UTX inhibits EMT-induced breast CSC properties by epigenetic repression of EMT genes in cooperation with LSD1 and HDAC1

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1 EMBO reports - Peer Review Process File - EMBOR Manuscript EMBOR UTX inhibits EMT-induced breast CSC properties by epigenetic repression of EMT genes in cooperation with LSD1 and HDAC1 Hee-Joo Choi, Ji-Hye Park, Mi-Kyung Park, Hee-Young Won, Hyeong-seok Joo, Chang Hoon Lee, Jeong-Yeon Lee and Gu Kong Corresponding author: Gu Kong and Jeong-Yeon Lee Review timeline: Submission date: 13 February 2015 Editorial Decision: 16 March 2015 Revision received: 12 June 2015 Editorial Decision: 08 July 2015 Revision received: 10 July 2015 Accepted: 14 July 2015 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.) Editor: Esther Schnapp 1st Editorial Decision 16 March 2015 Thank you for your patience while your manuscript was reviewed at EMBO reports. I am sorry for the slight delay in getting back to you; we have now received all enclosed reports and crosscomments from the referees. As you will see, while the referees acknowledge that the findings are potentially interesting, referee 1 raises a number of mostly technical concerns, which all need to be addressed. Both referees 2 and 3 (in her/his cross-comments) agree that qpcr should be performed instead of semi-quantitative PCR, and referee 3 also agrees that a FACS plot should be added to figure 1, that MCF-10A cells cannot be called CSC, that Snail and Zeb2 should be localized in the nucleus, and that several PCR bands look very similar. All these concerns need to be addressed. Regarding the similar bands, please do not crop the bands out of gels, but instead run all bands for one figure panel on the same gel. Please also send us the source data (entire gels) for all cropped gels and blots. Potentially duplicated bands are a very sensitive issue, and by providing source data we may prevent you from accusation of data manipulation post-publication. Given these constructive comments, we would like to invite you to revise your manuscript with the understanding that the referee concerns must be fully addressed and their suggestions taken on board. Acceptance of the manuscript will depend on a positive outcome of a second round of review. It is EMBO reports policy to allow a single round of revision only and acceptance or European Molecular Biology Organization 1

2 EMBO reports - Peer Review Process File - EMBOR rejection of the manuscript will therefore depend on the completeness of your responses included in the next, final version of the manuscript. Revised manuscripts should be submitted within three months of a request for revision; they will otherwise be treated as new submissions. Please contact us if a 3-months time frame is not sufficient for the revisions so that we can discuss the revisions further. For a short report, the revised manuscript should not exceed 35,000 characters (including spaces and references) and 5 main plus 5 supplementary figures. Please let me know if you would like to keep more than 5 main figures, as we also offer the publication of normal articles now, with no length limitations and separate Results and Discussion sections. However, I feel that your study would be more suitable for a typical short report. For a short report, please also combine the results and discussion section which may help to eliminate some redundancy that is inevitable when discussing the same experiments twice. Commonly used materials and methods can further be moved to the supplementary information, however, please note that materials and methods essential for the understanding of the experiments described in the main text must remain in the main manuscript file. Regarding data quantification, please specify the number "n" for how many experiments were performed, the bars and error bars (e.g. SEM, SD) and the test used to calculate p-values in the respective figure legends. This information is currently incomplete and must be provided in the figure legends. We now strongly encourage the publication of original source data with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure or per figure panel. I look forward to seeing a revised version of your manuscript when it is ready. Please let me know if you have questions or comments regarding the revision. REFEREE REPORTS: Referee #1: This manuscript focuses on UTX and its role in breast cancer cell lines. The authors claim that knockdown of UTX increases CSC-like populations, sphere formation, invasion and migration in both cancer cell lines and in an immortalized normal mammary epithelial cell line. This triggers downregulation of Cdh1 by upregulation of several EMT specific transcription factors (Snail, Zeb1 and Zeb2). Overexpression of Utx in cell line with low endogenouse Utx expression causes the opposite effect. Furthermore, they propose that downregulation of CDH1 is H3K27me3 independent. The authors try to establish a link between UTX, LSD1 and HDAC1 in repression of EMT specific transcription factors. The authors follow a scientifically very appealing theory about the regulation of cancer stem cells. Nevertheless they don't corroborate the hypothesis with enough experimental data and the provided data lacks confidence (e.g. not a single quantitative experiment concerning gene expression or chromatin occupancy). They use two cell lines for the main experiments which suitability I put in question. Indeed, MCF10A are a non-transformed mammary epithelial cell line, and MDA-MB-231 are derived from a pleural metastasis. Finding a CSC population in MCF10A cells is very questionable. Keeping that in mind the authors should at least use more markers for stem cell phenotypes (such as Aldh, cd49f, etc...). Moreover, it is unclear why they use different cell lines for the in vivo experiments. The localization of the EMT-TFs in the immunostaining are NOT nuclear but the ChIP analysis shows recruitment to the chromatin. Several PCR panels seem manipulated (contrast, etc...) and also duplicated in some cases. Finally, the language used in the article demands a revision for better understanding. I would thus not recommend publication at this stage. Specific Comments European Molecular Biology Organization 2

