Randomly incorporated genomic N6 ‐methyldeoxyadenosine delays zygotic transcription initiation in a cnidarian

  1. Febrimarsa
  2. Sebastian G Gornik
  3. Sofia N Barreira
  4. Miguel Salinas‐Saavedra
  5. Christine E Schnitzler
  6. Andreas D Baxevanis
  7. Uri Frank

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Abstract

N6‐methyldeoxyadenosine (6mA) is a chemical alteration of DNA, observed across all realms of life. Although the functions of 6mA are well understood in bacteria and protists, its roles in animal genomes have been controversial. We show that 6mA randomly accumulates in early embryos of the cnidarian Hydractinia symbiolongicarpus , with a peak at the 16‐cell stage followed by clearance to background levels two cell cycles later, at the 64‐cell stage—the embryonic stage at which zygotic genome activation occurs in this animal. Knocking down Alkbh1 , a putative initiator of animal 6mA clearance, resulted in higher levels of 6mA at the 64‐cell stage and a delay in the initiation of zygotic transcription. Our data are consistent with 6mA originating from recycled nucleotides of degraded m6A‐marked maternal RNA postfertilization. Therefore, while 6mA does not function as an epigenetic mark in Hydractinia , its random incorporation into the early embryonic genome inhibits transcription. In turn, Alkbh1 functions as a genomic 6mA “cleaner,” facilitating timely zygotic genome activation. Given the random nature of genomic 6mA accumulation and its ability to interfere with gene expression, defects in 6mA clearance may represent a hitherto unknown cause of various pathologies.

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  1. Version published to 10.15252/embj.2022112934
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    Reply to the reviewers

    1. General Statements [optional]

    We appreciate the efforts the two reviewers had invested in reviewing our manuscript. Their constructive comments will help improve the paper overall.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1 (Evidence, reproducibility and clarity):

    The main point of the current report is that 6mA is present in DNA of Hydractinia, and is introduced randomly into the genome by DNA polymerases, originating from degradation of maternally provided RNA via nucleotide salvage pathway. The authors observed that 6mA levels are changing over development and peak at 16-cell stage, with a sudden decrease to 'background levels' at 64 cell stage, a stage when zygotic genome gets activated. The 6mA drop is Alkbh1 dependent, since upon K/D of Alkbh1, 6mA levels were significantly higher than in control embryos. Authors also observed that AlkbH1 K/D delays zygotic genome activation (ZGA) to later stages, but without any noticeable consequences for the proper development. To demonstrate that 6mA is not controlled via direct DNA methylation, they show that K/D of two potential DNA methyl transferases N6amt1 and Mettl4 does not have any effect on 6mA levels. Supporting their hypothesis, authors demonstrate high activity and imperfect selectivity towards non-modified nucleotides of salvage pathway during embryo development using EU labeling experiments.
    In general, the provided data support their model, however, the paper needs some improvements to include missing information and controls before publication.

    Major comments:

    1. Fig 1A shows a schematic where D3-6mA is added to only QTRAP but not QQQ experiment, usually QQQ methods also require isotopic standards for each component quantified to normalize for ionization differences and provide true quantitative information. Why did authors not use dA isotope? The ionization suppression is more pronounced at high concentrations of the components, which is true for dA in the current set up. How do authors control or at least test this?

    We have limited resources of isotopic-labelled standards. Therefore, we initially used QQQ without these standards to obtain data that covered many time points in development to identify the general pattern and key time points of high and low 6mA. Once the QQQ indicated that the 16-cell stage has the highest 6mA and that this drops to background at the 64-cell stage (and remains so later on), we performed QTRAP with the isotope-labeled standard control only for these two stages. Looking at the data resulting from both techniques, it appears that they essentially revealed the same pattern. Since the main focus of the study is on 16- and 64-cell embryos, we feel that the contribution of performing all stages by QTRAP would be marginal. We have performed control experiments to assess ionization suppression for dA and found that it was insignificant. We will add the corresponding data to the Materials and Methods section.

    Fig S1 show that quantification works well, but were the total DNA amounts comparable to the gDNA amount used in actual samples? If yes, please indicate so.

    Yes, the amounts were the same (2mg). We will change the methods sections accordingly.

    1. In line 68 and in fig 1B, 1C there is a mysterious 'Neg. Ctrl 'sample. It is unclear what was the sample and more interestingly in fig 1B the levels in this sample are 0.015% but in fig 1C it is much below 0.001%. Why there is such a striking difference for the identical sample.

