ADAM interact with large protein complexes to regulate Histone modification, gene expression and splicing

  1. Ankit Pandey
  2. Helene Cousin
  3. Shiv Kumar
  4. Louis Taylor
  5. Ashmita Chander
  6. Kelsey Coppenrath
  7. Nikko-Ideen Shaidani
  8. Marko Horb
  9. Dominique Alfandari

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Abstract

Cranial neural crest (CNC) cells are key stem cells that contribute to most of the facial structures in vertebrates. ADAM ( A D isintegrin A nd M etalloprotease) proteins are essential for the induction and migration of the CNC. We have shown that Adam13 associates with the transcription factor Arid3a to regulate gene expression. Here we show that Adam13 modulates Histone modifications in the CNC. We show that Arid3a binding to the tfap2α promoter depends on the presence of Adam13. This association promotes the expression of one tfap2α variant expressed in the CNC that uniquely activates the expression of gene critical for CNC migration. We show that both Adam13 and human ADAM9 associate with proteins involved in histone modification and RNA splicing, a function critically affected by the loss of Adam13. We propose that ADAMs may act as extracellular sensors to modulate chromatin availability, leading to changes in gene expression and splicing.

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    Reply to the reviewers

    Overall Response.

    We would like to thank the reviewers for their analysis of the manuscript. From their comments it is clear that our manuscript was not. We completely rewrote the manuscript to focus on the central core question which was how does Adam13 regulates gene expression in general and TFap2a in particular leading to the expression of Calpain8 a protein required for CNC migration.

    The following model will be the central line of our story. It will address all of the proteins involved and mechanistical evidences that link Adam13 to one of its proven effector target Calpain8.

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)): **

    In this manuscript, Pandey et al. show that the ADAM13 protein modulates histone modifications in cranical neural crest and that the Arid3a protein binds the Tfap2a promoter in an Adam13-dependent manner and has promoter-specific effects on transcription. Furthermore, they show that the Adam13 and human ADAM9 proteins associated with histone modifiers as well as proteins involved in RNA splicing. Although the manuscript is mostly clearly written and the figures well assembled, it reads like a couple of separate and unfinished stories.*

    I believe that our story line was not clear and that the overarching questions was not well stated. We have made every effort to change this in the revised manuscript. I would like to include a figure that explains the story.

    In short:

    1 We knew that Adam13 could regulate gene expression in CNC via its cytoplasmic domain.

    2 We also knew that this required Adam13 interaction with Arid3a and that a direct target with the transcription factor TFAP2a which in turn regulates functional targets that we had identified including the protocadherin PCNS and the protease Calpain8.

    Our goal was to understand the mechanism allowing Adam13 to regulate gene expression.

    3 This first part of this manuscript shows how Adam13 modulates Histone modification in vivo in the CNC globally as well as specifically on the Tfap2a promoter. This results I an Open chromatin.

    4 Using Chip we show that Adam13 and Arid3a both bind to the Tfap2a promoter and that Arid3a binding to the first ATG depends on Adam13.

    5 Using Luciferase reporter we show that both Adam13 and Arid3a can induce expression at the first ATG.

    *They show using immunocytochemistry and qPCR that ADAM13 knockouts in CNCs afffects histone modifications. Here ChIP-seq or Cut-n-Run experiments would be more appropriate and would result in a more comprehensive understanding of the changes mediated. *

    I agree but we did not have the fund and now I have nobody working in the lab to do this experiment. These are also likely to overlap with the RNAseq data that we have and would simply add more open leads. We selected to go after the only direct target that we know which is TFAP2a and focus on this gene to understand the mechanism.

    We believe that the Chip PCR experiment are sufficient for this story.

    *The immunohistochemistry assays should at least be verified further using western blotting or other more quantiative methods. *

    Immunofluorescence and statistical analysis is a valid quantification method. Western blot of CNC explants is not trivial and requires a large amount of material. Given the small overall change we also would not expect to be able to detect the change over the noise of western blot. The Chip PCR confirms our finding in a completely independent manner.

