UTX mediates demethylation of H3K27me3 at musclespecific genes during myogenesis

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1 Manuscript EMBO UTX mediates demethylation of H3K27me3 at musclespecific genes during myogenesis Shayesta Seenundun, Shravanti Rampalli, Qi-Cai Liu, Arif Aziz, Carmen Palii, SunHwa Hong, Alexandre Blais, Marjorie Brand, Kai Ge and F. Jeffrey Dilworth Corresponding author: F. Jeffrey Dilworth, Ottawa Hospital Research Institute Review timeline: Submission date: 06 October 2009 Editorial Decision: 12 November 2009 Revision received: 26 January 2010 Editorial Decision: 18 February 2010 Revision received: 18 February 2010 Accepted: 19 February 2010 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.) 1st Editorial Decision 12 November 2009 Thank you for submitting your manuscript for consideration by The EMBO Journal. First, I would like to apologise for the delay in getting back to you with a decision. However, one of the referees was not able to get back to us with his/her report as quickly as initially expected. Your manuscript has now been seen by three referees whose comments to the authors are shown below. As you will see while referee 2 is not in favour of publication of the paper here the other two referees are considerably more positive and would support publication after adequate revision. Clearly, the concerns raised by referee 2 regarding the overall conceptual advance provided by this study result from taking a more general conceptual perspective. Still, because of the publications he/she cites it is clear that the main novelty of your findings is the recruitment of the histione demethylase UTX by a transcription factor, Six4. It is therefore important that this aspect of the study is particularly strong and fully convincing. In the view of referee 3 this has not been achieved yet and it will thus be indispensable to strengthen the study along the lines he/she suggests to his/her full satisfaction. A second major issue is put forward by referee 1. He/she feels that more direct evidence for UTX spreading in a pol II-dependent manner needs to be presented. All in all we have thus come to the conclusion that we will be able to consider a revised manuscript if you can address the referees' criticisms in an adequate manner and to their satisfaction. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript as well as on the final assessment by the referees. When preparing your letter of response to the referees' comments, please bear in mind that this will form part of the Review Process File, and will therefore be available online to the community. For European Molecular Biology Organization 1

2 more details on our Transparent Editorial Process initiative, please visit our website: Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely, Editor The EMBO Journal REFEREE COMMENTS Referee #1 (Remarks to the Author): Seenundun et al present work showing that the UTX histone lysine demethylase functions at myogenic genes during myogenesis. They indicate that there is a two-step process, with initial demethylation occurring in the regulatory sequence region of the genes analyzed and a subsequent step in which UTX activity in the coding sequences of the genes is tied to elongating RNA pol II. The topic is novel and the approach is sufficiently mechanistic to justify consideration at EMBO J. My main complaint is that while the data generally support the conclusions, there is no direct evidence that the UTX enzyme "spreads" into the gene from the regulatory sequences upstream in a pol II dependent manner. The authors primarily rely on the presence or absence of H3K27 and H3K4 methylation as indicators of UTX function, which is fair, but to argue that the enzyme spreads into the coding region requires a time course of UTX ChIPs across the gene loci being examined. If the authors are correct, this experiment will demonstrate progressive UTX occupancy in a 5' to 3' direction across the loci on a DRB sensitive manner as a function of time. If a different result is obtained, then the authors need to re-evaluate their conclusions. Since the main complaint can be addressed experimentally and since the list of other considerations below are likely things that the authors can address, I suggest that the authors be permitted to revise the manuscript. Other issues: On. p. 6 the authors indicate that in Fig. 2E, the H3K27me3 had not yet spread into CKm gene at 48 h because activation of the gene was delayed due to the lentiviral infection procedure. This is easily testable, and this hypothesis should be confirmed by monitoring mrna levels at 48 h and later timepoints and showing that complete spread of H3K27me3 occurred at later timepoints that correlated with activation of the gene under these conditions. Fig. 3C demonstrates that the shrna against Six4 decreases Six4 mrna levels. This should really be analyzed at the level of protein expression. Figs. 3E-F and Supp Figs. 2A-B do not include the scrambled shrna control. Supp. Fig. 4- the authors should provide western data to demonstrate that the Ash2L shrna did reduce Ash2L levels, especially since as in Figure 3C, there is no scrambled shrna control. Fig. 2A presents data about Ash2L occupancy but this is not mentioned in the text describing the figure on p. 5. Similarly Fig. 2E incorporates data about methylation of histones on the HoxB8 promoter, but there is no explanation of HoxB8 in the text or legend. Fig, 3H and Supp. Fig. 3A contain two graphs; the ones showing data about H3K4me3 are not discussed at all in the text. By enlarging Fig. 2D on screen, I can see the data. Printing on two different printers left me with images that could barely be seen, never mind be interpreted. Maybe it's my printers, but can this European Molecular Biology Organization 2