    Negative controls were the same amounts (2 µg) of oligonucleotides without 6mA, DNAse-treated exactly like the samples. Figure 1B shows that QQQ is not sensitive enough to reliably detect 6mA concentrations below 0.02%, incapable to distinguish the background 6mA in the negative control from the level of 6mA in the 64-cell stage and later. Therefore, we utilized D3-6mA labelled QTRAP (Figure 1C) and determined that the level in the 64 cells stage embryos was actually ~0.01%. In the negative control, the amount was considerably lower, around 6 ppm (0.0006%).

    1. As I can see authors measured natural isotopologue of 6mA, however traces of contaminant bacterial DNA originating even from recombinant DNA degradation enzymes also have 6mA, giving background signal. In their LC/MS experiments, did authors check if the 6mA comes truly from the gDNA and not from contaminant during DNA purification and processing before MS?

    Yes, we did. As control for the level of 6mA contamination from the enzymatic digestion (sourced from bacteria), we also performed digestion of the negative control (see also answer to previous comment).

    1. Fig 1D in the legend: authors should indicate that samples were already RNAse treated, and Line 80 in the text mentions a second RNase treatment (fig S1C) to confirm the specificity of the DNA staining.

    The samples were indeed RNase-treated. We will modify the legend and the reference to figure 1D on line 80 accordingly.

    1. In lines 86-87, authors compare the LC/MS and sequencing based quantifications, and say they are consistent. Can authors make a figure analogous to fig 1B but using sequencing data?

    The data are already provided in Figure S1E. However, we used a Venn diagram to denote that these figures were generated by a different type of analysis (SMRT-sequencing as opposed to QTRAP). They are consistent but not identical.

    1. Fig 3B and 3C, controls showing the validity of EU staining, are required, such as RNAse treated sample with a signals disappearing; or control embryos without EU, thus having only background signal.

    Indeed, Fig 3C shows an RNase treated sample in which the EU signal is abolished as expected.

    1. Fig 3D specificity control is missing, control embryos without EdU having only background signal.

    The control is provided in Figure 3B. It shows a sample without EdU (treated with EU) and shows the background signal.

    1. Fig 4A legend: 'rescue solution (see text)'. Please describe in the legend what the solution was. Moreover, I did not find clear explanation in the text either, my only guess was from the materials in methods section, where authors used both shAlkbh1 and Alkbh1 mRNA with silent mutations.

    The reviewer is right, this was indeed the control that was used. We will modify the text to clarify this point.

    1. Fig 4B shows many data points per condition and the legend says EU signals (in triplicate), was these triplicate animals with multiple cells, where EU signal from each cell was plotted as a point? Please specify in the legend.

    Yes, triplicate embryos and each cell used as point. The legend will be adapted.

    1. Lines 169-170 state 'the lack of premature ZGA following N6amt1/Mettl4 knockdown (Figure S7B) indicate a lack of methyl transferase that maintains 6mA through embryogenesis' while an experiment indeed demonstrates that these are not the major players in this process, it does not prove these are not DNA methyl transferases. The absence of evidence is not the evidence of absence. I think authors should at least soften this conclusion.

    We agree and will tone down the relevant statement.

    1. Discussion section describes many experimental data that belong to Results section.

    This is a point also raised by Reviewer #2. We will move these points to the results and expand the discussion.

    1. Fig S8 I think should be a part of the main figure since it is one of the important experiments to prove the high activity and somewhat low selectivity of salvage pathway in the embryos during the critical early stages.

    We had originally left it out to save space. We prefer to leave this decision with the editor.

    1. Fig 5C the model is confusing, authors should improve it.

    It is difficult to describe a complex story using a single static model. Therefore, we will add an animation to the supplemental material to clarify the model.

    1. Fig S8 negative controls showing the specificity of CuAAC staining are missing: control animals/ embryos without EU.

    We will redo these experiments and include appropriate controls.

    1. Authors may find this reference useful: PMID: 32355286.

    We will add this ref.

    1. It is known that in mammals ADAL protein is the one which demethylates m6A nucleotide to clear it from the nucleotide pools and prevent it entering into the salvage pathway (PMID: 29884623). Does Hydractinia Symbiolongicarpus have an ADAL analog? If yes then it would be important to see if knock down/overexpression of this enzyme has any effect on the timing of ZGA. In principle, passively introduced 6mA may be regulatory to proper time the ZGA, and is controlled via an activity of Adal and Alkbh1.

