MITF and TFEB cross-regulation in melanoma cells

Josué Ballesteros-Álvarez; Ramile Dilshat; Valerie Fock; Katrín Möller; Ludwig Karl; Lionel Larue; Margrét Helga Ögmundsdóttir; Eiríkur Steingrímsson

doi:10.1371/journal.pone.0238546

Peer Review History

Original SubmissionMay 21, 2020
19 Jun 2020 Decision Letter - Klaus Roemer, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-20-15262 MITF and TFEB cross-regulation in melanoma cells PLOS ONE Dear Dr. Steingrimsson, 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 respond to all critique, point-by-point. In particular: - Consult databases that provide insight into the expression of the MiT-TFE gene family. - Discuss pool of low-affinity bdg. sites of MITF and the possible influence of acetylation. Please submit your revised manuscript by Aug 03 2020 11:59PM. 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Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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. 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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: The manuscript is interesting and is well structured in general. The Authors conducted a number of assays using different techniques to reveal MITF and TFEB cross-regulation in melanoma cells. Moreover, in my opinion it could be interesting for a reasonable number of scientists since the studied cross-regulatory interactions need to be taken into account when considering pathways regulated by these proteins - e.g. as a potential mechanism underlying antimelnoma effect of the new treatment options. I recommend publication of this study in its present form. Reviewer #2: This manuscript sets out to investigate the potential for cross regulation of the MiT-TFE family of genes, specifically how the melanocyte master regulator MITF may be influenced by the other family members. Previous studies have shown that the MITF mRNA is expressed at much higher levels than the TFE3, TFEB and TFEC genes in human melanoma cell lines and tumors. The authors have chosen to look at two melanoma cell lines 501Mel and Skmel28 to conduct a series of experiments, and first exclude the potential of TFEC for regulation as its expression is undetectable. Through the examination of ChIP-seq data it was found that MITF can bind the MITF and TFEB loci regulatory regions but less likely interact with TFE3. From these initial experiments the focus was then on the cross regulation of MITF and TFEB. The interaction of these genes at both the mRNA and protein level was studied by transient overexpression of each protein in these melanoma lines. This found that the MITF protein induced TFEB but reduced the endogenous MITF transcript, with TFEB reducing MITF. The effect of TFEB on endogenous TFEB is not addressed? These results were validated using an siRNA KD approach. This negative feedback loop is illustrated in Figure 6. The binding of MITF to the non-consensus E-box CAGCTG sequence within the region of the first intron of TFEB was then demonstrated using a luciferase reporter construct and by DNA binding EMSA assay. This sequence element was then further analysed in the ChIP-seq dataset for MITF confirming it as an efficient binding site. The nuclear/cytoplasmic localisation of MITF and TFEB and effect of mTOR were examined by immunostaining and Torin-1 treatment of melanoma cells. This data clearly demonstrates MITF as a major presence in the nuclear fraction, whereas TFEB located in the cytoplasm and nucleus which the can move to the nucleus following inhibition of mTOR by Torin-1. Things to consider: 1. In the final sentence of the manuscript “… improving our understanding of their role in healthy tissue …” does not consider what the expression status of the MiT-TFE family may be in normal melanocytic cells. The authors may consider if there are any databases examining expression of these genes in different tissues could be consulted e.g. Melanocytes in the skin--comparative whole transcriptome analysis of main skin cell types. Reemann P, Reimann E, Ilmjärv S, Porosaar O, Silm H, Jaks V, Vasar E, Kingo K, Kõks S.PLoS One. 2014 Dec 29;9(12):e115717. This data was extracted from. S1 Table. RPKM values of genes we detected in MC, KC, FB and the whole skin. Melanocytes genes MC_1 MC_2 MC_3 MC_4 ENSG00000187098 199.197 156.316 191.509 167.668 MITF ENSG00000112561 0.400417 1.12329 0.798733 0.35943 TFEB ENSG00000068323 1.13973 1.78558 1.92091 1.13326 TFE3 ENSG00000105967 0.0451891 0.048543 0.0621599 0.0607503 TFEC 2. The recent paper describing acetylation of MITF, in which the senior author of this manuscript is a co-author, should at least be cited. Some discussion of how MITF has a pool of low affinity binding sites that may keep it in the nucleus and how may this differ or be similar for the other MiT-TFE family, and effects of acetylation on other family members such as TFEB could be included. Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution. Louphrasitthiphol P, Siddaway R, Loffreda A, Pogenberg V, Friedrichsen H, Schepsky A, Zeng Z, Lu M, Strub T, Freter R, Lisle R, Suer E, Thomas B, Schuster-Böckler B, Filippakopoulos P, Middleton M, Lu X, Patton EE, Davidson I, Lambert JP, Wilmanns M, Steingrímsson E, Mazza D, Goding CR.Mol Cell. 2020 Jun 4:S1097-2765(20)30345-2. ****** 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: No 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.0238546.r001
Revision 1
