From the journal RSC Chemical Biology Peer review history

23-Apr-2020

Dear Dr Kanai:

Manuscript ID: CB-COM-03-2020-000029
TITLE: Synthetic Hyperacetylation of Nucleosomal Histones

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

Both Reviewers #1 and #3 have suggested that you should describe acylation levels on other subunits and at other lysine sites, which is a critical point to address. Also, the revision should include some discussion/clarification in order to address the concerns from Reviewer #2. After careful evaluation of your manuscript and the reviewers’ reports, I will be pleased to encourage the submission of a revised manuscript.

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Yours sincerely,

Cai-Guang Yang, Ph.D
Associate Editor
RSC Chemical Biology

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Reviewer 1

The manuscript by Kajino et al. is an extension of the previous work by the Kanai Group that developed an artificial catalyst system for the generation of nucleosomes carrying acetylated histones. In this study, the authors described the discovery of phenyl acetate (PAc)-based acetyl donors, which showed improved stability and lowered non-specific acetylation background compared with the previously reported N-methoxydiacetamide (NMD)-based acetyl donor. Using one of the Pac-based acetyl donors, the authors generated Xenopus laevis sperm chromatin (XSC) containing hyperacetylated histone H3 and further showed that hyperacetylated XSC inhibited DNA replication by attenuating the chromatin binding of ELYS, a protein that initiates the nuclear pore complex formation to facilitate DNA replication. The development and characterization of Pac-based acetyl donors were well-designed and performed, while some additional examinations on the hyperacetylated nucleosomes should be done before I can fully support the publication of this manuscript.

1. In the characterization of the acetylated nucleosomes, the authors only monitored the acetylation yield of lysine residues in the H3 tail, including K9, K14, K18, and K23. How about the lysine residues located in the core globular region, for example, H3K56, K79, K122. The acetylation yield should also be calculated.
2. Additionally, the authors should determine if H3 is the prominent target, or other histones also result in similar levels of acetylation after the reaction. This can be done in a semi-quantitative way by western blot using a pan-Kac antibody.
3. For Fig 4d, should add a unit (min).
4. The abbreviation NMD should be fully spelled for the first time it used in the manuscript.

Reviewer 2

This appears to be careful, rigorous work, on which I feel confident to comment from the perspective of a biochemist/cell biologist with an interest in the epigenetic regulation of DNA function. I do not have adequate expertise in synthetic chemistry to comment in detail on this aspect of the work. I find it to be described clearly and logically in a way a non-expert can understand and be reasonably assured of rigour. However, the novelty of this aspect of the work in terms of priority for publication may be questionable. This is a refinement of chemistry published previously by the group (2017) that achieved synthetic histone acetylation at much lower efficiency. The improvement, however, is substantial.

I am not convinced of the utility of chromatin acetylated ex-vivo for the study of cell biology, including studies of the nature presented as Figure 4, as informative of cellular events in vivo. The experiment presented as Figure 4 appears to have generated high-quality data. However, the authors extend their interpretation into having relevance in vivo, which I challenge. I would be very cautious about such inferences, given the extreme over-simplification of the system. Even were such studies adequately representative of the true events in an intracellular environment, I question the advantage of generating chromatin acetylated ex-vivo, as opposed to extracting from biological sources. Highly-acetylated chromatin extracted from biological material would have patterns of acetylation be representative of the in vivo condition, which will not be the case for synthetically-acetylated chromatin.

Reviewer 3

This manuscript builds upon previous work from the authors developing methods for chemical acylation of histones using a supramolecular DMAP-like catalyst. The authors find that phenolacetate is more efficient compared to their previously used reagent, and they demonstrate that ex vivo acylated histones can be used in cell lysates to recapitulate the biological effects of hyperacetylation. While in vitro modification of histones certainly has limitations, this technology still represents an advance given how few tools are available for the manipulation of histones in living cells. The work is nicely done and the manuscript well written. The one critical point for the authors to address prior to publication is:

-The manuscript reports acylation levels at H3 lysines 9, 14, 18, 23. However, these are far from the only lysine residues on histones. The authors should describe acylation levels on other subunits and at other lysine sites.

April 27th, 2020

Dr. Cai-Guang Yang
Associate Editor
RSC Chemical Biology

Dear Dr. Cai-Guang Yang:

We are grateful to you and the reviewers for carefully examining our manuscript (“Synthetic Hyperacetylation of Nucleosomal Histones.”, Manuscript ID: CB-COM-03-2020-000029). We are pleased that the reviewers indicated that our study, if properly revised, is suitable for publication in RSC Chemical Biology.

We are now submitting a revised manuscript that has been modified to address all the concerns raised by the reviewers. A point-by-point response to the reviewers’ comments is attached.

Briefly, we evaluated and described acetylation levels on other subunits and at other lysine sites, which was suggested by both Reviewers #1 and #3. In addition, we made changes of sentences to address the concerns from Reviewer #2.

We believe additional data have improved our manuscript and strengthened our conclusions, and do hope that you find this version of the manuscript suitable for publication in RSC Chemical Biology.

Thank you for your great effort in handling this manuscript.

Yours sincerely,
Motomu Kanai
Professor
The University of Tokyo

29-Apr-2020

Dear Dr Kanai:

Manuscript ID: CB-COM-03-2020-000029.R1
TITLE: Synthetic Hyperacetylation of Nucleosomal Histones

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Cai-Guang Yang, Ph.D
Associate Editor
RSC Chemical Biology

Reviewer 3

The authors have addressed my concerns.