    *The authors then show that ADAM13 interacts with a number of histone modifiers such as KDM3B, KDM4B and KMT2A but strangely they do not follow up this interesting observation to map the interactions further (apart from a co-ip with KMT2A), the domains involved, the functional role of the interactions or how they mediate the changes in chromatin modifications. *

    We selected KMT2a because it is expressed in the Hek293T cells. KMT2D has been shown to regulate CNC development in Xenopus and is responsible for the Kabuki syndrome in human. We used aphafold to predict interaction and found that Adam13 interact with the Set domain. In addition we see multiple Set- containing domain protein in our mass spec data. The mass spec is done on Human hek293T cells that express a subset of KMT proteins. We now include evidence that Adam13 interact with the KMT2D SET domain (new figure 5D)

    The authors then show that ADAM13 affects expression of the TFAP2a gene in a promoter specific manner - affecting expression from S1 but not S2.

    It is the S1 but not S3. Adam13 has no effect on S2.

    • They further show that ADAM13 affects the binding of the Arid3 transcription fator to the S1-promoter but not to the S3 promoter. However, ADAM13 was present at both promoters. Absence of ADAM13 resulted in increased H3K9me2/3 and decreased H3K4me3 at the S1 promoter whereas only H3K4me3 was changed at the S2 promoter*

    S3 not S2*. Unfortunately, they do not show how this is mediated or through which binding elements this takes place. Why is ADAM13 present at both promoters but only affects Arid3 binding at S1? *

    We agree this is a very interesting question that could be the subject of an entire publication. Promoter deletion and mutation to identify which site are bound by and modulated by Adam13/Arid3a is not trivial.

    *The authors claim that transfecting Arid3a and Adam13 together further increases expression from a reporter (Fig 4E) but this is not true as no statistical comparison is done between the singly transfected and double transfected cells. *

    This is correct, there is a small increase that is not significant with both. The fact that both proteins can induce the promoter suggest but does not prove that they can have additive roles. The loss of function experiment shows that the human Arid3a expressed in Hek293T cells is important for Adam13 increases of S1. It is possible that the dose of the endogenous Arid3a is sufficient to get full activity of Adam13.* Then the authors surprisingly start investigating association of proteins with the two isoforms of TFAP2a which in the mind of this reviewer is a different question entirely. *

    We agree and have removed this part of the manuscript.

    *They find a number of proteins involved in splicing. And the observation that ADAM13 also interacts with splicing factors is really irrelevant in terms of the story that they are trying to tell. Transcription regulation and splicing are different processes and although both affect the final outcome, mRNA, they need to be investigated separately. The link is at least not very clear from the manuscript. Again, the effects on splicing are not further investigated through functional analysis and as presented the data presented is too open-ended and lacking in clarity. *

    We agree that beside the different activities of the TFap2a isoform, the rest of the splicing regulation could be a separate study. We were interested to understand how these two isoforms could activate Calpain8 so differently this is why we looked at LC/MS/MS. We have removed this part of the story from the manuscript.*

    Additional points:

    1. In the abstract they propose that the ADAMs may act as extracellular sensors. This is not substantiated by the results. *

    As an extracellular protein translocating into the nucleus it is a possibility that we propose, but I agree this is not investigated in this manuscript. We will modify the text.*

    1. Page 5, line 16: what is referred to by 6 samples 897 proteins? Were 6 samples analyzed for each condition? The number of repeats for the mass spec analysis is not clear from the text nor are the statistical parameters used to analyse the data. This is also true for the mass spec presented in the part on TFAP2aL-S1 and Adam13 regulate splicing. Statistics and repeats are not presented. *

    In general we provide biological triplicate and use the statistical function of Scaffold to identify proteins that are significantly enriched or absent in each samples.