3 EMBO reports - Peer Review Process File - EMBOR Figure 1: 1) Include a better specification of cell lines (tissue origin, marker expression). Since the blots shown are clearly different blot for loading control and samples, the authors should provide the original blots. 2) For the data presented in this figure, qpcr and/or Western Blot needs to be shown. A FACS plot to show the sorted population (and the gating used) has to be added. 3) In general the percentage of CD44+/CD24-/EpCam+ cells is very low and the increase to around 2.5% is not impressive. 4) In panel E, MCF10A cells show higher sphere and colony forming capacity than tumorigenic cell lines. How do the authors explain that? Additionally they should seed the same amount of starting cells for each cell line in order to proper compare results! Figure 2: 1) From the data presented there is no clear switch in morphology, particularly for MDA-MB-231 cells. 2) qpcr data should be included instead of the conventional RT-PCR. 3) Double staining has to be done to show that cells gaining E-cad loose vimentin. Quantification of the cells acquiring or losing the respective markers has to be included for figure 2C. Figure 3: 1) The reverse experiment (with MDA-MB-231 cells) is missing. 2) Again, qpcr needs to be provided for figure 3B. 3) Snail, Zeb2 should have nuclear localization, especially since the authors argue for chromatin occupancy of these factors!! 4) qpcr needs to be provided using set of primers for TSS, gene body, upstream region (figure 3E). Figure 4: 1) Since MCF10A are non-transformed mammary epithelial cells, it is unclear how the authors can isolate a CSC population. The authors should include additional markers, such as ALDH and CD49. 2) Please define "shtet" vector. Are both control and samples treated with doxycycline (at which concentration)? 3) qpcr should be included in figures 4D and 4E. Where does the additional band seen in the Zeb1 panel 4E come from? Figure 5: 1) Control IgG (rather then no antibody control) and analysis of additional regions should be included in figure 5B and 5D. 2) Endogenous IP and reciprocal IPs would be a nice control to include here!! Figure 6: 1) It seems that several panels in figure 6B are duplicated. 2) LSD1 knockdown is not very efficient in UTX overexpressing cells making difficult the interpretation of these results. Referee #2: This manuscript explores the role of UTX in breast cancer biology and the link between UTX and EMT. The authors show that UTX is involved in mediating EMT related genes and that this has an impact on stem cell properties. Functional links are provided between UTX and c-myc and between UTX and LSD1. The communication between UTX and the EMT transcription factors is provided. This is a comprehensive and detailed study. The experiments are well conducted and importantly both overexpression and knock down experiments complement each other. The findings provide novel mechanistic insight into the role of UTX and how it modulates cell behavior. The figures are clear and the paper is well written. I only have a couple of minor comments. - All the ChIP is based on semi-quantitative PCR, where small differences can make differences look more dramatic than they actually are. The paper would benefit enormously if a couple of key ChIP experiments were redone using qpcr as an endpoint. Maybe just the key findings from Figure European Molecular Biology Organization 3

4 EMBO reports - Peer Review Process File - EMBOR B and Figure 6D. - Is c-myc binding altered by inhibition of LSD1? Referee #3: Choi et al. (Kong) EMBO Reports The authors evaluate the role of the H3K27 demethylase UTX in the generation of breast cancer stem cells and epithelial-mesenchymal transition, and come up with a scenario that UTX epigenetically represses the expression of EMT transcription factors in part dependent on its role in controlling histone methylation, and consequently controls E-cadherin expression. This manuscript was rather difficult to read, in part because of difficult and overly shortened sentences. However, as remarkable as this may sound, I do not have any questions or problems with the science (and this is very unusual for me). The conclusions drawn are justified based on the data provided, and all data are convincing, as far as I am concerned. My only suggestion/request at this point is that the authors "relax" the language to make the manuscript more accessible to the reader. This is particularly needed for the Abstract. I realize that this will require that the authors go somewhat beyond the imposed word/character limit, but it is worth it. To give some examples: - Abstract line 5: "transactivating" is inappropriately used as it has particular connotations in terms of the function in transcription control. - page 7, last line is also difficult to understand. Explain things better, relax the language. - page 9, last full sentence: Again this is hard to understand, presumably because the authors try to be too concise. So, try to make the manuscript more readable. But the science is good. Cross-comments from Referee 3: - Indeed, many PCR bands are shown and many of these look similar, which is in my view not surprising considering the nature of these experiments with using the same types of gels and the ability to present good pictures with nice contrast. I do, however, not see a reason to assume that there is data fudging or duplication. Yet, considering the semi-quantitative nature of these experiments, and the possibility to provide exposures that result in the most pleasing presentation, it is fair to ask for qrt-pcr quantification of the data, including those for ChIP experiments. - It would indeed be nice to see a few FACS plots in Fig. 1, yet I am generally fine with having most data presented as bar graphs. - I am satisfied with the use of CD44+/CD24-/EpCAM+ as stem cell markers and do not see a reason for asking more markers. This is standard and appropriate. - Considering the nature of the cell lines used, I am not surprised that the % incidence of CSCs is as low as it is. Additionally, while a 2.5-fold increase in CSC generation is "not impressive", it is substantial. In fact, TGF-b-induced stimulation of CSC generation, which is a strong stimulation, is often in that range. I agree that some cell systems provide higher percentages. - It is indeed technically not correct to consider the CD44+/CD24-/EpCAM+ population of MCF- 10A cells as "cancer stem cells", since the MCF10A cells are not cancer cells, yet are easily driven into becoming cancer cells. On the other hand, this is also what was done with the discovery of the "CSC population" of HMLE cells, which are not transformed, and that paper clearly got away with it. As the CD44+/CD24-/EpCAM+ population also characterizes non-cancer breast epithelial stem cells, maybe it is worth naming these "breast epithelial stem cells" - Fig. 2: MDA-MB231 cells are unfortunately not changing much in phenotype. They keep having a morphology in between epithelial and mesenchymal, even when they start making more E-cadherin. I am satisfied with the apparent EMT change in morphology of MCF10A cells. This is fine, and typical for these cells. - I agree that it is surprising that the immunofluorescence of Snail and Zeb2 does not really show nuclear localization. Perhaps it is worth suggesting to do a separation of nuclear versus cytoplasmic fractions combined with immunoblotting. On the other hand, these data are not really needed for this story. 1st Revision - authors' response 12 June 2015 European Molecular Biology Organization 4

