Dear Dr Bergmann,

Thank you for submitting your manuscript entitled "Cell Fate Programming by Transcription Factors and Epigenetic Machineryin Stomatal Development" for consideration as a Research Article by PLOS Biology.

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Kind regards,

Ines

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Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Dear Dr Bergmann,

Thank you for your patience while your manuscript entitled "Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development" was peer-reviewed at PLOS Biology. Please also accept my apologies for the time it has taken us to provide you with a decision. The manuscript has now been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

The reviews are attached below. As you will see, the reviewers find the conclusions novel and interesting for the field, but they also raise several issues that would need to be addressed before we consider the manuscript for publication. Reviewer 1 mostly suggests minor improvements in the figurers and asks for several clarifications, whereas Reviewer 2 is more critical and suggests an experiment expressing FAMA to see if it has a similar effect to MUTE. While this reviewer could have misinterpreted the arguments considered, we think this experiment would be important to clarify whether or not the differences in binding profile between MUTE and FAMA translate into differences in the ability to displace nucleosomes. In addition, the Academic Editor thinks that the section describing the differences between FAMA and MUTE is hard to interpret, thus we would encourage you to improve the writing to make the argument clearer to readers. The Academic Editor is also concerned that the differences between FAMA and both MUTE and SCRM might be technical because SCRM and MUTE profiles come from previous datasets, whilst FAMA profile is from a database generated for this manuscript. Thus, we think you should present in the main results section some data to confirm that the new profiling data is comparable – in terms of coverage, signal to noise, background signal, number of peaks, etc. In an ideal situation, the MUTE or SCRM profiling could be repeated to demonstrate that technical variability does not account for the differences between their profiles and that of FAMA.

In light of the reviews, which you will find at the end of this email, we would like to invite you to revise the work to thoroughly address the reviewers' reports and Academic Editor's comments. Given the revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

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You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

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Please provide the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

d) *Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

----------------------------------------------

Reviewers' comments

Rev. 1:

The manuscript by Liu et al, "Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development", investigated how MUTE, SCRM and PAMA, key bHLH TFs in stomatal development, interplay with chromatin at their target genes and how they functionally interplay with other epigenetic factors to control gene expression.

The authors first analyzed transcriptional entropy in stomatal lineage single cell RNA-seq data, and identified different phases of transcriptional regulation during stomatal development, which they intended to use as indicators of differentiated vs undifferentiated cells . They further showed that MUTE, FAMA and SCRM could indeed overcome repressive chromatin and each has the potential to induce nucleosome repositioning during these phases. Lastly, the team used TurboID-based strategy to identify the proteins that are physically associated with FAMA by LC-MS and found BRM complex components and HAC1, a histone acetyl transferase. They then demonstrated that stomatal lineage-specific knock down of components of the BRM complex or HAC1 results in reduced expression of the bHLHs' targets and plants that are impaired in forming terminally differentiated stomata. It is a work of significant interest to the plant biology and epigenetics community; and in my view, the major contribution of this work is the identification of BRM and HAC1 and demonstration of their physical association with the bHLHs and their functional interplay at target genes in controlling stomata development. Overall, the manuscript is well written and the evidence presented support their conclusion.

I only have a few minor suggestions for improving the manuscript:

Figure 1a: what is the X-axis? Please label or explain.

Lines 147-156 and Figure 2 and Figure S3: the authors analyzed and compared previously published

ChIP-seq data with their newly generated ChIP-seq data, as well as scRNA-seq data. Please add some background information indicating that these experiments were conducted with similar plant materials including growth conditions so they are comparable and they are suitable for this manuscript. This also applies to other places in this manuscript where published data were used.

Table 1: about the TurboID-based MS identification of SCRM interactors: should the numbers of peptides identified by MS for each of the interactors listed? Such data would not only show the interaction, but can also show it in a quantitative manner.

Lines 241-244: the authors said "Interaction between the bHLHs and HAC1 was relatively weak in both Y2H (Fig S5a) and bimolecular fluorescence complementation (BiFC) assays (Fig S5c), suggesting that strong interaction with HAC1 may require the presence of the heterodimer or an additional linker protein (e.g. LUH, which interacts both with SCRM and FAMA and with HAC1 (Fig S5a and b))." This statement is true for Y2H, but not for BiFC as it is done in planta.