3 image be enlarged a little? Perhaps the editor will make a determination if this is an issue or not. In addition, the legend does not indicate the time of differentiation at which these samples were analyzed. The legend to Figure 1, which describes parts A and B, does not correspond to the Figure, which has parts A-D, or the text in the Results. Page 6, line 18 contains the phrase "in parallel with UTX (Fig. 2D)". I'm not sure what figure is being referred to, but Fig. 2D does not deal with UTX recruitment. Please check. Page 9 -lines 19-20: This is not a complete sentence. Please fix. Supp. Fig. 1 legend - next to last sentence - should the word "either" be dropped, or is the sentence incomplete? Referee #2 (Remarks to the Author): In this report, Seenundum et al. provide evidence for active demethylation of H3K27 and methylation of H3K4 following activation of Myog and Ckm. They report that the demethylation of H3K27 occurs via recruitment of UTX first via Six4 and then by traveling RNA polymerase II. COMMENTS: 1. The novelty of this study is tempered by previous reports. The contribution of Jmjd3 and UTX to cell lineage differentiation via H3K27me3 demethylation has been previously described (Sen et a. G&D : ; Burgold et al. PLoS One (8):e3034; Lan et al. Nature 2007, 449:689-94). Similarly, tracking of Jmjd3 and UTX with RNA II polymerase has been reported (Smith et al. MCB 2008, 28:1041-6; De Santa EMBO J September 24). 2. It is not clear what the authors means when they state that" the demethylase activity of UTX seems to be more tightly regulated". 3. Fig.3H. It seems as if SB prevents H3K27 demethylation at position 1 (-10Kb) and position 4 (+1Kb), much less at position 2 (-1Kb) and position 0 (TSS) of myogenin. There is no correlation with H3Kme3 at position 1 (no effect of SB) and position 3 (no or little effect of SB on H3K27me3 but decreased H3K4me3). Since UTX associates with MLL, these results are unexpected. Does SB somehow interfere with MLL recruitment at specific myogenin regions? 4. Because of co-tracking of UTX and PolII (Smith et al. MCB 2008, 28:1041-6), the results obtained with the inhibitor of transcriptional elongation DRB (i.e., accumulation of PolII at the 5'end of the genes, loss of UTX and increased in H3K27me3 within the genes) were predictable. Referee #3 (Remarks to the Author): Seenundun et al analyze changes in the chromatin state of the muscle-specific genes myogenin and CKm genes during in vitro differentiation of C2C12 myoblasts. Using chromatin immunoprecipitaiton assays, the authors monitored the levels of the post-translational modifications H3-K27me3 and H3-K4me3 as well as binding of the H3-K27-specific histone demethylase Utx at several regions within these two genes. They find that H3-K27me3 is present across the upstream, promoter and coding region of both genes in proliferating myoblasts. Upon differentiation of the cells, the H3-K27me3 modification is lost in a gene-specific pattern with gene-specific kinetics and eventually only persists at the 3' end of both genes. At both genes, H3-K4me3 starts to accumulate at the promoter and coding region and, at least at the CKm gene, this modification appears to transiently co-exist with H3-K27me3. The authors find that Utx becomes recruited to both genes, and RNAi-mediated knock-down of Utx reveals that removal of H3-K27me3 is indeed dependent on European Molecular Biology Organization 3

4 Utx. ChIP analyses show that Six4, a known activator of myogenin and CKm binds to both genes during differentiation and RNAi knock-down of Six4 suggests that Utx recruitment depends on Six4. The authors report that endogenous Six4 and Utx proteins can be co-immunoprecipitated. Finally, the authors induce RNA Pol II stalling by treating the cells with DRB and find that this results in lack of Utx binding and persistence of PRC2 recruitment at the promoter and concomittant maintenance of high H3-K27me3 levels in the 5' coding region. The authors propose a model where the initial recruitment of Utx occurs through the enhancer-binding Six4 protein and that Utx then "spreads" into the coding region to demethylate nucleosomes bearing the H3-K27me3 mark. Critique: This manuscript reports a number of interesting observations that should be of general interested to researchers in the chromatin/ transcription and stem cell biology fields. The paper is written in a concise and accessible style. However, in order for the paper to become publishable, the authors need to clarify several issues concerning the technical aspect of their ChIP analysis (see point 1 below) and they should provide more complete documentation of this analysis (see point 2 below). The ChIP analysis that the authors use to claim that Six4 is required for Utx targeting is very suggestive. However, the data that are shown to claim an (in-)direct association of Six4 with Utx are not so convincing; better biochemical evidence is needed to strengthen this point (see point 3 below). This is particularly critical because the recruitment of the histone demethylse Utx by a transcription factor, Six4, is one of the key novel claims of this study. Point 1 concerns the technical execution of the ChIP analysis: On page 13, the authors state that "Average values of duplicate samples are displayed with error bars corresponding to {plus minus} s.d. Each experiment was performed at least twice using independent chromatin samples, and yielded similar results. Each experiment was performed at least twice using independent chromatin samples, and yielded similar results." Does this mean that the bars in the graphs with error bars represent the results of two independent PCR reactions but only from one ChIP experiment (i.e. technical rather than biological replicates)? If yes, this is not acceptable. The authors have to perform at least three independent chromatin immunoprecipitation reactions from three independently prepared batches of chromatin/ nucleosomes (i.e. three independent biological replicates) and the bars in the graphs have to represent the results of these three (or more) independent chromatin IP reactions. A second issue here is the representation the qpcr analysis of the ChIP experiments. I am not sure whether I understand what the numbers on the Y-axis represent. Please explain. The most accessible way to represent qpcr results from ChIP analyses is to represent the immunoprecipitated DNA as percentage of input DNA. That is, for each DNA sequence analyzed by qpcr, one determines the fraction present in the immunoprecipitated material relative to the amount of this DNA present in the input material. To this end, serial dilutions of (de-crosslinked) and purified DNA from input chromatin are analyzed by qpcr together with the DNA recovered by chromatin immunoprecipitation. Point 2 concerns the ChIP results: Figure 1 should be extended to show the Utx binding profile at all 5 (in the case of Myog) or 6 (in the case of CKm) regions and at all three time points (i.e. as in the case of the H3-K27me3 and H3- K4me3 profiles). For example, on page 10 (para2) the authors wrote: "Examination of H3K27me3 enrichment across the CKm locus at 24 h (prior to expression of this gene) clearly demonstrates a localized demethylation that does not spread into the gene." So it would be good to have the information about Utx binding in CKm region #5 at this time point. Similarly, the authors propose that Six4 is required for the initial recruitment of Utx and they suggest that the subsequent demethylation of H3-K27me3 in the coding region might be mediated by a mechanism involving elongation by RNA polymerase. But how does this explain the demethylation of the region 10 kb upstream of Myog and CKm promoter? Does Utx ever associate with region #1 and can this be monitored by chromatin immunoprecipitation? A related point: What happens to Utx and H3-K27me3 levels in Six4 knock-down cells 48 hrs after induction of differentiation? Point 3 concerns the physical association of Six4 with Utx. European Molecular Biology Organization 4