    When we specify 6 samples it means 6 independent proteins samples were analyzed and used for our statistic. We use Scafold T-test with a p value less than 0.05. Peptides were identified with 95% confidence and proteins with 99% confidence.*

    1. Page 6, line 19: set domain should be SET domain. *

    Yes*

    1. The number of repeats in the RNA sequencing of the CNCs is not clear from the text. *

    Three biological replicates (Different batch of embryos from different females).*

    1. The explanation of Figure C is a bit lacking. There are two forms of TFAP2a, L and S, but only one is presented in the figure. Do both forms have the extra S1-3 exons? Also, at the top of the figure it is not clear that the boxes are part of a continuous DNA sequence. Also, it is not clear which codon is not coding. *

    Xenopus laevis are pseudo tetraploid giving in most cases L and S genes in addition to the 2 alleles from being diploid. The TFAP2a gene structure is conserved between both aloalleles and is similar to the human gene. For promoter analysis and Chip PCR we chose one of the alloallele (L), given that the RNAseq data showed that both genes and variant behave the same in response to Adam13. This only becomes important in loss of function experiment in which both L and S version need to be knock down or Knock out.

    In the sashimi plot there are green and pink shaded areas. What do they denote? What exactly is lacking in the MO13 mutant - seems that a particular exon is missing suggesting skipping?*

    MO13 is a morpholino that bocks the translation of Adam13 (Already characterized with >90% of the protein absent) but does not affect Adam13 mRNA expression.*

    1. Page 11, line 9: „with either MbC or MbC and MO13" needs to be rephrased. *

    Will do *8. Page 11, line 19: „the c-terminus of....and S3) and" should be „the C-terminus of...and S3 and". **

    1. Page 15, line 10: substrateS
    2. Page 16, line 23: the sentence „increases H3K9 to the promoter of the most upstream" needs revision.
    3. Page 26, line 12: Here the authors say: „for two samples two-tail unpaired". What does this mean? Statistics should not be performed on fewer than three samples. In legnd to Figure 6 it indicates that T-test was performed on two samples.
    4. The discussion should be shortened and simplified.
    5. Figure 1 legend. How many images were quantitated for each condition? *

    At least 3 images per condition. For 3 independent experiments. (9 images per condition).*

    1. Figure 2 has a strange order of panels where G is below B.
    2. Figure 6 legend, line 12. „proteins that were significantly enriched in either of the 2 samples" is not very clear. What exactly does this mean?

    Reviewer #1 (Significance (Required)):

    If the authors follow up on either the transcription-part of the story, or the splicing part of the story, they are likely to have important results to present. However, in the present format the paper is lacking in focus as both issues are mixed together without a clear end-result. *

    We have entirely changed the paper according to these comments.

    *Reviewer #2 (Evidence, reproducibility and clarity (Required)): **

    Panday et al seeks to determine the function of ADAM13 in regulating histone modifications, gene expression and splicing during cranial neural crest development. Specifically, the authors tested how Adam13, a metalloprotease, could modify chromatin by interaction with Arid3a and Tfap2a and RNA splicing and gene expression. They then utilize knockouts in Xenopus and HEK293T cells followed by immunofluorescence, IPs, BioID, luciferase assays, Mass spec and RNA assays. Although there is some strong data in the BioID and luciferase experiments, the manuscript tells multiple stories, linking together too many things to make a compelling story. The result is a paper that is very difficult to read and understand the take home message. In addition, some of the conclusions are not supported by the data. This unfortunately means it is not ready for publication. However, I have added below some suggestions that would strengthen the manuscript. My comments are below: *

    Clarity is clearly an issue here. The new version is entirely re-written.

    Here is the take home message:

    We knew that Adam13 could regulate gene expression via its cytoplasmic domain. One of the key targets was identified as Calpain8, a protein critical for CNC migration. We subsequently showed that Adam13 and Arid3a regulated Tfap2a expression which in turn regulated Calpain8.

    In this manuscript we investigated 1) how Adam13 regulates TFAP2a and 2) how Tfap2a controls Calpain8 expression.