Figure 5 and the related text: about the BRM and HAC1 amiRNAi lines, the authors did not show in this manuscript to what extent the BRM/HAC1 transcript or protein were reduced? It would be helpful to present such data. It seems that they were using their previously generated lines, but it would be helpful to confirm again, as it might change over time. However, I did notice that in the Materials and Methods section, they described the RNA extraction and qRT-PCR analysis of these lines, but I did not see any mentioning of such results in either the figures or the main text.

Line 298: cite references for "…a line we termed 'short-lived FAMA…',".

Rev. 2:

The ms by Liu et al. on cell fate reprogramming by TFs and their recruitment of epigenetic machinery in stomata provides thoughtful experiments on a key aspect of plant gene regulation—the identification of pioneering transcription factors that can access closed chromatin. This is a key mechanism in development that determines how cells embark on new fates. The paper contains a series of key experiments that provide evidence for what may be perhaps the second good case for pioneering transcription factors in plants. The experiments themselves are rigorous and appropriate tests for this major claim. The argument is strong, and, overall, the chain of evidence, the logic of the experiments, and the presentation and interpretation of the results are all excellent. I have one major question on the chain of evidence that I cannot reconcile and weakens the argument if my interpretation is correct. It may not be, in which case, I feel the ms needs some clarification. I also have several minor and easily addressed comments.

1. In the section on bHLHs having the potential to bind closed chromatin and remodel it (starting 178), the evidence showed that FAMA (but not SCRM or MUTE) had comparable affinity to nucleosome and non-nucleosome bound chromatin. This, together with binding co-occurrence data led to the conclusion that FAMA and SCRM together bind nucleosomal DNA. But then, in experiments that built on the evidence to show the potential to remodel, the authors focused on MUTE. The inducible MUTE experiment followed by MNase nucleosome occupancy analysis supported its role to change nucleosome density. To me, this was a disconnect. The binding affinity pointed to FAMA as the pioneer factor with SCRM implicated as a partner. But then the functional test was done on MUTE, for which affinity evidence did not implicate? So, this breaks the chain of evidence in my mind that build the case for FAMA as a pioneering factor. The remodeling test would need to be done on FAMA, not MUTE? Could the authors address this issue?

Minor Points

2. I struggled to try to understand how the entropy was measured until I carefully read the methods. But it's such a lengthy part of the set up that I think it should be clear in the text. E.g., I wondered whether entropy was being measured from perspective of a single gene among all the cells or among all the genes. The seems to be the case but clear explanation in the main text would help.

3. Fig. 2D, it looks like SPCH should be significant in the opposite direction from MUTE, FAMA, and SCRM. I couldn't see how these statistics were done, one tailed?

4. I got a bit hung up on the FAMA_LGK experiment. It's not clear what is progression of events is assumed to be happening in the reprogramming. Are cells never reaching the mature state or do they get there and slip back, such that remodeling would have occurred and then reverted?

Dear Dr Bergmann,

Thank you for your patience while we considered your revised manuscript entitled "Cell Fate Programming by Transcription Factors and Epigenetic Machinery in Stomatal Development" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and one of the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the data and other policy-related requests stated below.

In addition, we would like you to consider a suggestion to improve the title:

"bHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development"

As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

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*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

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DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

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Fig. 1A, C, D; Fig. 2A, D; Fig. 3A, C, D; Fig. 4A, D, E; Fig. 5A; Fig. S1B; Fig. S3A, B; Fig. S4A-D; Fig. S6A, C, D and Fig. S8B

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CODE POLICY

Per journal policy, if you have generated any custom code during the course of this investigation, please make it available without restrictions. Please ensure that the code is sufficiently well documented and reusable, and that your Data Statement in the Editorial Manager submission system accurately describes where your code can be found.

Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

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Reviewers' comments

Rev. 2: Ken Birnbaum - note that this reviewer has signed his review

The authors have addressed my comment. The revisions to the nucleosome vs. non-nucleosome binding section clarified my misinterpretation of the result.

Dear Dr Bergmann,

Thank you for the submission of your revised Research Article entitled "bHLH transcription factors cooperate with chromatin remodelers to regulate cell fate decisions during Arabidopsis stomatal development" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Peter Sarkies, I am delighted to let you know that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Many congratulations and thanks again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org