    The gene is present in the Hydractinia genome. We could perform the experiments recommended. We will knock the gene down and look at the effect of this manipulation on ZGA.

    1. Material and methods are missing information:
      a. Line 370-371 provide references to the protocols listed or describe the steps.
      b. Line 373 standard column based purification protocol, what is it either explain or provide a reference.

    References will be provided.

    Minor points:
    Line 79 : 'Fig 1D and S1B', Did authors meant 'Fig1D and S1C'?
    Fig 5A Y axis title is missing.
    Line 379: 3D1-6mA should be D3-6mA please correct the other appearances as well.
    Line 405: terms : dsDNA solutions and standard solutions are confusing please rephrase.
    Line 410: Cleaned embryos, what does cleaned mean, be specific.
    Line 413: PTx is mentioned, please explain what is it.
    Line 415 and line 440 : HCl was washed and embryos were neutralized, I guess it should state : HCl was neutralized and embryos were washed with...'
    Line 431: ' before fixed by incubation in PAGA-T..." did authors meant : 'before fixation with PAGA-T...?
    Line 435: Permeabilization was done by further washes the fixed embryos with...", did authors meant: Permeabilization was done by an additional wash of the fixed embryos with...?
    Line 440: The HCL was washed with what solution?
    Line 446: For how long were the PTx washes?
    Lines 458-460: the sentence is confusing.
    Line 500: 'then used detect' should be 'then used to detect'

    We will adopt all minor points above.

    Reviewer #1 (Significance):

    There are many high profile papers describing the existence of 6mA in gDNA of different organism including insects and mammals. However, there is no proof that it has any biological function. Indeed, recent reports (PMID: 32355286 and 32203414) indicate that in mammalian cells, 6mA is indeed primarily incorporated by DNA polymerases and originates from a salvage pathway. The present report is the first in vivo evidence that confirms this to be the case more generally and, importantly, demonstrates a 6mA effect on ZGA. Hence, this is an important and timely report, which will be interesting to the field, as well as a broad audience to clarify the role of 6mA and the mechanism whereby it is introduced into gDNA.
    My expertise: Biochemistry and biology of DNA and RNA modifications, including 6mA. Fair expertise: bioinformatics analysis.

    Reviewer #2 (Evidence, reproducibility and clarity):

    The manuscript reports developmental dynamics of DNA 6mA in the cnidarian Hydractinia symbiolongicarpus. The authors describe an event of a seemingly random accumulation of this DNA modification in 16-cell stage embryos of Hydractinia symbiolongicarpus followed by an apparent clearance of 6mA by the 64-cell stage. Interestingly, the depletion of cnidarian orthologue of the putative 6mA 'demethylase', Alkbh1, results in delay in zygotic transcription accompanied by high levels of DNA 6mA in 64-cell stage cnidarian embryos. The authors suggest that the 6mA they observe originates from random misincorporation of recycled degraded m6A-marked ribo-nucleotides during early cnidarian embryogenesis.
    Overall, most of the experiments are performed at high technical level and the paper is generally nicely written. Despite this, in my opinion, the manuscript would benefit from incorporation of several addition controls and answering a number of points on the description/presenation of the data.
    Major comments:

    1. In the present version of the manuscript, the authors demonstrate the negative correlation between the presence of 6mA in the genome of cnidarian embryos and transcription. Although, the depletion of Alkbh1 leads to the delay in ZGA, strictly speaking, this effect may be independent of the catalytic function of Alkbh1. Therefore, to make a statement that m6A "random incorporation into the early embryonic genome inhibits transcription" the authors should use a catalytically inactive form of this enzyme as a control in the corresponding experiments and/or (ideally) perform in vitro transcription assays using 6mA-containing substrates.

    We could perform shRNA-mediated Alkbh1 KD and try rescue ZGA by co-injecting a catalytically-inactive Alkbh1 mRNA.

    The suggested in vitro experiment would be inconclusive for two reasons: first, Hydractinia polymerase may respond differently to 6mA; second, 6mA-mediated transcription inhibition could be indirect, requiring the in vivo context. We would like to add that transcription inhibition of 6mA has been demonstrated in vitro using yeast DNA polymerase as cited in the paper.

    1. Despite several experiments suggesting that random incorporation of recycled ribonucleotides occurs in cnidarian embryos, the source of 6mA in their DNA seems currently unclear. Would it be possible to directly test the author's hypothesis by comparing the levels of 6mA upon maternal (and possibly zygotic) depletion of the cnidarian orthologue of RNA m6A methyltransferase Mettl3 in cnidarian embryos? Alternatively, the authors could incubate the embryos in medium supplemented with labeled ribo-m6A followed by checking the levels of DNA 6mA in the embryonic DNA?