5 Below are our point-by-point responses to the Editorial board and the reviewers comments Editorial requirements: As you will see, while the referees acknowledge that the findings are potentially interesting, referee 1 raises a number of mostly technical concerns, which all need to be addressed. Both referees 2 and 3 (in her/his cross-comments) agree that qpcr should be performed instead of semi-quantitative PCR, and referee 3 also agrees that a FACS plot should be added to figure 1, that MCF-10A cells cannot be called CSC, that Snail and Zeb2 should be localized in the nucleus, and that several PCR bands look very similar. All these concerns need to be addressed. In this revised manuscript, all concerns raised by the referees have been addressed as follows. i) Instead of semi-quantitative PCR, the real-time qpcr have been assessed for analysis of gene expression and chromatin occupancy in Figures 1-5 and Supplementary Figures 2-4. ii) The FACS plot for analysis of CD44 + /CD24 - /ESA + cells has been added in Figure 1. iii) The stem-like cell population in MCF-10A cells has been redefined as breast epithelial stem cells instead of CSC. iv) The cellular localization of EMT-TFs, Snail, ZEB1, and ZEB2 has been re-evaluated. v) To avoid any doubts for similar PCR bands, most experiments regarding RT-PCR and ChIP analysis have been re-analyzed using real-time qpcr. For the gel image data in Supplementary Figure 4, we also provide the entire gel image. In addition, the quality of the writing in the manuscript has been improved by two professional editors in our field. Regarding the similar bands, please do not crop the bands out of gels, but instead run all bands for one figure panel on the same gel. Please also send us the source data (entire gels) for all cropped gels and blots. Potentially duplicated bands are a very sensitive issue, and by providing source data we may prevent you from accusation of data manipulation post-publication. As mentioned above, most PCR bands have been displayed as bar graphs by performing real-time qpcr in

6 the revised manuscript. For the gel image data, we ran all bands on the same gel for one figure panel (Supplementary Figure 4). We also provide the source data for all cropped blots and gels. For a short report, the revised manuscript should not exceed 35,000 characters (including spaces and references) and 5 main plus 5 supplementary figures. Please let me know if you would like to keep more than 5 main figures, as we also offer the publication of normal articles now, with no length limitations and separate Results and Discussion sections. However, I feel that your study would be more suitable for a typical short report. For a short report, please also combine the results and discussion section which may help to eliminate some redundancy that is inevitable when discussing the same experiments twice. Commonly used materials and methods can further be moved to the supplementary information, however, please note that materials and methods essential for the understanding of the experiments described in the main text must remain in the main manuscript file. To comply with the journal format for a short report, the manuscript has been shortened within 35,000 characters, and the results and discussion sections have been combined. This revised manuscript contains 5 main plus 4 supplementary figures and 1 supplementary table. Regarding data quantification, please specify the number "n" for how many experiments were performed, the bars and error bars (e.g. SEM, SD) and the test used to calculate p-values in the respective figure legends. This information is currently incomplete and must be provided in the figure legends. As pointed out, the information for number of samples and experiments, bars and error bars, and statistical test for calculating values have been specified in the figure legends in more detail. Furthermore, each P-value has been provided in all figure data. We now strongly encourage the publication of original source data with the aim of making primary data more accessible and transparent to the reader. The source data will be published in a separate source data file online along with the accepted manuscript and will be linked to the relevant figure. If you would like to use this opportunity, please submit the source data (for example scans of entire gels or blots, data points of graphs in an excel sheet, additional images, etc.) of your key experiments together with the revised manuscript. Please include size markers for scans of entire gels, label the scans with figure and panel number, and send one PDF file per figure or per figure panel. We now submit all source data for immunoblotting and PCR analysis in one PDF file format. In the scans of the entire blots and gels, size markers have been included and the figure and panel numbers have been labeled. We also provide the source data (excel sheets) for all of in vivo xenograft experiments. Referees comments: Referee #1: This manuscript focuses on UTX and its role in breast cancer cell lines. The authors claim that knockdown of UTX increases CSC-like populations, sphere formation, invasion and migration in both cancer cell lines and in an immortalized normal mammary epithelial cell line. This triggers downregulation of Cdh1 by upregulation of several EMT specific transcription factors (Snail, Zeb1 and Zeb2). Overexpression of Utx in cell line with low endogenouse Utx expression causes the opposite effect. Furthermore, they propose that downregulation of CDH1 is H3K27me3 independent. The authors try to establish a link between UTX, LSD1 and HDAC1 in repression of EMT specific transcription factors. The authors follow a scientifically very appealing theory about the regulation of cancer stem cells.

7 Nevertheless they don't corroborate the hypothesis with enough experimental data and the provided data lacks confidence (e.g. not a single quantitative experiment concerning gene expression or chromatin occupancy). Thank you for the reviewer s thorough comments. We have addressed all of the concerns to make our data more reliable. The data in original manuscript has been extensively revised following the reviewer s suggestions. In particular, by performing real-time quantitative PCR analysis, we quantified and statistically analyzed the gene expression and chromatin occupancy levels in each figure (Fig 1B, Fig 2C, Fig 3C and 3F, Fig 4A, Fig 5B and 5D, Fig S2A and 2F-G, Fig S3B-D, and Fig S4C-D). In the Fig S2A, Fig S3C-D, and Fig S4A-B, we also provide the ChIP assay data for additional sites of target chromatins including promoter, TSS, and gene body regions according to the reviewer s request. We believe that these improved data are sufficient to support our hypothesis in this revised manuscript. They use two cell lines for the main experiments which suitability I put in question. Indeed, MCF10A are a non-transformed mammary epithelial cell line, and MDA-MB-231 are derived from a pleural metastasis. Finding a CSC population in MCF10A cells is very questionable. Keeping that in mind the authors should at least use more markers for stem cell phenotypes (such as Aldh, cd49f, etc...). Moreover, it is unclear why they use different cell lines for the in vivo experiments. Previous reports have provide evidence that MCF10A cells, a spontaneously immortalized normal human breast epithelial cells, also have a subset of cells having stem cell-like properties of which dysregulation can induce neoplastic transformation, supporting the possibility of an existence of tumor-initiating cells in immortalized normal cells (Yu et al, Genes Dev 2009, 23: ; Nishi et al, Oncogene 2014, 33: ; Hollier et al, Cancer Res 2013, 73: ). Moreover, CD44 + /CD24 - /ESA + stem cell population of mammary epithelial cells has been shown to have ability to undergo EMT (Mani et al, Cell 2008, 133: ; Yu et al, Genes Dev 2009, 23: ; Lim et al, PLoS One 2013, 8:e66558). Although these studies have demonstrated the properties of stem-like cells having a capacity to initiate tumor in non-cancerous cell lines, we agree with the reviewers opinion that the term of CSC should be carefully used in our manuscript. Therefore, we re-defined CD44 + /CD24 - /ESA + cells in MCF10A cell lines as breast epithelial stem cells instead of CSC in the revised manuscript. We also described the spheres formed from MCF10A cells as mammosphere instead of tumorsphere in this manuscript. Results and Discussion (page 5, lines and 18; page 6, line 24; page 7, lines 21-23; page 8, lines 1, 3, and 12), Materials and Methods (page 13, lines 10 and 13), Figure legends (page 21, line 13; page 22, line 24; page 23, line 17), and Supplementary figures (page 10, line 1; page 11, lines 1 and 5) sections have been revised accordingly. The use of CD44 + /CD24 - /ESA + as one of representative breast stem cell markers seems to be reasonable as referred in the cross-comments from Referee 3. To further prove the induction of a CSC-like phenotype by the loss of UTX in the immortalized cells, we performed in vivo xenograft using UTX knockdown MCF10A cells. As shown in the figure below, mice bearing MCF10A/UTX knockdown cells remarkably initiated tumors, as well as accelerated the tumor growth, while control mice could not form any tumors, suggesting that UTX loss induces neoplastic transformation of breast epithelial cells by stimulating CSC-like cells. This data was newly added in Supplementary Figure 1E. Changes in the Results and Discussion (page 5, lines 23-24; page 6, lines 1-3) section, and Supplementary methods (page 6, lines 20-23) and figures (page 9, lines 9-12) sections have been made accordingly.