5 The Six4/Utx co-immunoprecipitation data are not very convincing. It would be desirable if the authors could show a silver-stained gel of the material immunopurified with Six4 or Utx antibody, respectively. Alternatively, the authors should at least extend the analysis by western blotting, i.e. by performing IPs with antibodies against other, unrelated proteins and, in parallel, WB analysis of the IP-ed material with antibodies against other, unrelated proteins. Here the only control is an IP with anti-myogenin antibody probed for presence of Six4 and IP with anti-six4 antibody probed for presence of Myogenin. Moreover, the input lanes in Fig. 3A are separated from the IP-ed material. Do the western blot signals in Fig. 3A come from the same western blot membrane and were they exposed to film for the same period? Input and immunoprecipitate should be blotted onto the same membrane, processed together and subjected to the same period of exposure to film. 1st Revision - authors' response 26 January 2010 Referee #1 (Remarks to the Author): Seenundun et al present work showing that the UTX histone lysine demethylase functions at myogenic genes during myogenesis. They indicate that there is a two-step process, with initial demethylation occurring in the regulatory sequence region of the genes analyzed and a subsequent step in which UTX activity in the coding sequences of the genes is tied to elongating RNA pol II. The topic is novel and the approach is sufficiently mechanistic to justify consideration at EMBO J. My main complaint is that while the data generally support the conclusions, there is no direct evidence that the UTX enzyme "spreads" into the gene from the regulatory sequences upstream in a pol II dependent manner. The authors primarily rely on the presence or absence of H3K27 and H3K4 methylation as indicators of UTX function, which is fair, but to argue that the enzyme spreads into the coding region requires a time course of UTX ChIPs across the gene loci being examined. If the authors are correct, this experiment will demonstrate progressive UTX occupancy in a 5' to 3' direction across the loci on a DRB sensitive manner as a function of time. If a different result is obtained, then the authors need to re-evaluate their conclusions. To address this point brought forward by the reviewer, we have performed UTX ChIPs across both the myogenin and CKm loci at three time points during differentiation. The results of this experiment are now presented in a new figure (Figure 2) to highlight their importance. We observe that UTX first associates with the two muscle-specific enhancers of the CKm gene at 24 hrs of differentiation. The association with the enhancers is decreased at 48 hrs, and at the same time we see increased association with the coding region of the gene. In addition, we have performed experiments with C2C12 cells differentiated for 48 hrs that have been treated with (or without) DRB for 1 h (See Supplemental Figure 5A). In this case, we see that levels of UTX decrease within the CKm gene and again accumulate on the two enhancers. Similarly, we observe that enrichment of UTX in the coding region of the Myog gene is lost in the presence of DRB, and we begin to see UTX accumulate on the promoter of this gene. We agree with reviewer 1 that it would be ideal to show a progressive movement of UTX across the CKm gene with the RNA Pol II. Such experiments are possible with inducible systems such as activation of ER target genes by the addition of Estrogen where the addition of ligand induces gene expression very quickly. However, in our case, we are looking at the activation of gene expression that occurs over 2 days during muscle differentiation. Thus, using the system of myogenesis, it is not possible to identify a window of minutes where we would be able to observe progressive movement of UTX across the CKm gene in synchronous manner. Nevertheless, we feel that the new experimentation described above provides strong new data that further supports the conclusions that UTX spreads across the gene with the elongating RNA Pol II. Since the main complaint can be addressed experimentally and since the list of other European Molecular Biology Organization 5

6 considerations below are likely things that the authors can address, I suggest that the authors be permitted to revise the manuscript. Other issues: On. p. 6 the authors indicate that in Fig. 2E, the H3K27me3 had not yet spread into CKm gene at 48 h because activation of the gene was delayed due to the lentiviral infection procedure. This is easily testable, and this hypothesis should be confirmed by monitoring mrna levels at 48 h and later timepoints and showing that complete spread of H3K27me3 occurred at later timepoints that correlated with activation of the gene under these conditions. We agree completely with the reviewer. To address this possibility, we have repeated the UTX knock-downs and extracted RNA for qpcr analysis at various times of differentiation. The results of this qpcr analysis are provided for the reviewers (Reviewer Only Figure 1), and demonstrate that maximal activation of the CKm gene is not obtained at 48 h of differentiation but occurs at a point between 48 and 72 hrs. As such, we have repeated our Native ChIP analyses across the CKm and Myog loci at 72 h of differentiation. Under these conditions, we observe the removal of H3K27me3 at positions 1 through 5 (but not 6) in differentiating cells infected with scrambled shrna, but this removal is not observed in cells infected with UTX shrna. These results are shown in Figure 3D, and replace the similar analysis that was performed at 48 h of differentiation in the initial manuscript that was submitted. As a similar infection procedure was used to examine the recruitment of UTX and H3K27me3 levels in the absence of Six4 at 24 h of differentiation (Figure 4F and G), we have also repeated these experiments at 48 hrs of differentiation. The efficiency of the Six4 knockdown at the protein level is now included in the manuscript as Figure 4D. Under these conditions, we observe a similar (or slightly better) effect of Six4 knock-down on impaired UTX recruitment and enrichment of H3K27me3 at the Myog and CKm regulatory regions. This suggests that recruitment of UTX and Six4 to the CKm and Myog genes occurs prior to 24 h, and indeed, we observe UTX associated with the gene regulatory region of these genes as early as 12 h under normal differentiation conditions (data not shown). As these results are relatively similar, we have maintained Figure 4F and 4G in its original form. However, if the reviewer prefers, we could replace the data in the manuscript with the data generated for 48 h of differentiation that is provided in "Reviewer Only Figure 2". Fig. 3C demonstrates that the shrna against Six4 decreases Six4 mrna levels. This should really be analyzed at the level of protein expression. To confirm that loss of Six4 occurs at the protein level as well as the RNA level, we have performed Western blots on cell extracts obtained from C2C12 cells that have been infected with shrna targeting Six4. In the new Figure 4D, we now demonstrate that Six4 protein levels are strongly decreased by the knock-down, but that levels of UTX, Tubulin, and global H3K27 trimethylation in the cell are not significantly affected by the knock-down. Figs. 3E-F and Supp Figs. 2A-B do not include the scrambled shrna control. We apologize for creating this confusion. Figure 3E-F and Supp Figs 2A-B (now combined as Figure 4F and 4G in the revised manuscript) were performed with scrambled shrna controls. To save space, we left out the line containing the + and - corresponding to cell conditions treated with scrambled shrna. To clarify this result the figures have additional labeling indicating which cells have been infected with Six4-targeted shrna, and which cells have been infected with a scrambled shrna. Supp. Fig. 4- the authors should provide western data to demonstrate that the Ash2L shrna did reduce Ash2L levels, especially since as in Figure 3C, there is no scrambled shrna control. To address this concern, we now show the qpcr experiment that was done at the time of the knock-down experiment showing that the sirna decreases Ash2L levels at the mrna level (Supplemental Figure 4A). We understand that the reviewer asked specifically for a Western blot, however we note that the knock-down of Ash2L was performed using sirna exactly as we have previously done to study Ash2L recruitment to muscle-specific promoters European Molecular Biology Organization 6