    The take home message is that Adam13 bind to Histone methyl transferase and changes the histone methylation code overall in the CNC and in particular at the TFAP2a promoter. This results in more open chromatin. We further find that Adam13 binds to the Tfap2a promoter in vivo and is important for Arid3a binding to the first start. Tfap2a that include this N-terminus sequence regulates Capn8 expression.*

    Major comments:

    1. I think it would be better to split out the chromatin modification function from the splicing in two separate papers. While there is a connection, having it all together makes the story difficult to follow. *

    Agree but I believe that the S1 vs S3 story of Tfap2a is important for the overall story. The new paper does not emphasize splicing.*

    1. The immunofluorescence of H3K9me2/3, in Figure 1, 2, 3 following Adam13 knockdown is not convincing. There seems to be a strong edge effect especially in Figure 2 and 3. *

    The statistical analysis shows that the results, while modest, are significant (Three independent experiments using 3 different females and 3 explants for each condition were analyzed). The edge effect observed is eliminated by the mask that we use that normalize the expression to either DAPI or Snai2. The edge effect is seen in both control and KD as well. These are further confirmed by the Chip PCR on one direct target.

    Similarly the Arid3a expression in Supp Figure 1 if anything seems increased.

    We have previously shown that Arid3a expression is not affected by Adam13 KD (Khedgikar et al). Our point here is simply that the difference in Tfap2a cannot be explained by a decrease in Arid3a expression. It is not a critical figure and was eliminated in the new manuscript.

    *It would be better to quantify by western blot and not by fluorescent intensity since it is difficult to determine what a small change in fluorescent intensity means in vivo. *

    Not all antibodies used here work by western blot and the quantity of material required for western blot is much larger than IF. Given the small overall changes and the variability observed in Western blot it is not a viable alternative.

    IF is a quantitative method that has been used widely to assert increase or decrease of protein level or post translational modification. The fact that the same post translational modification that we see in cranial neural crest explants can also be seen by ChipPCR on the Tfap2a promoter confirm this observation.

    *Also, it does not say in the text or the figure legend what these are, Xenopus explants of CNC? *

    These are CNC explants. It is now clearly stated in the figure legend.*

    1. The rationale for isolating KMT2A from the other chromatin modifiers in the dataset is not clear. *

    The new manuscript is clarifying that point. Because we are using Hek293T cells in this assay, which are human embryonic kidney derived instead of Xenopus Cranial neural crest cells, we are not interested in a specific protein but rather a family of protein that can modify histones (KMT and KDM). Our rational is if Adam13 can bind to KMT2 via the SET domain, it is likely to interact with KTM2 that are expressed in the CNC. KMT2A and D are expressed in the CNC. This is why we selected KMT2a here (Hek293T). We now include 1 co-IP with the Set domain of Xenopus KMT2D (new figure 5D)

    From the RNA-seq in Supp Figure 2 it is not changed as much as likely some of the others.

    The new manuscript addresses this point. We did not show or expect that the loss of Adam13 would affect mRNA expression of Kmt2.

    *Also, the arrow seems to indicate that it is right above the cutoff. What about other proteins with ATPase activity? That is the top hit in the Dot plot nuclear function. Would be helpful to write out Adam13 cytoplasm/nucleus here. *

    We have used another set of proteomics data that does not include the cytoplasmic/nuclear extract to simplify the results. We hope that the changes make it more obvious.

    Given that we are looking at Chromatin remodeling enzyme here we did not chose to investigate further in this report the ATPase. This is such a wide category that it could lead us away from the main story here.*

    1. The splicing information, while interesting would be better as a different manuscript. The sashimi plot requires more explanation as written. *

    We agree and think that a simple representation of the fold change of the different isoform is more obvious. It is now a minor part of figure 1 and the legend has been improved to describe the method here.

    How do you tell if the interactions are changed from this?

    I do not understand this question. The sashimi plot indicate the read through from the mRNA that goes from one exon to the next quantifying the specific exon usage. It can therefore be quantified and compared between different conditions.