8 Figure S1E, revised ver. For in vivo breast tumor xenograft experiments in the original manuscript, we chose MDA-MB-231 and SK-BR-3 cells, as representative models of UTX overexpression and knockdown, respectively. In the case of the UTX knockdown xenograft model, we preferentially considered SK-BR-3 cells than T-47D cells, since the estrogen receptor-positive T-47D breast cancer cell lines require artificial injection of estradiol for inducing tumorigenesis in mouse model that may not be able to provide intrinsic force of UTX for tumor initiation. Together with our new in vivo data using MCF-10A cells, these in vivo findings support our hypothesis that UTX is crucial for inhibiting tumor initiation as a negative regulator of stem-like cells in both immortalized and cancerous cells. In the revised manuscript, the issues above were described in the Results and Discussion sections (page 5, lines 18-24; page 6, lines 1-6) accordingly. The localization of the EMT-TFs in the immunostaining are NOT nuclear but the ChIP analysis shows recruitment to the chromatin. It seems that the merged images of immunostaining for the EMT-TFs with DAPI staining had masked the nuclear staining of the EMT-TFs due to a strong nuclear DAPI signal, because the paired unmerged data exhibited both nuclear and cytoplasmic staining of the EMT-TFs. Moreover, the overall signal intensities for IF of EMT-TFs were stronger in UTX-knockdown cells compared with control cells, supporting our finding that UTX inhibits the expression of EMT-TFs. Consistent with our observations, several previous reports have shown that SNAIL, ZEB1, and ZEB2 are expressed in both nucleus and cytoplasm (Du C et al, Cancer Res, 2010, 15;70(20):7810-9, Domingues et al, Mol. Cell Biol. 2003, 23, , Oztas et al, Experimental and Molecular Pathology, 2010, 89, , Acun T et al, BMC Cancer :223. doi: / ). Figure 3, original ver. (red box, unmerged images not provided in the original ver.)

9 To clarify these results more carefully, we re-performed this experiment in both MCF10A and MDA-MB- 231 cells and quantified the results. As shown in the figure below, EMT-TFs are preferentially expressed in the nucleus although they are also located in the cytoplasm. It might be reasonable that UTX loss increases both nuclear and cytoplasmic EMT-TFs, since UTX inhibits total expression levels of the EMT-TFs by modulating their epigenetic expression. In this revised manuscript, we displayed both merged and unmerged IF images for EMT-TFs, together with graphs showing the quantification of the data, in Figure 3E. To supply more convincing results for enhanced expression of nuclear EMT-TFs, we also included an immunoblotting data using cytoplasmic and nuclear fractions following the Referee 3 s suggestion. Data was included in the Figure 3D. Changes in the Results and Discussion (page 7, line 10), Figure legends (page 22, lines 17-19), and Supplementary methods (page 5, lines 6-14) sections were revised accordingly. Figure 3, revised ver. Several PCR panels seem manipulated (contrast, etc...) and also duplicated in some cases. Although the ChIP-PCR data of UTX, c-myc, and H3K4me2 in Figure 3B seem to be similar with those in Figure 6B due to a same experimental condition in the original manuscript, we assure that the Figure 3B and Figure 6B are originated from different batch of samples and experiments. As mentioned by Referee 3 in cross-comments, sometimes many PCR bands are shown similar. To avoid any doubts, we re-analyzed the all RT-PCR data for mrna expression and the main results of chromatin occupancy by performing quantitative real-time PCR in the revised manuscript (Fig 1B, Fig 2C, Fig 3C and 3F, Fig 4A, Fig 5B and 5D, Fig S2A and 2F-G, Fig S3B-C, and Fig S4C-D). For some of the ChIP data represented using agarose gel (Fig 5D, in original ver, which is now Fig S4A-B), we provide the uncut gel image. Changes in the Materials and Methods (page 13, lines 16-20), Figure legends (page 21, lines 7 and 23; page 22, line 16), and Supplementary methods (page 3, lines 7-11, 14, and 19) and figures (page 11, line 8) sections were revised accordingly. Detailed results for these experiments in each figure are displayed in the Specific Comments of Referee #1. Finally, the language used in the article demands a revision for better understanding. I would thus not recommend publication at this stage. For better understanding of the language used in article, our manuscript has been revised by two professional editors in our field from a professional scientific English editing service. Specific Comments

10 Figure 1: 1) Include a better specification of cell lines (tissue origin, marker expression). Since the blots shown are clearly different blot for loading control and samples, the authors should provide the original blots. As suggested by the referee, we described the specification of cell lines in more detail in Figure 1A. Because the molecular size of EZH2 protein (~100 kda) almost overlaps with those of UTX (~150 kda), detecting the EZH2 and UTX blots in a same gel was impossible. Thus, we ran a same sample in different gels. The blot for loading control (~42 kda) was from the same gel with UTX blot. For better reliability, we also provide the original uncut blot as source data. 2) For the data presented in this figure, qpcr and/or Western Blot needs to be shown. A FACS plot to show the sorted population (and the gating used) has to be added. As suggested, all PCR data showed by the band intensity in Figures 1B were reassessed by quantitative real-time PCR. The western blots which related with Figure 1 have already been presented in Supplementary Figures 1A and 1B due to a limitation for space in the main figure. We also added FACS plots at the top of quantified graphs in Figure 1C. Changes in the Figure legends (page 21, lines 7-8 and 10-11) and Supplementary methods (page 3, lines 7-11 and 14) sections were revised accordingly. Figure 1B, original ver. Figure 1B, revised ver. Figure 1C, original ver. Figure 1C, revised ver.