7 (Rampalli et al. Nature Struct Mol Biol 14: ). In this publication, we showed that the knock-down is efficient at the protein level (a copy of the published figure has been included for the reviewers - Reviewer Only Figure 3). Since we have previously demonstrated the efficiency of this sirna at diminishing Ash2L expression, we have only confirmed the knockdown at the RNA level in our subsequent studies. Repeating the knock-down to generate material for a Western blot would require the purchase of additional sirna. As such, we hope that the reviewer will be convinced with our previously published data, along with the qpcr analysis (Supplemental Figure 4A) that was performed to accompany the Native ChIP analysis demonstrated in Supplemental Figure 4B. Fig. 2A presents data about Ash2L occupancy but this is not mentioned in the text describing the figure on p. 5. Similarly Fig. 2E incorporates data about methylation of histones on the HoxB8 promoter, but there is no explanation of HoxB8 in the text or legend. Fig, 3H and Supp. Fig. 3A contain two graphs; the ones showing data about H3K4me3 are not discussed at all in the text. Thank you for pointing out the fact that we have provided data that was not discussed. The Ash2L occupancy in Figure 2A was to demonstrate that recruitment coincides with the addition of H3K4me3 at the Myog and CKm genes. In Figure 2E (now Figure 3D), the ChIP of H3K27me3 at the HoxB8 promoter was used as a negative control to show a gene which doesn t change its methylation status during muscle differentiation. Finally, the data in Figure 3H (Now Figure 5B) was used to demonstrate that H3K4me3 of the myogenin and CKm loci were dependent on p38 MAPK activity. We have now made reference to all these data points in the text. By enlarging Fig. 2D on screen, I can see the data. Printing on two different printers left me with images that could barely be seen, never mind be interpreted. Maybe it's my printers, but can this image be enlarged a little? Perhaps the editor will make a determination if this is an issue or not. In addition, the legend does not indicate the time of differentiation at which these samples were analyzed. We apologize for the difficulty the reviewer had viewing this figure. We have submitted a new TIF version of the same figure for further evaluation. If the converted TIF file is not of sufficient quality, we can provide the power point version of the figure for production. The confocal microscopy images shown in Figure 2D (now Figure 3C in the revised manuscript) were obtained at 72 hrs of differentiation since we were looking for extensive myotube formation. This information as well as the magnification (40X) is now included in the figure legend. The legend to Figure 1, which describes parts A and B, does not correspond to the Figure, which has parts A-D, or the text in the Results. Thank you for pointing out this error that happened when we reconfigured Figure 1 prior to our initial submission. We have now modified the text, Figure and Figure legend to ensure that all three correspond. Page 6, line 18 contains the phrase "in parallel with UTX (Fig. 2D)". I'm not sure what figure is being referred to, but Fig. 2D does not deal with UTX recruitment. Please check. This should have been Fig. 2A. We have modified this item in the text. Page 9 -lines 19-20: This is not a complete sentence. Please fix. Sentence has been modified to complete the idea. Supp. Fig. 1 legend - next to last sentence - should the word "either" be dropped, or is the sentence incomplete? Sentence has been modified to complete the idea. European Molecular Biology Organization 7