    • The authors argue there is a reduction of Tfap2a in Figure 3H but half the explant is not expressing sox9 in the Adam13 knockdown. How is this kind of experiment controlled when measure areas that don't have any fluorescence because of the nature of the explants? *

    We have removed this figure as we had already shown previously by western blot that Tfap2a protein decreased in MO13 embryos. As noted on the histogram, the fluorescence is only measured in Sox9 positive cells in each explant. Three independent experiments with 3 explants for each. We also have seen a decrease by Western blot and mRNA expression (Both RNAseq and realtime PCR). In most of our explants, the vast majority of the cells are positive for Snai2 and Sox9, while those that are negative are positive for Sox3 (data not shown here). There is always less signal in the center of the explant possibly due to the penetration of antibody or interference with the signal by the cells pigment or yolk autofluorescence. Our control explants have the same effect so our quantification is valid.*

    1. The use of a germ line Xenopus mutant for Adam13 is great but how were these knockouts validated? *

    All of the KO were validated by sequencing, RNAseq and protein expression. These are now included in the supplemental figure 1.

    *More information is required here. The Chip-qPCR has a lot of variability between the samples, especially in the H3K9me2/3. *

    All ChipPCR were performed on Xenopus embryos. The variability is tested by statistical analysis and is either significant or not.

    Because these are in cell lines, this should be more consistent.

    They are not in cell lines but in Xenopus embryos.

    • In addition, it is difficult to understand what this means for cranial neural crest cells when assaying in HEK293T cells with the luciferase assay. *

    We use Luciferase assay in Hek293T cells to test if Xenopus protein can induce a specific reporter (Gain of function). We also use luciferase reporter in Xenopus to test if they can perceive the loss of a specific protein (For example Adam13).

    Our result show that Adam13 or Arid3a expression in Hek293T cells can induce the TFAP2S1 reporter. *

    1. The migration assay shows only an example of what it looks like to have defective migration. But it would be better to show control embryos, embryos with Adam13 knockdown and what the rescues look like so the reader can make their own conclusion.*

    We can certainly include this but have published this assay in multiple publication before. The picture is a single example, the histogram shows that statistical validation.

    • The argument from the section above suggests the S1 isoform is the primary one but S3 in this assay also rescues, please explain what this result means since it seems to suggest that even though these isoforms have different activity the function is similar in terms of the ability to rescue defective migration. *

    The result in Hek293T cells shows that only TFAP2aS1 can induce Calpain8, while both S1 and S3 can partially rescue CNC migration in embryos lacking Adam13. The issue here is the dose of mRNA injected for each variant might be too high. Adam13 proteolytic activity is also critical, so we do not expect a complete rescue. The fact that S1 is significantly better at rescuing than S3 is relevant here. It is possible that if we were to decrease the dose of each mRNA we would find one in which S3 no longer rescues but S1 does.

    The next section again talks about yet another protein Calpain-8. Here the authors use MO13 for luciferase assays instead of HEK293 cells. The authors do not explain why they decided to switch from cells to MO.*

    Calpain8 is one of the validate target of Adam13 that can rescue CNC migration (Cousin et al Dev Cell). We use the luciferase reporter corresponding to the Xenopus Capn8 reporter to show 1 in vivo that loss of Adam13 reduce its expression (Similar to the Capn8 gene). We then went in vitro using Hek293T cells for gain of function experiment that shows that only the Tfaps2S1 variant can induce it while S3 does not.

    We hope that the graphical summary and the new manuscript make this clear.*

    1. The experiment to IP RNA supports only the correlation that Adam9 and Adam13 bind RNA and RNA binding proteins to regulate splicing. This conclusion presented is not supported by the data presented here. While there is a sentence about why Adam9 was chosen here, it would be preferred to focus on Adam13 as the rest of the manuscript is focused on Adam13. The conclusions are generalized to all ADAMs, but ADAM13 and ADAM9 are the only ADAMs investigated in the manuscript *

    This figure is no longer included. For each of the protein classes that we identify by Masspec we try to find a validation. RNA-IP is simply a validation that Adam13 and Adam9 can bind to complexes that include RNA in a cytoplasmic domain dependent fashion. The conclusion that Adam13 and possibly ADAM9 might be involved in regulating splicing is 1) that the protein associated with Adam13 are include multiple splicing factors, 2) that the RNAseq analysis shows abnormal splicing in CNC missing Adam13 and 3) that the form of TFAP2a induced by Adam13 (S1) associate significantly more with splicing factor than the S3 isoform.