11 3) In general the percentage of CD44+/CD24-/EpCam+ cells is very low and the increase to around 2.5% is not impressive. Since the CSCs are very small subpopulations that normally exist around 1% of cancer cells, we believe that the very low % of CD44 + /CD24 - /ESA + cells in our data is reasonable. Indeed, previous studies reported by other groups also showed similar % of CD44 + /CD24 - /ESA + cell population within these breast cancer cell lines (Fillmore CM et al, Proc Natl Acad Sci U S A. 2010, 107(50): , Zhang et al, Cancer Res, 2012, 72(17), Samanta S et al, Oncogene. 2015, Fillmore CM et al, Breast Cancer Res. 2008;10(2):R25, SM Farabaugh et al, Oncogene (2012) 31, ). As mentioned by referee 3 in the cross-comments, several CSC regulators, which are well-known to stimulate CSC strongly, including TGF-beta, have been shown to expand the CSC population in a range of 2-folds increase, and thus, about fold changes in the CSC population by UTX overexpression or knockdown are meaningful. 4) In panel E, MCF10A cells show higher sphere and colony forming capacity than tumorigenic cell lines. How do the authors explain that? Additionally they should seed the same amount of starting cells for each cell line in order to proper compare results! As described in the Materials and Methods (page 13, lines 11-13) and Supplementary methods (page 5, lines 18-20) sections, we performed these experiments using different amount of starting cells in each cell line based the previous reports (Zhang et al, Cancer Res, 2012, 72(17); Deng H et al, PLoS One., 2014, 9(2); Qian et al, Cancer Res, 2012, 72(22); Park et al, Cancer Res, 2009, 69(6); Ochieng et al, Exp Cell Res., 2009, 315(11); Ozbay et al, Cancer Chemother Pharmacol, 2010, 65(4) for adjusting the best suitable experimental condition of each cell line. The abilities for spheres and colony formation of MCF10A cells are generally lower compared with those of cancer cells, so that many studies have used a large amount of MCF10A cells to observe the formation of spheres and colonies. Indeed, our ancillary mammosphere data using same amount of starting cells of MCF10A and other cancer cells (5,000 cells) showed no formation of spheres from MCF10A cells as shown in the figure below. We would like to note that the purpose of these experiments in our manuscript is to investigate the role of UTX knockdown or overexpression in the regulation of self-renewal properties of stem cells within an each cellular context rather than comparing the abilities for spheres and colony formation between different of cell lines. Thus, to clarify the effect of UTX on these self-renewal properties in MCF10A cells, the use of more amount of starting cells than other cancer cells was necessary. Due to a strict limit of the number of supplemental materials to display (up to 5 figures), the ancillary data above was only provided in this letter. Figure 2: 1) From the data presented there is no clear switch in morphology, particularly for MDA-MB-231 cells. As pointed out, the morphological change of UTX-overexpressing MDA-MB-231 cells into MET, the reversible process of EMT, was not as clear as those of UTX knockdown MCF10A cells into EMT, although the EMT/MET marker expressions were remarkably changed by UTX in MDA-MB-231 cells. These data may imply that UTX is involved in the initiation of MET, but UTX alone is not sufficient to reverse the EMT

12 morphologies completely in the mesenchymal cells. From the long-term maintenance of stably UTX-overexpressing MDA-MB-231 cells, we now have more convincing data for MET morphology caused by UTX over expression, possibly suggesting the requirement of long-term passages of these cells for observing complete MET. We changed the original data in Figure 2A with these new images (now Figure 2A, in the revised manuscript). Figure 2A, original ver. Figure 2A, revised ver. 2) qpcr data should be included instead of the conventional RT-PCR. As suggested, we reassessed the RT-PCR in Figure 2B (which is now Figure 2C, in revised ver.) using quantitative real-time PCR. Consistent with the previous conventional RT-PCR data, epithelial and mesenchymal markers were positively and inversely regulated by UTX expression, respectively. Changes in the Figure legends (page 21, line 23) and Supplementary methods (page 3, lines 7-11 and 14) sections were revised accordingly. Figure 2B, original ver. Figure 2C, revised ver. 3) Double staining has to be done to show that cells gaining E-cad loose vimentin. Quantification of the cells acquiring or losing the respective markers has to be included for figure 2C. By performing double staining for E-cadherin and Vimentin in UTX knockdown MCF10A cells, we confirmed loss of E-cadherin and gain of Vimentin in a same cell. In the same way, UTX overexpressing MDA-MB-231 cells have acquired E-cadherin expression and loss of Vimentin. The quantifications of these results were represented as bar graphs in this figure. Changes in the Figure legends (page 21, lines 24; page 22,

13 lines 1-2) and Supplementary methods (page 5, lines 3-4 and 6-8) sections were revised accordingly. Figure 2C, original ver. Figure 2D, revised ver. Figure 3: 1) The reverse experiment (with MDA-MB-231 cells) is missing. As suggested, we performed reverse experiments with MDA-MB-231 cells as follows. In Figure 3A, the luciferase reporter assay for measuring UTX-dependent CDH1 promoter activity was also performed in UTXoverexpressing MDA-MB-231 cells. Consistent with the results in UTX knockdown MCF10A cells, UTX overexpression upregulated CDH1 promoter activity and this effect was abolished by mutation of the E-box site within its promoter. In addition, the immunofluorescence staining of EMT-TFs, SNAIL, ZEB1, and ZEB2, was carried out in UTX-overexpressing MDA-MB-231 cells, and the data was displayed together with the results from UTX knockdown cells in the Figure 3E in the revised manuscript. Changes in the Results and Discussion (page 7, lines 5 and 10) and Figure legends (page 22, lines 12 and 19) sections were revised accordingly. Figure 3, revised ver.