8 Referee #2 (Remarks to the Author): In this report, Seenundum et al. provide evidence for active demethylation of H3K27 and methylation of H3K4 following activation of Myog and Ckm. They report that the demethylation of H3K27 occurs via recruitment of UTX first via Six4 and then by traveling RNA polymerase II. COMMENTS: 1. The novelty of this study is tempered by previous reports. The contribution of Jmjd3 and UTX to cell lineage differentiation via H3K27me3 demethylation has been previously described (Sen et a. G&D : ; Burgold et al. PLoS One (8):e3034; Lan et al. Nature 2007, 449:689-94). Similarly, tracking of Jmjd3 and UTX with RNA II polymerase has been reported (Smith et al. MCB 2008, 28:1041-6; De Santa EMBO J September 24). While we agree with the reviewer that several exciting papers have been published describing UTX and JMJD3 function, we feel that our manuscript provides novel mechanistic insight into how UTX is targeted to tissue-specific genes, and how it acts to remove H3K27me3 marks across the locus. While there have been papers suggesting UTX associates with elongating polymerase (the Smith paper is cited several times in our manuscript), these papers do not show co-tracking of UTX with elongating RNA Pol II. The Smith paper demonstrates that 1) UTX co-localizes with elongating (Ser2-P) Pol II on polytene chromosomes (by immunofluorescence), 2) UTX and Pol II co-localize by ChIP at a single position on the heat-shock gene after heat shock, and 3) that an anti-utx antibody can co-immunoprecipitate Pol II (the Western blot wasn t resolutive enough to differentiate between the phosphorylated and non-phosphorylated forms of Pol II). Thus, we believe that the Smith paper does not provide evidence that UTX spreads with the elongating Pol II. Similarly, the De Santa paper shows that JMJD3 associates with genes that have Pol II at their promoter. This does not demonstrate that JMJD3 (or UTX) moves with Pol II. Thus, none of the experiment in either study can be interpreted as co-tracking of UTX and RNA Pol II. Therefore the data provided in our manuscript, while being consistent with the observations made by these two researcher groups, provides compelling new evidence to support the notion that UTX migrates across the gene with the elongating RNA Pol II. Beyond this interesting finding, we have also demonstrated for the first time that UTX is targeted to specific genes through the interaction with a transcriptional activator. This targeting of UTX to genes through something other than a direct interaction with elongating polymerase helps explain the interesting finding of Lan et al. that UTX is enriched at certain HOX genes that remain marked by H3k27me3 and others that lack RNA Pol II (Lan et al. Nature 449: ). Finally, we suggest a mechanism by which bivalent chromatin domains might be generated during development. Thus, we feel that our manuscript provides several novel findings with respect to the role of UTX in mediating activation of tissue specific gene expression. 2. It is not clear what the authors means when they state that" the demethylase activity of UTX seems to be more tightly regulated". The manuscript by Agger looked at knock-down of UTX and JMJD3 in Hela cells. The knockdown of JMJD3 leads to a global increase in H3K27me3 levels in the cell while knock-down of UTX did not demonstrate a significant change in bulk cellular H3K27me3 levels. This led us to hypothesize that UTX might act at a restricted number of genes. We have modified the text to better reflect our initial thinking that led us towards UTX rather than JMJD3. 3. Fig.3H. It seems as if SB prevents H3K27 demethylation at position 1 (-10Kb) and position 4 (+1Kb), much less at position 2 (-1Kb) and position 0 (TSS) of myogenin. There is no correlation with H3K4me3 at position 1 (no effect of SB) and position 3 (no or little effect of SB on H3K27me3 but decreased H3K4me3). Since UTX associates with MLL, these results are unexpected. Does SB somehow interfere with MLL recruitment at specific myogenin regions? European Molecular Biology Organization 8

9 a) Firstly, with respect to the question of SB interfering with MLL recruitment at myogenin, we have previously published that Ash2L1/MLL2(KMT2B) mediates the trimethylation of H3K4 at the Myog and Ckm genes leading to transcriptional activation. Furthermore, we demonstrated that recruitment of the Ash2L1/MLL2(KMT2B) complex to these promoters is mediated by Mef2D in a p38-dependent manner (Rampalli et al. Nature Struct Mol Biol 14: ). Thus, SB does interfere with the recruitment of the MLL complex that mediates H3H4me3. b) Addressing the issue of SB differentially affecting H3K4me3 and H3K27me3 at -10,000 bp, H3K4me3 is a mark that has been reported to be highly enriched in the 5 -end of genes (either transcribed, or bivalently marked), thus we would not expect to see this chromatin mark enriched in the presence or absence of SB at -10,000 bp of the Myog or CKm genes. In contrast, H3K27me marks have been shown to extend over extended chromatin domains (Barski et al. Cell 129: ). While we propose that the establishment of a transcriptionally poised promoter in the presence of SB leads to a block in demethylation in the coding region of the gene, it is not clear at this time why the spreading of the UTXdependent demethylase activity away from the gene is also inhibited by SB. The mechanism of UTX activity spreading away from the gene will be the subject of future studies. c) The reviewer is correct in his comment that UTX has been shown to complex with members of the MLL family - specifically MLL3(KMT2C) and MLL4(KMT2D). However, it has not been shown to complex with MLL1(KMT2A) or MLL2(KMT2B). Indeed, mounting evidence suggests that the MLL1(KMT2A)/MLL2(KMT2B)-containing complexes and the MLL3(KMT2C)/MLL4(KMT2D)-containing complexes differ both in their composition and their function. Purification of the MLL4(KMT2D) complex showed that this protein associates with a set of proteins that includes PTIP and UTX (Issaeva et al. Mol Cell Biol 27: ) Similarly, purification of a UTX complex showed an association with MLL3(KMT2C) and MLL4(KMT2D) as well as PTIP (Cho et al. J Biol Chem 282: ). These papers also described an association of components of the Ash2L core complex (Ash2L, WDR5, and RBBP5) with the MLL4(KMT2D)/UTX/PTIP complex. However, examination of the coomassie blue stained gels show that components of the Ash2L complex (Ash2L, WDR5, and RBBP5) are present in substoichiometric ratios compared to the MLL4, PTIP and UTX subunits (Issaeva et al. Mol Cell Biol 27: Figure 1B). In addition, the Kingston group has demonstrated that MLL1 and MLL2 (called MLL4 in the paper due to a confused nomenclature in the MLL field) contain a histone H3-like motif that allows a strong interaction with WDR5 (a core component of the Ash2L complex), and that this histone H3-like motif is not conserved in MLL3 and MLL4 (Song and Kingston, J Biol Chem 283: 35258). Finally, we have purified a TAP-tagged Ash2L1 complex under highly stringent conditions, and show that it does include UTX (Reviewer Only Figure 4). This TAP-tagged Ash2L1 (the major isoform expressed in C2C12 cells - see Supplemental Figure 2) was purified from K562 cells (the same cells used by Issaeva et al. Mol Cell Biol 27: ) using a stringent wash buffer (washed with 20 mm Hepes ph 7.6, 1 M NaCl, 1 M Urea, 10% glycerol, 0.2 mm EDTA, 0.5 mm DTT, and 0.5% NP-40). Western blot clearly shows that subunits of the core Ash2L complex (defined by Roeder s group - Dou et al. Nat Struct Mol Biol 13: and more recently by the Cosgrove group - Patel et al. J Biol Chem 284: ) co-purify with the TAP-tagged protein, while UTX is clearly absent from the purification (See Reviewer Only Figure 4). Consistent with this, our results clearly demonstrate differential recruitment properties of the Ash2L and UTX at muscle specific promoters - both temporally and pharmacologically. Taken together, these findings suggest that UTX/MLL3(KMT2C)/PTIP are a core complex that can associate as a module with the Ash2L core complex. Indeed, several transcriptional complexes are modular in structure including the Mediator complex which has a module that associates and dissociates to regulate gene expression (Pavri et al. Mol Cell 18: 83-96). The modular nature of this association needs to be confirmed, and the mechanism that would regulate the assembly of the modules remains to be delineated. Further strengthening the difference between MLL1(KMT2A)/MLL2(KMT2B)-containing complexes and the MLL3(KMT2C)/MLL4(KMT2D)-containing complexes, newly published data from the Shilatifard group show that MLL3-/- MEFs show "few or no changes in H3K4me3 across the Hox clusters" whereas the loss of H3K4me3 was extensive at the Hox locus in MLL1-/- MEFs, though both types of MEFs demonstrated changes in gene expression within European Molecular Biology Organization 9