    We show that maternal mRNAs are already methylated in the early embryo (Figure 5). Therefore, it would indeed make sense to ablate Mettl3 from the maternal tissue while maternal mRNAs are methylated. However, in the absence of a conditional knockout technique in Hydractinia, this would require generation of CRISPR-Cas9 mutants that would likely die early in their development, long before reaching sexual maturity.

    Instead, we are happy to perform the other experiment suggested by the reviewer to directly demonstrate m6A to 6mA transition.

    Minor comments:

    1. It would be nice to complement Fig. 4, 5, and S7 with immunostaining of the corresponding embryos for 6mA.

    6mA immunostaning is not compatible with EU labeling because, first, they require different types of fixation (PAGA-T vs formaldehyde); second, immunostaining requires RNase treatment to remove m6A which would also remove the EU signal.

    1. The current Discussion contains references for several figures with experimental results. I suggest separating these experimental data from the Discussion. The authors should, in my opinion, make an additional Results chapter and, if possible, expand the Discussion section (that is currently minimal) speculating on significance of their results for different biological systems.

    This has also been requested by Reviewer #1. We will follow the reviewer's recommendation.

    1. The present Title reads like a clear overstatement (at least currently, please see major comments above). The Title should also reference the organism where the observations have been made.

    Following the revision, we believe that both random incorporation of 6mA and a delay in zygotic transcription will be well supported by our data. We will add the organism's name to the title as suggested.

    Reviewer #2 (Significance):

    The presence and significance of DNA 6mA in animal genomes is a very interesting and highly controversial topic. Although a number of studies suggest that relatively high levels of this DNA modification occur in multicellular eukaryotes in different biological/functional contexts, other reports challenged these observations attributing them to different experimental artifacts. In this context, the current paper that provides high quality novel experimental data on the developmental dynamics of DNA 6mA in cnidarian is extremely interesting and timely. Moreover, the author's results and the hypotheses on the function/origin of 6mA in cnidarian embryogenesis may provide a conceptual framework for the interpretation of other 6mA/m6A-related studies performed on different experimental models. Thus, this manuscript will definitely be of interest for a wide range of researchers working in the fields of epigenetics, cancer biology and developmental biology.
    I strongly believe that this is an interesting and important study that definitely deserves to be published in a high impact journal.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    Reviewer #2 suggested four experiments, three of which are either impossible in our system or expected to reveal insignificant information. First, the reviewer suggests ablating Mettl3 from the maternal tissue. While being a good idea in principle, there is no conditional ablation technique available for Hydractinia. Generating CRISPR-Cas9 mutants would likely result in embryonic lethality, long before sexual maturation has been reached.

    Second, the reviewer proposed to perform in vitro experiments with m6A-containing substrates. These experiments are unlikely to reveal useful data since the Hydractinia polymerase may respond differently to methylated adenine than commercially available polymerases. Also, transcription inhibition may be indirect, depending on the in vivo context that cannot be mimicked in vitro.

    Finally, the reviewer suggested expressing a catalytically-dead Alkbh1 in the background of endogenous Alkbh1 knockdown to demonstrate that its function depends on the enzymatic activity to remove 6mA from the genome. While we could perform the experiment (see our reply above), the information emanating from it would arguably be outside the scope of this study.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript reports developmental dynamics of DNA 6mA in the cnidarian Hydractinia symbiolongicarpus. The authors describe an event of a seemingly random accumulation of this DNA modification in 16-cell stage embryos of Hydractinia symbiolongicarpus followed by an apparent clearance of 6mA by the 64-cell stage. Interestingly, the depletion of cnidarian orthologue of the putative 6mA 'demethylase', Alkbh1, results in delay in zygotic transcription accompanied by high levels of DNA 6mA in 64-cell stage cnidarian embryos. The authors suggest that the 6mA they observe originates from random misincorporation of recycled degraded m6A-marked ribo-nucleotides during early cnidarian embryogenesis.

    Overall, most of the experiments are performed at high technical level and the paper is generally nicely written. Despite this, in my opinion, the manuscript would benefit from incorporation of several addition controls and answering a number of points on the description/presenation of the data.