14 2) Again, qpcr needs to be provided for figure 3B As suggested, we performed quantitative real-time PCR for Figure 3B (which is now Figure 3C), and obtained consistent results with the previous data. The original data have been replaced with these new data. Changes in the Figure legends (page 22, line 16) and Supplementary methods (page 3, lines 7-11 and 14)

15 sections were revised accordingly. Figure 3B, original ver. Figure 3C, revised ver. 3) Snail, Zeb2 should have nuclear localization, especially since the authors argue for chromatin occupancy of these factors!! As mentioned above, it seems that the merged images in the original data do not reflect the nuclear localization of these EMT-TFs, since the pair of unmerged images of the original data showed preferential nuclear expression of these protein, as well as some of cytoplasmic expression. To provide more convincing data for these figures, we re-performed immunofluorescence in a condition avoiding any over-stain of antibodies against the EMT-TFs. As shown in the figure below, the data quality was improved by visualizing the nuclear expression of these EMT-TFs more clearly, compared with the previous data, but EMT-TFs were still stained not only nucleus but also cytoplasm area. From previous reports, we found that SNAIL, ZEB1, and ZEB2 are expressed in both nucleus and cytoplasm, even though they preferentially function in the nucleus as transcriptional regulators (Du C et al, Cancer Res, 2010, 15;70(20):7810-9, Domingues et al, Mol. Cell Biol. 2003, 23, , Oztas et al, Experimental and Molecular Pathology, 2010, 89, , Acun T et al, BMC Cancer :223. doi: / ), possibly suggesting that UTX loss may enhance the functions of both nuclear and cytoplasmic EMT-TFs by increasing the total expression of EMT- TFs. We also confirmed this result by immunoblot analysis using cytoplasmic and nuclear fractions following the Referee 3 s suggestion. Changes in the Results and Discussion (page 7, line 10), Figure legends (page 22, lines 17-19), and Supplementary methods (page 5, lines 6-14) sections were revised accordingly. Figure 3C, original ver. Figure 3D and 3E, revised ver. 4) qpcr needs to be provided using set of primers for TSS, gene body, upstream region (figure 3E).

16 Following the reviewer s suggestion, we additionally performed the ChIP assay using primers for proximal promoter containing TSS, gene body (about +1kb of CDH1 gene) and upstream (-3kb of CDH1 promoter) regions of CDH1. These primers were designed based on the presence of E-box sites, because SNAIL, ZEB1, and ZEB2 have been known to recognize the E-boxes to repress CDH1. At upstream region of CDH1, H3K27me3 level was enriched by loss of UTX, while EMT-TFs were not recruited. Similar with the results in the upstream region, there were no recruitments of EMT-TFs in the gene body region. Together, these data indicate that the SNAIL, ZEB1, and ZEB2 EMT-TFs preferentially bind to the proximal promoter regions around TSS of CDH1 and regulate its expression. Data were included in Figure 3F (for proximal promoter) and Supplementary Figure S2A (for gene body and upstream) in the revised manuscript. Changes in the Results and Discussion (page 7, lines 15-18), Materials and Methods (page 13, lines 16-20), and Supplementary methods (page 3, line 19; page 4, lines 1-5) and figures (page 10, line 4) sections were revised accordingly. Figure 3D, original ver. Figure 3F and Supplementary figure S2A, revised ver. Figure 4: 1) Since MCF10A are non-transformed mammary epithelial cells, it is unclear how the authors can isolate a CSC population. The authors should include additional markers, such as ALDH and CD49. As we mentioned in the section of the responses to the overall summary of reviewer #1 s comment in detail, the CD44 + /CD24 - /ESA + cell population and spheres formed from MCF10A cells were carefully defined as breast epithelial stem cells and mammosphere, instead of CSC and tumorsphere. Previous reports have shown the dysregulation of CD44 + /CD24 - /ESA + cells in MCF10A cells could lead to malignant transformation of these immortalized cells (Yu et al, Genes Dev 2009, 23: ; Nishi et al, Oncogene 2014, 33: ; Hollier et al, Cancer Res 2013, 73: ). Although we did not include other stem cell markers for our present study based on the cross-comments of Referee 3, we confirmed that UTX loss confers tumorigenic potential on the MCF10A immortalized breast epithelial cells by performing in vivo xenograft experiment using UTX knockdown MCF10A cells. This data was newly included in the Supplementary Figure 1E and discussed in the Results and Discussion section (page 5, lines 23-24; page 6, lines 1-3). Changes in the Supplementary methods (page 6, lines 20-23) and figures (page 9, lines 9-12) sections have been made accordingly.

17 Supplementary figure S1E, revised ver. 2) Please define "shtet" vector. Are both control and samples treated with doxycycline (at which concentration)? We apologize for the poor explanation of shtet vector in the Figure 4 and manuscript. After a generation of cell lines expressing either Tet-inducible EZH2 shrna or control shrnas, both control and EZH2 shrna cells were treated with 2 ug/ml doxycline. Thus, the shtet means the Tet-treated control shrna-expressing cells. To avoid confusion, we altered labeling of shtet to shcon in Figure 4 (Supplementary Figure 2, in revised ver.) and described in more detail in the Supplementary figure legends (page 11, lines 2-3). 3) qpcr should be included in figures 4D and 4E. Where does the additional band seen in the Zeb1 panel 4E come from? As suggested by the Referee, we included quantitative real-time PCR results instead of the conventional RT-PCR and ChIP data in Figures 4D and 4E (which are now Supplementary Figures 2F and 2G). Changes in the Materials and Methods (page 13, lines 16-20), Supplementary methods (page 3, lines 7-11, 14, and 19) and figures (page 11, lines 8 and 10) sections were revised accordingly. We assume that the additional band in the original figure is possibly a non-specific band or rest of primers. Figure 4D and 4E, original ver.