10 the locus (Wang et al. Mol Cell Biol 29: ). Consistent with these observations, our studies show that recruitment of UTX to both the Myog and CKm genes in the presence of the p38 MAPK inhibitor does not lead to an enrichment in H3K4me3 though we do observe demethylation of H3K27me3 within the promoter. Thus, the accumulated literature cited above, and the results presented in this manuscript suggest that Ash2L1/MLL2(KMT2B) and UTX/MLL3(KMT2C) are differentially regulated in their recruitment to muscle-specific genes to mediate the transition from a repressive to an activated chromatin environment during myogenesis. 4. Because of co-tracking of UTX and PolII (Smith et al. MCB 2008, 28:1041-6), the results obtained with the inhibitor of transcriptional elongation DRB (i.e., accumulation of PolII at the 5'end of the genes, loss of UTX and increased in H3K27me3 within the genes) were predictable. As mentioned in the section above, the Smith paper only showed co-localization of Pol II and UTX by immunofluorescence on polytene chromosomes, and an association of RNA Pol II by Western (using an antibody that recognizes all forms of Pol II). None of these experiments can be interpreted as co-tracking of UTX and RNA Pol II. Therefore that the idea proposed by the reviewer that Smith et al. showed co-tracking of UTX with the polymerase is an extrapolation of that paper s findings. Referee #3 (Remarks to the Author): Seenundun et al analyze changes in the chromatin state of the muscle-specific genes myogenin and CKm genes during in vitro differentiation of C2C12 myoblasts. Using chromatin immunoprecipitaiton assays, the authors monitored the levels of the post-translational modifications H3-K27me3 and H3-K4me3 as well as binding of the H3-K27-specific histone demethylase Utx at several regions within these two genes. They find that H3-K27me3 is present across the upstream, promoter and coding region of both genes in proliferating myoblasts. Upon differentiation of the cells, the H3-K27me3 modification is lost in a genespecific pattern with gene-specific kinetics and eventually only persists at the 3' end of both genes. At both genes, H3-K4me3 starts to accumulate at the promoter and coding region and, at least at the CKm gene, this modification appears to transiently co-exist with H3- K27me3. The authors find that Utx becomes recruited to both genes, and RNAi-mediated knock-down of Utx reveals that removal of H3-K27me3 is indeed dependent on Utx. ChIP analyses show that Six4, a known activator of myogenin and CKm binds to both genes during differentiation and RNAi knock-down of Six4 suggests that Utx recruitment depends on Six4. The authors report that endogenous Six4 and Utx proteins can be co-immunoprecipitated. Finally, the authors induce RNA Pol II stalling by treating the cells with DRB and find that this results in lack of Utx binding and persistence of PRC2 recruitment at the promoter and concomittant maintenance of high H3-K27me3 levels in the 5' coding region. The authors propose a model where the initial recruitment of Utx occurs through the enhancer-binding Six4 protein and that Utx then "spreads" into the coding region to demethylate nucleosomes bearing the H3-K27me3 mark. Critique: This manuscript reports a number of interesting observations that should be of general interested to researchers in the chromatin/ transcription and stem cell biology fields. The paper is written in a concise and accessible style. However, in order for the paper to become publishable, the authors need to clarify several issues concerning the technical aspect of their ChIP analysis (see point 1 below) and they should provide more complete documentation of this analysis (see point 2 below). The ChIP analysis that the authors use to claim that Six4 is required for Utx targeting is very suggestive. However, the data that are shown to claim an (in-)direct association of Six4 with Utx are not so convincing; better biochemical evidence is needed to strengthen this point (see point 3 below). This is particularly critical because the recruitment of the histone demethylse Utx by a transcription factor, Six4, is one of the key novel claims of this study. Point 1 concerns the technical execution of the ChIP analysis: European Molecular Biology Organization 10

