figures 2A and 2D.

Reviewer 2 Report

Comments and Suggestions for Authors

The work by Cruz et al. describes the role of the histone demethylase JMJD1B on genome stability and malignancy of human melanoma cells. The work is mostly clearly presented and well written. The results and conclusions add on to the authors’ previous work and are thus a valuable contribution to the field. However, I have several comments that the authors need to address before I can recommend their manuscript for publication in IJMS.

1.      The Introduction and Discussion sections should be extended. The authors are of course familiar with what they study but not all readers are so well acquainted with their work and the enzyme of interest. For example, it is not described anywhere what NASP is and what the function of this protein is. What is the difference between tNASP and sNASP?

2.      I have a problem with the authors’ interpretation of the Western blots presented in Figure 1.

-          There is no increased expression of H4 in the cytoplasm visible in the Western blot, but rather an increase in the nuclear expression.

-          The same is true for both tNASP and sNASP. There is a slight increase in cytoplasmic expression but the increase in nuclear localization seems to be more prominent. Maybe a more quantitative representation of the data would be more convincing?

-          Why are there two bands for tNASP? I checked your previous work, and this was not the case.

3.      Related to Figure 1, which shows the Western blots:

-          What is S100? One can figure out it stands for the cytosolic extract, but this should be mentioned in the legend and in the Methods part.

-          The preparation of nuclear extracts used for the Western blots is not described in the Methods part.

-          Can you also briefly mention why clones 7, 12, 13, 17 and 22 are missing in the Western blot shown in Figure 1?

4.      The COMET assay results are in my view not convincing. The images are not clear and the violin plots for WT and clones 16 and 18 basically look, if not identical, then highly similar, although they are supposedly significantly different.

5.      In the Results section concerning MNase, the authors say they perform it on nuclei derived from the cells. How these nuclei were isolated is not described in the Methods part. It is written there that cells were treated with MNase. This issue should be clarified.

6.      I consider the statistical analyses in the last part of the work a nice addition but in my opinion, they are also quite speculative, especially since none of these correlations was verified experimentally. Considering this and the fact that the results on genome stability, particularly those of the COMET assay, are not substantial, I would tone down the conclusions.

Author Response

We thank the reviewer for all the very constructive comments. We believe that those comments have improved the quality of our work. Here is our response to the comments:

1. The Introduction and Discussion sections should be extended. The authors are of course familiar with what they study but not all readers are so well acquainted with their work and the enzyme of interest. For example, it is not described anywhere what NASP is and what the function of this protein is. What is the difference between tNASP and sNASP?

Thanks for highlighting the need for additional information. In this revised version, we have expanded the Introduction to provide a more detailed explanation of the histone maturation pathway, and the key proteins involved.

2. I have a problem with the authors’ interpretation of the Western blots presented in Figure 1. (a) There is no increased expression of H4 in the cytoplasm visible in the Western blot, but rather an increase in the nuclear expression. (b) The same is true for both tNASP and sNASP. There is a slight increase in cytoplasmic expression but the increase in nuclear localization seems to be more prominent. Maybe a more quantitative representation of the data would be more convincing? (b) Why are there two bands for tNASP? I checked your previous work, and this was not the case.

Thank the reviewer for pointing out this. We have now included the quantitation of each band, so the result is shown as fold changes to wild type. The bands were quantified, normalized to β-actin, and then expressed as fold changes compared to the wild-type. As for the band below tNASP in NE, we do not know what it is, but it is specific to mouse nuclear extract. We added a comment about this in the figure legend: Of note: the band below tNASP is only observed in nuclear extract derived from mouse cells.

3. Related to Figure 1, which shows the Western blots:

(a)What is S100? One can figure out it stands for the cytosolic extract, but this should be mentioned in the legend and in the Methods part.

We have fixed this mistake in the Methods section and Figure legend.

(b) The preparation of nuclear extracts used for the Western blots is not described in the Methods part.

We have fixed this mistake in the Methods section.

(c) Can you also briefly mention why clones 7, 12, 13, 17 and 22 are missing in the Western blot shown in Figure 1?

We have added this information in the figure legend.

4. The COMET assay results are in my view not convincing. The images are not clear and the violin plots for WT and clones 16 and 18 basically look, if not identical, then highly similar, although they are supposedly significantly different.

According to the statistical analysis, clone #16 is similar to the wild-type. On the other hand, clones #18 and #19 are statistically different when compared to wild-type. We mentioned this in the manuscript.

5. In the Results section concerning MNase, the authors say they perform it on nuclei derived from the cells. How these nuclei were isolated is not described in the Methods part. It is written there that cells were treated with MNase. This issue should be clarified.

Thanks to the reviewer for pointing out this mistake in the figure legend. We have fixed the error in the revised version of the manuscript. We used cells for MNase.

6. I consider the statistical analyses in the last part of the work a nice addition but in my opinion, they are also quite speculative, especially since none of these correlations was verified experimentally. Considering this and the fact that the results on genome stability, particularly those of the COMET assay, are not substantial, I would tone down the conclusions.

We have revised the conclusion of that figure as follows: Taken together, our findings suggest that JMJD1B plays an important role in maintaining genome stability, potentially acting as a safeguard against oncogenic mutations. Additionally, we have made this change throughout the text.