18 Supplementary figure S2F and S2G, revised ver. Figure 5: 1) Control IgG (rather than no antibody control) and analysis of additional regions should be included in figure 5B and 5D. As pointed out, we re-performed all of the ChIP assays using control IgG as negative controls, instead of no antibody control, in this revised manuscript. Furthermore, we analyzed the occupancy of the indicated molecules and the status of histone modifications in additional regions, in Figure 5B and 5D (which are now Figure 4A and Supplementary figure S3B-S3D, and Supplementary figure S4A-B, in revised ver.). Similar results were shown in these additional regions. Changes in the Figure legends (page 22, line 23) section, and Supplementary methods (page 3, lines 19-26; page 4, line 1) and figures (page 13, lines 3-9) sections were made accordingly. Figure 5B, original ver. Figure 4A and Supplementary Figure S3B-D, revised ver.

19

20 Figure 5D, original ver. Supplementary figure S4, revised ver. 2) Endogenous IP and reciprocal IPs would be a nice control to include here!! To provide suitable controls for the IP result, we additionally performed endogenous IP and reciprocal IPs in Figure 5C (which is now Figures 4B and 4C, in revised ver.). Together with the previous data, these results clearly suggest that HDAC1 and DNMT1 physically interact with UTX regardless of c-myc expression and that c-myc can bind to UTX, HDAC1 and DNMT1. Data was newly displayed in Figures 4B (for exogenous IP) and 4C (for endogenous IP). Changes in the Results and Discussion (page 9, lines 14-15), Figure legends (page 23, lines 13-15), and Supplementary methods (page 4, lines 9-11) sections were made accordingly. Figure 5C, original ver.

21 Figure 6: Figure 4B and 4C, revised ver.

22 1) It seems that several panels in figure 6B are duplicated. As we mentioned above, the ChIP-PCR data of UTX, c-myc, and H3K4me2 in Figure 6B and Figure 5B originated from different batch of samples and experiments and were not duplicated. To avoid any doubts and to quantify the ChIP results, the data in Figure 6B (which is now Figure 5B) has been changed with real-time qpcr. We also displayed the overlapped molecules between Figure 5B and 6B (now Fig 5B) only in the Figure 5B (now Fig 4A and Fig S3B-D, in revised ver.) following the story flow. Changes in the Materials and Methods (page 13, lines 16-20), Figure legends (page 24, line 1), and Supplementary methods (page 3, lines 19-26) and figures (page 13, lines 6-9) sections were revised accordingly. Figure 6B, original ver. Figure 5B, revised ver. 2) LSD1 knockdown is not very efficient in UTX overexpressing cells making difficult the interpretation of these results. To provide more convincing data, we replicated this experiment carefully and obtained more clear results. The original data was replaced with this new data. We also provide the source data (uncut blots) for these blots. Figure 6C, original ver. Figure 5C, revised ver. Referee #2: This manuscript explores the role of UTX in breast cancer biology and the link between UTX and EMT. The authors show that UTX is involved in mediating EMT related genes and that this has an impact on stem cell properties. Functional links are provided between UTX and c-myc and between UTX and LSD1. The communication between UTX and the EMT transcription factors is provided.

23 This is a comprehensive and detailed study. The experiments are well conducted and importantly both overexpression and knock down experiments complement each other. The findings provide novel mechanistic insight into the role of UTX and how it modulates cell behavior. The figures are clear and the paper is well written. I only have a couple of minor comments. We appreciate the thoughtful comments. We have addressed the remaining issues raised by the reviewer in the revised manuscript. - All the ChIP is based on semi-quantitative PCR, where small differences can make differences look more dramatic than they actually are. The paper would benefit enormously if a couple of key ChIP experiments were redone using qpcr as an endpoint. Maybe just the key findings from Figure 5B and Figure 6D. As suggested, we re-performed ChIP assay by using quantitative real-time PCR in key findings from Figures 5B and 6D (which are now Figures 4A and 5D, respectively, and Supplementary Figures S3 and S4), as well as Figures 3D (now Fig. 3F and Fig. S2A), 4E (now Fig. S2G), and 6B (now Fig. 5B and Fig. S4C-D), and obtained consistent results with the previous PCR data. Changes in the Materials and Methods (page 13, lines 16-20), Figure legends (page 21, lines 7 and 23; page 22, line 16), and Supplementary methods (page 3, lines 7-11, 14, and 19) and figures (page 11, line 8) sections were revised accordingly. Figure 5B, original ver. Figure 4A and Supplementary Figure S3B-D, revised ver.

24

25 Figure 6D, original ver. Figure 5D and Supplementary figure S4C-D, revised ver.

26 Figure 3D, original ver. Figure 3F and Supplementary figure S2A, revised ver. Figure 4E, original ver. Supplementary figure S2F and S2G, revised ver. - Is c-myc binding altered by inhibition of LSD1 We observed that the binding of c-myc to the promoter regions of EMT-TFs was not altered by inhibition of LSD1, since UTX-mediated dissociation of c-myc from these promoters was not recovered by subsequent LSD1 knockdown in MDA-MB-231 cells. This result may imply that UTX-mediated recruitment of LSD1- containing repressive complex to these regions might occur sequentially after dissociation of c-myc from these sites by UTX. The data was newly included in Figure 5D and Supplementary Figures S4C-D, and described in the Result (page 11, lines 13-14) section accordingly. Figure 6D, original ver.

27 Figure 5D and Supplementary figure S4C-D, revised ver. Referee #3: Choi et al. (Kong) EMBO Reports The authors evaluate the role of the H3K27 demethylase UTX in the generation of breast cancer stem cells and epithelial-mesenchymal transition, and come up with a scenario that UTX epigenetically represses the expression of EMT transcription factors in part dependent on its role in controlling histone methylation, and consequently controls E-cadherin expression. This manuscript was rather difficult to read, in part because of difficult and overly shortened sentences. However, as remarkable as this may sound, I do not have any questions or problems with the science (and this is very unusual for me). The conclusions drawn are justified based on the data provided, and all data are convincing, as far as I am concerned. My only suggestion/request at this point is that the authors "relax" the language to make the manuscript more accessible to the reader. This is particularly needed for the Abstract. I realize that this will require that the authors go somewhat beyond the imposed word/character limit, but it is worth it. To give some examples: - Abstract line 5: "transactivating" is inappropriately used as it has particular connotations in terms of