11 On page 13, the authors state that "Average values of duplicate samples are displayed with error bars corresponding to {plus minus} s.d. Each experiment was performed at least twice using independent chromatin samples, and yielded similar results. Each experiment was performed at least twice using independent chromatin samples, and yielded similar results." Does this mean that the bars in the graphs with error bars represent the results of two independent PCR reactions but only from one ChIP experiment (i.e. technical rather than biological replicates)? If yes, this is not acceptable. The authors have to perform at least three independent chromatin immunoprecipitation reactions from three independently prepared batches of chromatin/ nucleosomes (i.e. three independent biological replicates) and the bars in the graphs have to represent the results of these three (or more) independent chromatin IP reactions. We are sorry for creating confusion with our wording. The values +/- s.d. presented in each graph corresponds to 2 biological replicates that were performed in parallel on the same day, and qpcr reactions were performed in duplicate. In addition, for each condition, we have also repeated the experiment (with 2 independent biological replicates done in parallel) at least once. This number was chosen initially because we were examining multiple time points of differentiation. Importantly, as all experiments have been reproduced at least once, we have observed the same effects in at least four biological samples. It would be too costly to go back and redo all the experiments from the manuscript with triplicate biological samples. However, in all the experiments that were performed to respond to the reviewers comments, the data was performed using triplicate biological samples and analyzed using duplicate qpcr reactions as requested by the reviewer. To eliminate the confusion, we have changed the statement in the materials and methods to read - "Average values of an experiment represent biological replicates that are displayed with error bars corresponding to ± s.d. Each experiment was performed at least twice, and yielded similar results." A second issue here is the representation the qpcr analysis of the ChIP experiments. I am not sure whether I understand what the numbers on the Y-axis represent. Please explain. The most accessible way to represent qpcr results from ChIP analyses is to represent the immunoprecipitated DNA as percentage of input DNA. That is, for each DNA sequence analyzed by qpcr, one determines the fraction present in the immunoprecipitated material relative to the amount of this DNA present in the input material. To this end, serial dilutions of (de-crosslinked) and purified DNA from input chromatin are analyzed by qpcr together with the DNA recovered by chromatin immunoprecipitation. Our data is actually measured as a percentage of input as described by the reviewer and has been published in our peer reviewed Nature Protocols paper (Brand et al. Nature Protocols 3: ). The only difference is that we have not corrected for the dilution of the input because it provided us with the opportunity to present the y-axis with whole numbers instead of fractions. We have now divided these whole numbers by the dilution of the input DNA to obtain the true % of input to provide further clarity. Thus we have changed the labeling of the axis to "relative enrichment (% input)". We have chosen to keep the term relative enrichment to remind readers that ChIP is not a technique that provides absolute data, but instead data that must be interpreted relative to other points within the genome. We hope that this will be acceptable to the reviewer. Point 2 concerns the ChIP results: Figure 1 should be extended to show the Utx binding profile at all 5 (in the case of Myog) or 6 (in the case of CKm) regions and at all three time points (i.e. as in the case of the H3-K27me3 and H3-K4me3 profiles). For example, on page 10 (para2) the authors wrote: "Examination of H3K27me3 enrichment across the CKm locus at 24 h (prior to expression of this gene) clearly demonstrates a localized demethylation that does not spread into the gene." So it would be good to have the information about Utx binding in CKm region #5 at this time point. Similarly, the authors propose that Six4 is required for the initial recruitment of Utx and they suggest that the subsequent demethylation of H3-K27me3 in the coding region might be mediated by a mechanism involving elongation by RNA polymerase. But how does this explain the demethylation of the region 10 kb upstream of Myog and CKm promoter? Does Utx ever associate with region #1 and can this be monitored by chromatin immunoprecipitation? European Molecular Biology Organization 11

12 A related point: What happens to Utx and H3-K27me3 levels in Six4 knock-down cells 48 hrs after induction of differentiation? As requested, we have generated a new figure which shows UTX binding at all positions across the Myog and Ckm loci at the 3 different time points of differentiation. We have provided this information as a separate figure (Figure 2) since the Figure 1 is already very complex. What we found is that at 24 h, the region of localized demethylation is enriched for UTX. Upon activation of the gene, the association of UTX with the enhancer decreases, and an increase in UTX levels in the coding region is observed - and this can be blocked by DRB (see Supplemenatry Figure 5A). It is not clear to us how demethylation occurs in the region that lies -10 kb upstream of the gene. As seen in Figure 2, we can observe slight enrichments for UTX at this distal region of the gene. This demethylation is inhibited by DRB treatment. Furthermore, knock-down of UTX causes a renewed enrichment of H3K27me3 in this region suggesting that this demethylase is responsible for removing the repressive mark. While it is not clear how UTX migrates to this region of the gene, we could speculate that it moves with the Polymerase during intergenic transcription that is acting to maintian an open chromatin state at the locus. However, due to a lack of direct evidence, we have refrained from speculating as to how the H3K27me3 mark is removed in the distal region of the gene. We next addressed the related point of what happens to UTX and H3K27me3 levels in Six4 knock-down at 48 h. For this we repeated the knock-down of Six4 and demonstrate that the knock-down occurs at the protein level too (See Figure 4D). This does not affect protein levels of UTX or global levels H3K27me3 in the differentiating myoblasts. However, we believe that the reviewer was probably asking us to perform ChIP experiments at a second time point because differentiation is delayed by our procedure for lentiviral infection. As described above, we have performed this ChIP for UTX and H3K27me3 at 48 h of differentiation (See Reviewer Only Figure 2). Under these conditions, we observe a similar (or slightly better) effect of Six4 knock-down on impaired UTX recruitment and enrichment of H3K27me3 at the Myog and CKm regulatory regions. This suggests that recruitment of UTX and Six4 to the CKm and Myog genes occurs prior to 24 h, and indeed, we observe UTX associated with the gene regulatory region of these genes as early as 12 h under normal differentiation conditions (data not shown). As these results are relatively similar, we have maintained Figure 4F and 4G in its original form. However, if the reviewer prefers, we could replace the data in the manuscript with the data provided in "Reviewer Only Figure 2". Point 3 concerns the physical association of Six4 with Utx. The Six4/Utx co-immunoprecipitation data are not very convincing. It would be desirable if the authors could show a silver-stained gel of the material immunopurified with Six4 or Utx antibody, respectively. Alternatively, the authors should at least extend the analysis by western blotting, i.e. by performing IPs with antibodies against other, unrelated proteins and, in parallel, WB analysis of the IP-ed material with antibodies against other, unrelated proteins. Here the only control is an IP with anti-myogenin antibody probed for presence of Six4 and IP with anti-six4 antibody probed for presence of Myogenin. Moreover, the input lanes in Fig. 3A are separated from the IP-ed material. Do the western blot signals in Fig. 3A come from the same western blot membrane and were they exposed to film for the same period? Input and immunoprecipitate should be blotted onto the same membrane, processed together and subjected to the same period of exposure to film. Indeed, the strength of the interaction between Six4 and UTX suggests that the interaction could be indirect (through TLE1 - Grbavec et al. Biochem J 337 ( Pt 1): 13-17), or modulated through post-translational modifications (as we have previously observed for the interaction between Mef2d and Ash2L - Rampalli et al. Nature Struct Mol Biol 14: ). To further confirm the specificity of interaction between Six4 and UTX, we have now performed additional Six4 immunoprecipitations in a second cell type that endogenously expressed both Six4 and UTX. In the new Figure 4B, we demonstrate that Six4 and UTX also interact in human erythroleukemia cells (K562 cells), and furthermore, we show that Six4 doesn t interact with the TFIID subunit TAF10, nor the GCN5 associate protein Spt3. This provides European Molecular Biology Organization 12

