Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p

Taylor Stewart; Benjamin E. Wolfe; Stephen M. Fuchs

doi:10.1371/journal.pone.0247285

Peer Review History

Original SubmissionJanuary 31, 2021
22 Feb 2021 Decision Letter - Alvaro Galli, Editor PONE-D-21-03428 Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p PLOS ONE Dear Dr. Fuchs, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Apr 08 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." Please include your amended statements within your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript presents an analysis of the role of the poly-asparagine (polyN) domain of the yeast protein Azf1p. The authors report a role of the polyN domain in mediating Azf1p's function as a transcriptional regulator of carbon metabolism. They show that the polyN domain is not required for prion-like formation in yeast and provide a bioinformatics analysis across diverse yeast strains revealing diversity in length of the repeat across different yeast strains. Overall the experiments are performed to a high standard and are rigorous and the conclusions are supported by the data. I therefore recommend publication of this study in PLoS One because it fulfills the requirements for publication in this journal. I have one suggestion for the authors to consider. They may want to to perform rescue experiments with WT Azf1p (e.g., from a plasmid) to make sure the growth phenotypes and the changes in gene expression that they report in their ∆N mutants are definitely caused by the mutation and not some other perturbation. Reviewer #2: Dear Editor In the present paper by Stewart et al, the authors analyze the function of azf1 protein, a Zinc-finger transcription factor, regulating the expression of genes involved in growth, carbon metabolism and in cell wall integrity depending on carbon source present in growth medium. The protein contains polyN and polyQ domain typical of prions. Indeed it has been shown that azf1 exists in prion state and confer resistance to the drug radicicol. The aim of the paper is to characterize the polyN domain of azf1 and define its role in transcription and prion formation. The authors demonstrate that this domain has a subtle role in transcription but not in prion formation. Investigations on yeast prion has an important spill-over on the improvement of yeast strains for fermentation applications and small molecule production. The paper is well written and clear and experiments are carefully designed and mapped out. Moreover Data are convincing, well presented and discussed. I have only a minor drawbacks that need to be addressed before publication. 1- Pag 4 at the end of introduction “cell well integrity” must be corrected in “cell wall integrity” 2- Pag 10 paragraph “Differential roles of the polyN domain during growth in different carbon sources”: The concept expressed in line 3-4 referring to the phenotype of azf1delta strain is then repeated in line 6 with the sentence “ Consistent with previous reports, azf1∆ does not support growth on glycerol”. I think that the latter sentence can be omitted. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Aaron Gitler (Stanford University) Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0247285.r001
Revision 1
8 Apr 2021 Author Response Response to Reviewers: Reviewer #1: This manuscript presents an analysis of the role of the poly-asparagine (polyN) domain of the yeast protein Azf1p. The authors report a role of the polyN domain in mediating Azf1p's function as a transcriptional regulator of carbon metabolism. They show that the polyN domain is not required for prion-like formation in yeast and provide a bioinformatics analysis across diverse yeast strains revealing diversity in length of the repeat across different yeast strains. Overall the experiments are performed to a high standard and are rigorous and the conclusions are supported by the data. I therefore recommend publication of this study in PLoSOne because it fulfills the requirements for publication in this journal. Many thanks I have one suggestion for the authors to consider. They may want to to perform rescue experiments with WT Azf1p (e.g., from a plasmid) to make sure the growth phenotypes and the changes in gene expression that they report in their ∆N mutants are definitely caused by the mutation and not some other perturbation. This was a fantastic suggestion by Reviewer 1 and one which we spent the past several weeks trying to implement. We have included now an additional figure (S3 Fig) and updated the text to show that indeed the growth phenotypes are caused by the ∆N mutations. Reviewer #2: In the present paper by Stewart et al, the authors analyze the function of azf1 protein, a Zinc-finger transcription factor, regulating the expression of genes involved in growth, carbon metabolism and in cell wall integrity depending on carbon source present in growth medium. The protein contains polyN and polyQ domain typical of prions. Indeed it has been shown that azf1 exists in prion state and confer resistance to the drug radicicol. The aim of the paper is to characterize the polyN domain of azf1 and define its role in transcription and prion formation. The authors demonstrate that this domain has a subtle role in transcription but not in prion formation. Investigations on yeast prion has an important spill-over on the improvement of yeast strains for fermentation applications and small molecule production. The paper is well written and clear and experiments are carefully designed and mapped out. Moreover Data are convincing, well presented and discussed. Much appreciated I have only a minor drawbacks that need to be addressed before publication. 1- Pag 4 at the end of introduction “cell well integrity” must be corrected in “cell wall integrity” Fixed 2- Pag 10 paragraph “Differential roles of the polyN domain during growth in different carbon sources”: The concept expressed in line 3-4 referring to the phenotype of azf1delta strain is then repeated in line 6 with the sentence “ Consistent with previous reports, azf1∆ does not support growth on glycerol”. I think that the latter sentence can be omitted. Also corrected in the text Attachments Attachment Submitted filename: ResponsetoReviewers.docx https://doi.org/10.1371/journal.pone.0247285.r002
13 Apr 2021 Decision Letter - Alvaro Galli, Editor PONE-D-21-03428R1 Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p PLOS ONE Dear Dr. Fuchs, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please note that reviewer 2 has still some minor concerns that need to be addressed to accept the manuscript Please submit your revised manuscript by May 28 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. 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. We look forward to receiving your revised manuscript. Kind regards, Alvaro Galli Academic Editor PLOS ONE Journal Requirements: Please 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 rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: N/A ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors addressed all comments, but I still have two minor concerns: -I could not see the new figure S3 because, as the other supplementary figures, was not loaded -The authors expressed AZF1 from a plasmid where the gene is under control of GAL promoter. However, by qPCR, they observed expression of the gene also in glucose and this determined the rescue of the phenotype. It would be interesting to know the expression level of azf1 in glucose so I would add in the figure s3 the results of qPCR because, although it is known that gal promoter is leaky, the use of it to express protein in medium with glucose is not common. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Aaron Gitler Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0247285.r003
Revision 2
19 Apr 2021 Author Response Reviewer #1: (No Response) Reviewer #2: The authors addressed all comments, but I still have two minor concerns: -I could not see the new figure S3 because, as the other supplementary figures, was not loaded We have uploaded new versions of the supplemental figures (which include the additional data asked for below). The manuscript should now be complete. -The authors expressed AZF1 from a plasmid where the gene is under control of GAL promoter. However, by qPCR, they observed expression of the gene also in glucose and this determined the rescue of the phenotype. It would be interesting to know the expression level of azf1 in glucose so I would add in the figure s3 the results of qPCR because, although it is known that gal promoter is leaky, the use of it to express protein in medium with glucose is not common. We have added the qPCR data as reviewer 2 suggested to Figure S3B. We agree, this is not common, but the best we were able to use under our current working circumstances. Attachments Attachment Submitted filename: ResponsetoReviewers2.pdf https://doi.org/10.1371/journal.pone.0247285.r004
26 Apr 2021 Decision Letter - Alvaro Galli, Editor Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p PONE-D-21-03428R2 Dear Dr. Fuchs, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Alvaro Galli Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Aaron Gitler Reviewer #2: No https://doi.org/10.1371/journal.pone.0247285.r005
Formally Accepted
14 May 2021 Acceptance Letter - Alvaro Galli, Editor PONE-D-21-03428R2 Defining the role of the polyasparagine repeat domain of the S. cerevisiae transcription factor Azf1p Dear Dr. Fuchs: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Alvaro Galli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247285.r006

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