    Major comments:

    1. In the present version of the manuscript, the authors demonstrate the negative correlation between the presence of 6mA in the genome of cnidarian embryos and transcription. Although, the depletion of Alkbh1 leads to the delay in ZGA, strictly speaking, this effect may be independent of the catalytic function of Alkbh1. Therefore, to make a statement that m6A "random incorporation into the early embryonic genome inhibits transcription" the authors should use a catalytically inactive form of this enzyme as a control in the corresponding experiments and/or (ideally) perform in vitro transcription assays using 6mA-containing substrates.
    2. Despite several experiments suggesting that random incorporation of recycled ribonucleotides occurs in cnidarian embryos, the source of 6mA in their DNA seems currently unclear. Would it be possible to directly test the author's hypothesis by comparing the levels of 6mA upon maternal (and possibly zygotic) depletion of the cnidarian orthologue of RNA m6A methyltransferase Mettl3 in cnidarian embryos? Alternatively, the authors could incubate the embryos in medium supplemented with labeled ribo-m6A followed by checking the levels of DNA 6mA in the embryonic DNA?

    Minor comments:

    1. It would be nice to complement Fig. 4, 5, and S7 with immunostaining of the corresponding embryos for 6mA.
    2. The current Discussion contains references for several figures with experimental results. I suggest separating these experimental data from the Discussion. The authors should, in my opinion, make an additional Results chapter and, if possible, expand the Discussion section (that is currently minimal) speculating on significance of their results for different biological systems.
    3. The present Title reads like a clear overstatement (at least currently, please see major comments above). The Title should also reference the organism where the observations have been made.

    Significance

    The presence and significance of DNA 6mA in animal genomes is a very interesting and highly controversial topic. Although a number of studies suggest that relatively high levels of this DNA modification occur in multicellular eukaryotes in different biological/functional contexts, other reports challenged these observations attributing them to different experimental artifacts. In this context, the current paper that provides high quality novel experimental data on the developmental dynamics of DNA 6mA in cnidarian is extremely interesting and timely. Moreover, the author's results and the hypotheses on the function/origin of 6mA in cnidarian embryogenesis may provide a conceptual framework for the interpretation of other 6mA/m6A-related studies performed on different experimental models. Thus, this manuscript will definitely be of interest for a wide range of researchers working in the fields of epigenetics, cancer biology and developmental biology.
    I strongly believe that this is an interesting and important study that definitely deserves to be published in a high impact journal.

  4. Review Commons

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

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The main point of the current report is that 6mA is present in DNA of Hydractinia, and is introduced randomly into the genome by DNA polymerases, originating from degradation of maternally provided RNA via nucleotide salvage pathway. The authors observed that 6mA levels are changing over development and peak at 16-cell stage, with a sudden decrease to 'background levels' at 64 cell stage, a stage when zygotic genome gets activated. The 6mA drop is Alkbh1 dependent, since upon K/D of Alkbh1, 6mA levels were significantly higher than in control embryos. Authors also observed that AlkbH1 K/D delays zygotic genome activation (ZGA) to later stages, but without any noticeable consequences for the proper development. To demonstrate that 6mA is not controlled via direct DNA methylation, they show that K/D of two potential DNA methyl transferases N6amt1 and Mettl4 does not have any effect on 6mA levels. Supporting their hypothesis, authors demonstrate high activity and imperfect selectivity towards non-modified nucleotides of salvage pathway during embryo development using EU labeling experiments.

    In general, the provided data support their model, however, the paper needs some improvements to include missing information and controls before publication.

    Major comments:

    1. Fig 1A shows a schematic where D3-6mA is added to only QTRAP but not QQQ experiment, usually QQQ methods also require isotopic standards for each component quantified to normalize for ionization differences and provide true quantitative information. Why did authors not use dA isotope? The ionization suppression is more pronounced at high concentrations of the components, which is true for dA in the current set up. How do authors control or at least test this? Fig S1 show that quantification works well, but were the total DNA amounts comparable to the gDNA amount used in actual samples? If yes, please indicate so.
    2. In line 68 and in fig 1B, 1C there is a mysterious 'Neg. Ctrl 'sample. It is unclear what was the sample and more interestingly in fig 1B the levels in this sample are 0.015% but in fig 1C it is much below 0.001%. Why there is such a striking difference for the identical sample.
    3. As I can see authors measured natural isotopologue of 6mA, however traces of contaminant bacterial DNA originating even from recombinant DNA degradation enzymes also have 6mA, giving background signal. In their LC/MS experiments, did authors check if the 6mA comes truly from the gDNA and not from contaminant during DNA purification and processing before MS?
    4. Fig 1D in the legend: authors should indicate that samples were already RNAse treated, and Line 80 in the text mentions a second RNase treatment (fig S1C) to confirm the specificity of the DNA staining.
    5. In lines 86-87, authors compare the LC/MS and sequencing based quantifications, and say they are consistent. Can authors make a figure analogous to fig 1B but using sequencing data?
    6. Fig 3B and 3C, controls showing the validity of EU staining, are required, such as RNAse treated sample with a signals disappearing; or control embryos without EU, thus having only background signal.
    7. Fig 3D specificity control is missing, control embryos without EdU having only background signal.
    8. Fig 4A legend: 'rescue solution (see text)'. Please describe in the legend what the solution was. Moreover, I did not find clear explanation in the text either, my only guess was from the materials in methods section, where authors used both shAlkbh1 and Alkbh1 mRNA with silent mutations.
    9. Fig 4B shows many data points per condition and the legend says EU signals (in triplicate), was these triplicate animals with multiple cells, where EU signal from each cell was plotted as a point? Please specify in the legend.
    10. Lines 169-170 state 'the lack of premature ZGA following N6amt1/Mettl4 knockdown (Figure S7B) indicate a lack of methyl transferase that maintains 6mA through embryogenesis' while an experiment indeed demonstrates that these are not the major players in this process, it does not prove these are not DNA methyl transferases. The absence of evidence is not the evidence of absence. I think authors should at least soften this conclusion.
    11. Discussion section describes many experimental data that belong to Results section.
    12. Fig S8 I think should be a part of the main figure since it is one of the important experiments to prove the high activity and somewhat low selectivity of salvage pathway in the embryos during the critical early stages.
    13. Fig 5C the model is confusing, authors should improve it.
    14. Fig S8 negative controls showing the specificity of CuAAC staining are missing: control animals/ embryos without EU.
    15. Authors may find this reference useful: PMID: 32355286.
    16. It is known that in mammals ADAL protein is the one which demethylates m6A nucleotide to clear it from the nucleotide pools and prevent it entering into the salvage pathway (PMID: 29884623). Does Hydractinia Symbiolongicarpus have an ADAL analog? If yes then it would be important to see if knock down/overexpression of this enzyme has any effect on the timing of ZGA. In principle, passively introduced 6mA may be regulatory to proper time the ZGA, and is controlled via an activity of Adal and Alkbh1.
    17. Material and methods are missing information:
    • a. Line 370-371 provide references to the protocols listed or describe the steps.
    • b. Line 373 standard column based purification protocol, what is it either explain or provide a reference.

    Minor points:

    Line 79 : 'Fig 1D and S1B', Did authors meant 'Fig1D and S1C'?

    Fig 5A Y axis title is missing.

    Line 379: 3D1-6mA should be D3-6mA please correct the other appearances as well.

    Line 405: terms : dsDNA solutions and standard solutions are confusing please rephrase.

    Line 410: Cleaned embryos, what does cleaned mean, be specific.

    Line 413: PTx is mentioned, please explain what is it.

    Line 415 and line 440 : HCl was washed and embryos were neutralized, I guess it should state : HCl was neutralized and embryos were washed with...'

    Line 431: ' before fixed by incubation in PAGA-T..." did authors meant : 'before fixation with PAGA-T...?

    Line 435: Permeabilization was done by further washes the fixed embryos with...", did authors meant: Permeabilization was done by an additional wash of the fixed embryos with...?

    Line 440: The HCL was washed with what solution?

    Line 446: For how long were the PTx washes?

    Lines 458-460: the sentence is confusing.

    Line 500: 'then used detect' should be 'then used to detect'

    Significance

    There are many high profile papers describing the existence of 6mA in gDNA of different organism including insects and mammals. However, there is no proof that it has any biological function. Indeed, recent reports (PMID: 32355286 and 32203414) indicate that in mammalian cells, 6mA is indeed primarily incorporated by DNA polymerases and originates from a salvage pathway. The present report is the first in vivo evidence that confirms this to be the case more generally and, importantly, demonstrates a 6mA effect on ZGA. Hence, this is an important and timely report, which will be interesting to the field, as well as a broad audience to clarify the role of 6mA and the mechanism whereby it is introduced into gDNA.

    My expertise: Biochemistry and biology of DNA and RNA modifications, including 6mA. Fair expertise: bioinformatics analysis.

  5. Version published to 10.1101/2022.10.27.514014v1 on bioRxiv