13 more confidence in the specificity of the interaction between Six4 and UTX. Furthermore, we provide for reviewers evidence that UTX does not interact with Mef2 (Author Only Figure 5). We prefer to provide this data only for the reviewers since Mef2 co-migrates on SDS-Page gels with antibody heavy chain, and therefore the dark band from the IgG detracts the viewers attention. We hope that this new data will satisfy the reviewer about the interaction between Six4 and UTX. As for the point about the gel processing, input and eluates from the immunoprecipitation are run on the same gel and exposed for the same amount of time. The reason we have spliced the gel is to remove blank lanes and FT fractions. To demonstrate this point we provide the unspliced version of the UTX and Six4 panels in Reviewer Only Figure 6. For the other figures, the white box that separates the two sections of the gel was added in powerpoint to cover the molecular weight marker lane. The gels are run in this way to ensure that the input does not spill over into eluate lanes. To show that these gels are not spliced, we have now removed this white box from the second panel of Figure 4A, and 4B. European Molecular Biology Organization 13

14 Seenundun et al. Reviewer Only Figure 1 Expression Relative to DDX Myog CKm Differentiation (h) Differentiation (h) Control Scrambled UTX Reviewer Only Figure 1. Expression of CKm is delayed during differentiation in cells infected with lentivirus that have been selected using puromycin. C2C12 cells were infected with lentivirus at 20% confluency in growth media. Infected cells were then re-infected 24 h later with fresh lentivirus, and selection with puromycin began 36 h after the initial infection. Differentiation was induced 48 h after the start of the infection, and continued for an additional 72 h. RNA was extracted from cells at times indicated in the x-axis, converted to cdna and the amount of Myog and CKm transcript at the various times was quantitated relative to those of the ubiquitously expressed DDX5 using qpcr. Note: We believe this is caused by slightly delayed differentiation due to the Fact that it was induced at suboptimal confluence. We have thus re-examined the effect of UTX knock-down on H3K27me3 at 72 h of differentiation when CKm is strongly expressed. This H3K27me3 result is now displayed in Figure 3D of the revised manuscript.

15 0.2 Seenundun et al. Reviewer Only Figure 2 Relative UTX Enrichment (% input) 0 Differentiated shrna-scrm shrna-six4 1.8 Relative H3K27me3 Enrichment (% input) 0.0 Differentiated Myog (Region 3) CKm (Region 2) shrna-scrm shrna-six4 + + Reviewer Only Figure 2. Knock-down of Six4 leads to decreased recruitment of UTX to the Myog and Ckm genes and results in an increase in H3K27me3 enrichment within the regulatory region of the gene. Cross-linked chromatin from growing (-), or 48 h differentiated (+) C2C12 cells was subjected to ChIP analysis using antibodies directed against UTX. Immunopurified DNA was quantitated by qpcr using probes that recognize the promoter (Region 3) of Myog, or the upstream enhancer (Region 2) of CKm. G) Native chromatin from growing (-), or 48 h differentiated (+) C2C12 cells was subject to ChIP analysis using antibodies directed against H3K27me3. Immunopurified DNA was quantitated by qpcr using probes that recognize the promoter (Region 3) of the Myog gene, or the upstream enhancer (Region 2) of CKm. + +

16 Seenundun et al. Reviewer Only Figure 3 sirna 48 h Control Ash2L Ash2L β-actin Reviewer Only Figure 3. sirna mediated knock-down of Ash2L results in decreased levels of the protein in differentiating C2C12 cells. Western blot analysis of whole cell extracts taken from C2C12 cells that have been transfected with sirna targeting mash2l. Note: This figure is taken from our published paper (Rampalli et al. Nat Struc Mol Biol 14: ), and therefore cannot be used in this manuscript.

17 Seenundun et al. Reviewer Only Figure 4 IB: MLL2 IB: UTX IB: RbBP5 IB: Ash2L IB: CGBP Reviewer Only Figure 4. UTX is not stably complexed to Ash2L1 purified from K562 cells. The human erythroleukemia cell line K562 was stably transfected with the pcep4 plasmids expressing a cdna encoding Tap-tagged human Ash2L1 isoform. A large scale nuclear extract (protein concentration 10 mg/ml) was prepared from 2.5x10 8 cells, and subjected to purification by incubating for 4 h with rabbit IgG agarose. Proteins complexed with Ash2L1 were purified by washing with a stringent buffer containing 20 mm Hepes ph 7.6, 1 M NaCl, 1 M Urea, 10% glycerol, 0.2 mm EDTA, 0.5 mm DTT, and 0.5% NP-40. Proteins were then eluted by boiling in 1X loading dye, and subjected to Western blot analysis using the antibodies indicated. Note: Both TAP-tagged Ash2L1 and endogenous Ash2L1 are present in the elution though the TAP-tagged protein was specifically encoded Ash2L1 suggesting that there are at least two Ash2L molecules in the purified complex. Furthermore, mass-spec analysis of a full TAP-tag purification (IgG purification followed by Calmodulin binding) of Ash2L1 identified MLL2, HCF1C1, CGBP, Ash2L, RBBP5, and WDR5 all components of the KMT2A/ KMT2B complex. However, we found no evidence of UTX, PTIP, MLL4 or MLL3 by mass spec.