    We agree that the generalization to other ADAM is not demonstrated here but only suggested. We selected ADAM9 and ADAM19 because we have shown that they can each rescue Adam13 function in the CNC. Unfortunately there are no ADAM19 antibody that work by IP on the market. We have tested multiple company and multiple cell lines.

    We believe that the ADAM9 experiment is critical to show that the protein associated with Adam13 are not simply the result of overexpressing a different species protein sin ADAM9 is the endogenous protein.*

    Minor comments

    1. The manuscript using a lot of abbreviations (PCNS, NI, MO, SH3) and lingo that are unclear to a general reader. Please define acronyms when first used, as well as be clear on which model is being used in each experiment. *

    We have corrected this*

    1. Similarly, the figures are not labeled such that a reader would be able to understand ie MO13 should be Adam13 knockdown etc. *

    We have corrected this in the legend

    • Please identify the genes on the heatmap and some highlighted genes from volcano plot from the RNA-seq.*

    The volcano plot is from MS/MS not RNAseq. We have list of all of the genes and/or proteins corresponding to each figure in tables

    We now have a figure from the RNAseq and a subset of genes of interest are show. *4. Why use the flag tag in Figure 5? *

    We used Flag-tagged construct to only immunoprecipitated the variants and not the endogenous TFPA2a in these experiments. Also we used RFP-Flag to eliminate any protein that bound to the tag or the antibody.

    This figure is no longer in the manuscript.*

    1. Is the data in figure 4A-D the same as Supp. Figure 4A-D? *

    These are independent biological replicates of the same experiment.*

    1. Please italicize gene symbols - e.g. "key transcription factors that exemplify CNC, such as the SOX9, FOXD3, SNAI1, SNAI2, and TFAP2 family". *

    We clearly have missed some, we are using italicized for gene, and regular for proteins. It might not be clear in the text when we are referring to genes and proteins. We will correct this in the rewrite.*

    1. Please review the manuscript for grammatical and typographical errors. * We have used all available software including Word and Grammarly. We will try to improve on the next version.* **Cross-commenting**

    I think the two reviewers on one the same page on this manuscript.

    Reviewer #2 (Significance (Required)):

    If more solid, would be a conceptual advance in role of Adam13 in mediating chromatin modification and transcription factors, adds to exiting work from this lab, good for a specialize audience, my expertise is in in neural crest development, non-mammalian modes, epigenetic regulators.*

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    Referee #2

    Evidence, reproducibility and clarity

    Panday et al seeks to determine the function of ADAM13 in regulating histone modifications, gene expression and splicing during cranial neural crest development. Specifically, the authors tested how Adam13, a metalloprotease, could modify chromatin by interaction with Arid3a and Tfap2a and RNA splicing and gene expression. They then utilize knockouts in Xenopus and HEK293T cells followed by immunofluorescence, IPs, BioID, luciferase assays, Mass spec and RNA assays. Although there is some strong data in the BioID and luciferase experiments, the manuscript tells multiple stories, linking together too many things to make a compelling story. The result is a paper that is very difficult to read and understand the take home message. In addition, some of the conclusions are not supported by the data. This unfortunately means it is not ready for publication. However, I have added below some suggestions that would strengthen the manuscript. My comments are below:

    Major comments:

    1. I think it would be better to split out the chromatin modification function from the splicing in two separate papers. While there is a connection, having it all together makes the story difficult to follow.
    2. The immunofluorescence of H3K9me2/3, in Figure 1, 2, 3 following Adam13 knockdown is not convincing. There seems to be a strong edge effect especially in Figure 2 and 3. Similarly the Arid3a expression in Supp Figure 1 if anything seems increased. It would be better to quantify by western blot and not by fluorescent intensity since it is difficult to determine what a small change in fluorescent intensity means in vivo. Also, it does not say in the text or the figure legend what these are, Xenopus explants of CNC?
    3. The rationale for isolating KMT2A from the other chromatin modifiers in the dataset is not clear. From the RNA-seq in Supp Figure 2 it is not changed as much as likely some of the others. Also, the arrow seems to indicate that it is right above the cutoff. What about other proteins with ATPase activity? That is the top hit in the Dot plot nuclear function. Would be helpful to write out Adam13 cytoplasm/nucleus here.
    4. The splicing information, while interesting would be better as a different manuscript. The sashimi plot requires more explanation as written. How do you tell if the interactions are changed from this? The authors argue there is a reduction of Tfap2a in Figure 3H but half the explant is not expressing sox9 in the Adam13 knockdown. How is this kind of experiment controlled when measure areas that don't have any fluorescence because of the nature of the explants?
    5. The use of a germ line Xenopus mutant for Adam13 is great but how were these knockouts validated? More information is required here. The Chip-qPCR has a lot of variability between the samples, especially in the H3K9me2/3. Because these are in cell lines, this should be more consistent. In addition, it is difficult to understand what this means for cranial neural crest cells when assaying in HEK293T cells with the luciferase assay.
    6. The migration assay shows only an example of what it looks like to have defective migration. But it would be better to show control embryos, embryos with Adam13 knockdown and what the rescues look like so the reader can make their own conclusion. The argument from the section above suggests the S1 isoform is the primary one but S3 in this assay also rescues, please explain what this result means since it seems to suggest that even though these isoforms have different activity the function is similar in terms of the ability to rescue defective migration.
    7. The next section again talks about yet another protein Calpain-8. Here the authors use MO13 for luciferase assays instead of HEK293 cells. The authors do not explain why they decided to switch from cells to MO.
    8. The experiment to IP RNA supports only the correlation that Adam9 and Adam13 bind RNA and RNA binding proteins to regulate splicing. This conclusion presented is not supported by the data presented here. While there is a sentence about why Adam9 was chosen here, it would be preferred to focus on Adam13 as the rest of the manuscript is focused on Adam13. The conclusions are generalized to all ADAMs, but ADAM13 and ADAM9 are the only ADAMs investigated in the manuscript

    Minor comments

    1. The manuscript using a lot of abbreviations (PCNS, NI, MO, SH3) and lingo that are unclear to a general reader. Please define acronyms when first used, as well as be clear on which model is being used in each experiment.
    2. Similarly, the figures are not labeled such that a reader would be able to understand ie MO13 should be Adam13 knockdown etc.
    3. Please identify the genes on the heatmap and some highlighted genes from volcano plot from the RNA-seq.
    4. Why use the flag tag in Figure 5?
    5. Is the data in figure 4A-D the same as Supp. Figure 4A-D?
    6. Please italicize gene symbols - e.g. "key transcription factors that exemplify CNC, such as the SOX9, FOXD3, SNAI1, SNAI2, and TFAP2 family".
    7. Please review the manuscript for grammatical and typographical errors.

    Cross-commenting

    I think the two reviewers on one the same page on this manuscript.

    Significance

    If more solid, would be a conceptual advance in role of Adam13 in mediating chromatin modification and transcription factors, adds to exiting work from this lab, good for a specialize audience, my expertise is in in neural crest development, non-mammalian modes, epigenetic regulators

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    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Pandey et al. show that the ADAM13 protein modulates histone modifications in cranical neural crest and that the Arid3a protein binds the Tfap2a promoter in an Adam13-dependent manner and has promoter-specific effects on transcription. Furthermore, they show that the Adam13 and human ADAM9 proteins associated with histone modifiers as well as proteins involved in RNA splicing.