6 Jul 2020 Author Response Dear editor, We thank PLOS One for the opportunity to resubmit our manuscript after addressing the concerns of reviewers and editor. We also thank the reviewers for their critical comments and analysis. Following are the specifics of the changes we made: 1. We have made sure that the manuscript fits the PLOS ONE style requirements and renamed the files appropriately. 2. The uncropped and unadjusted images are provided for all images as supporting information. 3. The data previously referred to as “data not shown” is now available on Figshare (DOI: 10.6084/m9.figshare.12568646). Reviewer #2: Comment 1: In the final sentence of the manuscript “… improving our understanding of their role in healthy tissue …” does not consider what the expression status of the MiT-TFE family may be in normal melanocytic cells. The authors may consider if there are any databases examining expression of these genes in different tissues could be consulted e.g. Melanocytes in the skin--comparative whole transcriptome analysis of main skin cell types. Reemann P, Reimann E, Ilmjärv S, Porosaar O, Silm H, Jaks V, Vasar E, Kingo K, Kõks S.PLoS One. 2014 Dec 29;9(12):e115717. This data was extracted from. S1 Table. RPKM values of genes we detected in MC, KC, FB and the whole skin. Melanocytes genes MC_1 MC_2 MC_3 MC_4 ENSG00000187098 199.197 156.316 191.509 167.668 MITF ENSG00000112561 0.400417 1.12329 0.798733 0.35943 TFEB ENSG00000068323 1.13973 1.78558 1.92091 1.13326 TFE3 ENSG00000105967 0.0451891 0.048543 0.0621599 0.0607503 TFEC We have consulted our own array-based gene expression data where the expression of MITF, TFEB and TFE3 was determined in melanocytes and melanoma cells. We have introduced the following into the results section, page 6, lines 72-76: “Furthermore, analysis of gene expression in 23 human melanoma cell lines as well as in normal human epidermal melanocytes (NHEM) using a microarray platform revealed that the expression of TFEB and TFE3 was roughly 50-fold lower than that of MITF, whereas expression of TFEC mRNA was about 850-fold lower than that of MITF [21,31].” We also added the following in the Discussion section, page 17, lines 317-317: “MITF, TFEB and TFE3 are expressed to some extent across melanoma tumors and cell lines, whereas TFEC is not [21,31]. Similarly, TFEB and TFE3 mRNA expression can be detected in normal melanocytes, albeit at lower levels than MITF [31,41].” Comment 2. The recent paper describing acetylation of MITF, in which the senior author of this manuscript is a co-author, should at least be cited. Some discussion of how MITF has a pool of low affinity binding sites that may keep it in the nucleus and how may this differ or be similar for the other MiT-TFE family, and effects of acetylation on other family members such as TFEB could be included. Tuning Transcription Factor Availability through Acetylation-Mediated Genomic Redistribution. Louphrasitthiphol P, Siddaway R, Loffreda A, Pogenberg V, Friedrichsen H, Schepsky A, Zeng Z, Lu M, Strub T, Freter R, Lisle R, Suer E, Thomas B, Schuster-Böckler B, Filippakopoulos P, Middleton M, Lu X, Patton EE, Davidson I, Lambert JP, Wilmanns M, Steingrímsson E, Mazza D, Goding CR.Mol Cell. 2020 Jun 4:S1097-2765(20)30345-2. We have entered a paragraph discussing this interesting study in the page 20, between lines 374-383, of the Discussion that reads the following: “Recent studies have shown that the acetylation status of MITF impacts genomic occupancy as a means to modulate its transcriptional activity. Non-acetylated high DNA-binding-affinity MITF is able to bind a large pool of DNA loci including non-canonical degenerate motifs. In contrast, K243-acetylated MITF or the acetyl-mimetic K243Q mutant has low DNA-binding-affinity, yet robustly activates expression of melanocyte and melanoma target genes (54). It is possible that acetylation of MITF affects binding to the non-canonical CAGCTG motifs found in TFEB. Furthermore, mTORC1 has been shown to phosphorylate and positively regulate the p300 acetyltransferase (55), which in turn acetylates MITF (49, 54, 56), suggesting that the mTOR pathway might be capable of modulating not only the subcellular localization of the MiT-TFE factors, but also shift their genomic occupancy towards high-affinity sites.” In addition, we reason that this study may be relevant to the observation that although the expression of TFEB is low in melanocytes and in some melanoma tumor and cell lines, the protein levels of this factor may be sufficient for binding and activation for a subet of high-affinity binding sites. This statement can be found in the Discussion section, pages 17-18 between lines 317-320: “Although the expression of TFEB is low at the mRNA level, there may be sufficient protein in the cells to have major effects, especially taking into consideration the biological role of TFEB and recent findings regarding low affinity vs high affinity binding sites across the genome for a given transcription factor [42].“ Best regards Attachments Attachment Submitted filename: Response to Reviewers.doc https://doi.org/10.1371/journal.pone.0238546.r002
19 Aug 2020 Decision Letter - Klaus Roemer, Editor MITF and TFEB cross-regulation in melanoma cells PONE-D-20-15262R1 Dear Dr. Steingrimsson, 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, Klaus Roemer Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0238546.r003
Formally Accepted
21 Aug 2020 Acceptance Letter - Klaus Roemer, Editor PONE-D-20-15262R1 MITF and TFEB cross-regulation in melanoma cells Dear Dr. Steingrímsson: 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. Klaus Roemer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0238546.r004

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