28 the function in transcription control. - page 7, last line is also difficult to understand. Explain things better, relax the language. - page 9, last full sentence: Again this is hard to understand, presumably because the authors try to be too concise. So, try to make the manuscript more readable. But the science is good. We appreciate for the reviewer s thoughtful comments. As suggested, we have tried to make the manuscript more accessible to the readers and to resolve any English language problems. We changed the sentences in the Abstract, and Results and Discussion sections better readable, as pointed by the reviewer. For instance, the transactivating in the Abstract has been replaced with increasing the expression (page 2, line 6). The last sentences in the Results and Discussion (pages 7 and 9 in original manuscript) have been described in more detail. In addition, for better understanding, our manuscript has been edited by two qualified native-english editors in our research field from a professional scientific English editing service. Cross-comments from Referee 3: - Indeed, many PCR bands are shown and many of these look similar, which is in my view not surprising considering the nature of these experiments with using the same types of gels and the ability to present good pictures with nice contrast. I do, however, not see a reason to assume that there is data fudging or duplication. Yet, considering the semi-quantitative nature of these experiments, and the possibility to provide exposures that result in the most pleasing presentation, it is fair to ask for qrt- PCR quantification of the data, including those for ChIP experiments. We truly appreciate the cross-comments from Referee 3. As suggested, to avoid any doubt or debate, we have extensively re-performed all PCR and ChIP results using real-time qpcr analysis in the revised manuscript (Fig 1B, Fig 2C, Fig 3C and 3F, Fig 4A, Fig 5B and 5D, Fig S2A and 2F-G, Fig S3B-D, and Fig S4C-D). Changes in the Materials and Methods (page 13, lines 16-20), Figure legends (page 21, lines 7 and 23; page 22, line 16), and Supplementary methods (page 3, lines 7-11, 14, and 19) and figures (page 11, line 8) sections were revised accordingly. - It would indeed be nice to see a few FACS plots in Fig. 1, yet I am generally fine with having most data presented as bar graphs. As suggested by the Referee 1 and 3, we included FACS plots in the Figure 1B. Changes in the Figure legends (page 21, lines 10-11) section were made accordingly. Figure 1C, original ver.

29 Figure 1C, revised ver. - I am satisfied with the use of CD44+/CD24-/EpCAM+ as stem cell markers and do not see a reason for asking more markers. This is standard and appropriate. Thank you for the comment. We did not include additional stem cell markers for MCF10A cells in our study. Instead, we provided in vivo xenograft results showing the UTX loss-induced tumorigenesis of MCF10A cells to suggest a tumor-initiating ability of MCF10A cells having UTX loss. The data was included in Supplementary Figure 1E. - Considering the nature of the cell lines used, I am not surprised that the % incidence of CSCs is as low as it is. Additionally, while a 2.5-fold increase in CSC generation is "not impressive", it is substantial. In fact, TGF-b-induced stimulation of CSC generation, which is a strong stimulation, is often in that range. I agree that some cell systems provide higher percentages. Thank you for the supportive comments about our CSC population data. Based on this suggestion, we emphasized the meaning of our data to the Referee 1. - It is indeed technically not correct to consider the CD44+/CD24-/EpCAM+ population of MCF-10A cells as "cancer stem cells", since the MCF10A cells are not cancer cells, yet are easily driven into becoming cancer cells. On the other hand, this is also what was done with the discovery of the "CSC population" of HMLE cells, which are not transformed, and that paper clearly got away with it. As the CD44+/CD24-/EpCAM+ population also characterizes non-cancer breast epithelial stem cells, maybe it is worth naming these "breast epithelial stem cells" Following the Referee 3 s suggestion, we re-defined the CD44 + /CD24 - /ESA + cell population in MCF10A

30 cells as breast epithelial stem cells instead of CSCs in our revised manuscript. Results and Discussion (page 5, lines and 18; page 6, line 24; page 7, lines 21-23; page 8, lines 1, 3, and 12), Materials and Methods (page 13, lines 10 and 13), Figure legends (page 21, line 13; page 22, line 24; page 23, line 17), and Supplementary Materials (page 10, line 1; page 11, lines 1 and 5) sections have been revised accordingly. - Fig. 2: MDA-MB231 cells are unfortunately not changing much in phenotype. They keep having a morphology in between epithelial and mesenchymal, even when they start making more E-cadherin. I am satisfied with the apparent EMT change in morphology of MCF10A cells. This is fine, and typical for these cells. We agree that the reversible morphological change in the MDA-MB-231 following the UTX overexpression was not as clear as those of MCF10A cells depleting UTX. We now provide an improved image for MET morphology of UTX-overexpressing MDA-MB-231 cells, which were obtained from longterm culture of the stable cells. Figure 2A, original ver. Figure 2A, revised ver. - I agree that it is surprising that the immunofluorescence of Snail and Zeb2 does not really show nuclear localization. Perhaps it is worth suggesting to do a separation of nuclear versus cytoplasmic fractions combined with immunoblotting. On the other hand, these data are not really needed for this story. Due to no supply for unmerged images for immunofluorescence of EMT-TFs and too strong DAPI signal (blue) in merged images in the original manuscript, the subcellular EMT-TF expression levels seem to be misunderstood. Indeed, the paired unmerged data exhibited both nuclear and cytoplasmic staining of the EMT- TFs as shown in the figure below (red box). Figure 3, original ver. (red box, unmerged images not provided in the original ver.)

31 To further clarify these results, we re-analyzed the immunofluorescence staining of these EMT-TFs in both MCF10A and MDA-MB-231 cells, and additionally performed the immunoblotting with nuclear and cytoplasmic fractions as suggested by the Referee 3. These data indicated that EMT-TFs are preferentially expressed in the nucleus but also slightly expressed in the cytoplasm, and that UTX loss increases total expression of EMT-TFs (both nuclear and cytoplasmic EMT-TFs) by activating their gene transcriptions. These new data were displayed in Figure 3E and F. Changes in the Results and Discussion (page 7, line 10), Figure legends (page 22, lines 17-19), and Supplementary methods (page 5, lines 6-14) sections were revised accordingly. Figure 3C, original ver. Figure 3D and 3E, revised ver. Once again, thank you for reviewing our manuscript.