    Although the manuscript is mostly clearly written and the figures well assembled, it reads like a couple of separate and unfinished stories. They show using immunocytochemistry and qPCR that ADAM13 knockouts in CNCs afffects histone modifications. Here ChIP-seq or Cut-n-Run experiments would be more appropriate and would result in a more comprehensive understanding of the changes mediated. The immunohistochemistry assays should at least be verified further using western blotting or other more quantiative methods. The authors then show that ADAM13 interacts with a number of histone modifiers such as KDM3B, KDM4B and KMT2A but strangely they do not follow up this interesting observation to map the interactions further (apart from a co-ip with KMT2A), the domains involved, the functional role of the interactions or how they mediate the changes in chromatin modifications.

    The authors then show that ADAM13 affects expression of the TFAP2a gene in a promoter specific manner - affecting expression from S1 but not S2. They further show that ADAM13 affects the binding of the Arid3 transcription fator to the S1-promoter but not to the S3 promoter. However, ADAM13 was present at both promoters. Absence of ADAM13 resulted in increased H3K9me2/3 and decreased H3K4me3 at the S1 promoter whereas only H3K4me3 was changed at the S2 promoter. Unfortunately, they do not show how this is mediated or through which binding elements this takes place. Why is ADAM13 present at both promoters but only affects Arid3 binding at S1? The authors claim that transfecting Arid3a and Adam13 together further increases expression from a reporter (Fig 4E) but this is not true as no statistical comparison is done between the singly transfected and double transfected cells.

    Then the authors surprisingly start investigating association of proteins with the two isoforms of TFAP2a which in the mind of this reviewer is a different question entirely. They find a number of proteins involved in splicing. And the observation that ADAM13 also interacts with splicing factors is really irrelevant in terms of the story that they are trying to tell. Transcription regulation and splicing are different processes and although both affect the final outcome, mRNA, they need to be investigated separately. The link is at least not very clear from the manuscript. Again, the effects on splicing are not further investigated through functional analysis and as presented the data presented is too open-ended and lacking in clarity.

    Additional points:

    1. In the abstract they propose that the ADAMs may act as extracellular sensors. This is not substantiated by the results.
    2. Page 5, line 16: what is referred to by 6 samples 897 proteins? Were 6 samples analyzed for each condition? The number of repeats for the mass spec analysis is not clear from the text nor are the statistical parameters used to analyse the data. This is also true for the mass spec presented in the part on TFAP2aL-S1 and Adam13 regulate splicing. Statistics and repeats are not presented.
    3. Page 6, line 19: set domain should be SET domain.
    4. The number of repeats in the RNA sequencing of the CNCs is not clear from the text.
    5. The explanation of Figure C is a bit lacking. There are two forms of TFAP2a, L and S, but only one is presented in the figure. Do both forms have the extra S1-3 exons? Also, at the top of the figure it is not clear that the boxes are part of a continuous DNA sequence. Also, it is not clear which codon is not coding.
    6. In the sashimi plot there are green and pink shaded areas. What do they denote? What exactly is lacking in the MO13 mutant - seems that a particular exon is missing suggesting skipping?
    7. Page 11, line 9: „with either MbC or MbC and MO13" needs to be rephrased.
    8. Page 11, line 19: „the c-terminus of....and S3) and" should be „the C-terminus of...and S3 and".
    9. Page 15, line 10: substrateS
    10. Page 16, line 23: the sentence „increases H3K9 to the promoter of the most upstream" needs revision.
    11. Page 26, line 12: Here the authors say: „for two samples two-tail unpaired". What does this mean? Statistics should not be performed on fewer than three samples. In legnd to Figure 6 it indicates that T-test was performed on two samples.
    12. The discussion should be shortened and simplified.
    13. Figure 1 legend. How many images were quantitated for each condition?
    14. Figure 2 has a strange order of panels where G is below B.
    15. Figure 6 legend, line 12. „proteins that were significantly enriched in either of the 2 samples" is not very clear. What exactly does this mean?

    Significance

    If the authors follow up on either the transcription-part of the story, or the splicing part of the story, they are likely to have important results to present. However, in the present format the paper is lacking in focus as both issues are mixed together without a clear end-result.

  4. Version published to 10.1101/2024.08.18.608474 on bioRxiv