Repression of SRF target genes is critical for Mycdependent apoptosis of epithelial cells

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1 Manuscript EMBO Repression of SRF target genes is critical for Mycdependent apoptosis of epithelial cells Katrin E. Wiese, Heidi M. Haikala, Bjoern von Eyss, Elmar Wolf, Cyril Esnault, Andreas Rosenwald, Richard Treisman, Juha Klefstrom and Martin Eilers Corresponding author: Martin Eilers, Theodor Boveri Institute Review timeline: Submission date: 05 November 2014 Editorial Decision: 11 December 2014 Revision received: 08 March 2015 Editorial Decision: 25 March 2015 Revision received: 28 March 2015 Accepted: 30 March 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: Andrea Leibfried 1st Editorial Decision 11 December 2014 Thank you for submitting your manuscript entitled 'Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells'. I have now received the reports from all referees. As you can see below, all referees find your manuscript potentially interesting. However, they raise various concerns that need to be addressed. More specifically, the role of Akt in SRF-mediated competition against the Myc/Miz effect needs to be further confirmed, also at the genetic level. Furthermore, the text needs some amendments to better comment on the actual findings, and the referees ask for additional controls and experiments to further support your conclusions. Given the clear comments provided, I would like to invite you to submit a revised version of the manuscript, addressing all concerns of the referees. Please contact me in case of further questions. I should also add that it is EMBO Journal policy to allow only a single round of revision and that it is therefore important to address all concerns raised at this stage. Thank you for the opportunity to consider your work for publication. I look forward to your revision Referee #1: European Molecular Biology Organization 1

2 Manuscript EMBOJ Repression of SRF target genes is critical for Myc-dependent apoptosis of epithelial cells By Wiese et al. The authors investigated the molecular mechanism mediating induction of apoptosis by supraphysiological levels of the oncogene Myc in a model of breast epithelial cells. They report that induction of apoptosis by Myc in this model depends on the association of Myc with Miz1. They went on to demonstrate that high levels of Myc together with Miz1 invade low-affinity target sites and repress transcription of genes that are enriched for binding sites of the serum response factor (SRF). This suppression of SRF-dependent transcriptional programs contributes to cell survival of epithelial cells. Major points: Figure 1: To confirm that high expression levels of wildtype Myc are counterselected more rapidly than the mutant variant of Myc, i.e. McyVD, it is recommended to quantify protein levels of Myc, e.g. by densitometric analysis, and to provide also a quantitative analysis of protein expression with data derived from several independent experiments. Figure 2: Data shown in panel C of figure 2 demonstrate that expression of Myc-ER results in induction of primarily Annexin V single-positive cells. By comparison, expression of wildtype Myc or MycVD leads to an increase in both Annexin V single-positive as well as Annexin V/propidium iodide double-positive cells. Therefore, the question arises whether the different variants of Myc differ in their ability to induce apoptosis compared to other forms of cell death. Figure 6: It is unclear why the authors used different ways to present data on caspase activation. For example, in panel D they present the percentage of cleaved caspase-3 cells, whereas they show fold increase in caspase-3/-7 cleavage in panels E, F and H. To allow a better comparison of the different sets of experiments, it is recommended to consistently use similar parameters to assess caspase activation. Furthermore, it is recommended to determine cell death induction in addition to caspase activation for the different types of apoptotic stimuli that were used, i.e. doxorubicin, TRAIL, glutamine depletion of glucose depletion. Figure 7: While data presented in panel B demonstrate that Akt inhibition using the pharmacological inhibitor MK-2206 diminishes the ability of ΔN-MAL to alleviate Myc-induced apoptosis in glutaminedeprived cells. This effect is however only partial. It is therefore suggested to further investigate the role of Akt in mediated survival by using a genetic approach to the pharmacological approach. Referee #2: This study by Wiese, et. al explores the role of Myc as a regulator of apoptotic processes in epithelial cells, and how excess Myc can sensitize cells to apoptotic signaling. It is asserted that these apoptotic processes act, in part, through Miz1 protein as the authors use a MycVD mutant unable to bind and repress genes through Miz1 interaction. The group shows how, overall, high levels of Myc (similar to those in HeLa or HCT116 cell lines) results in increased levels of apoptosis/p53/cleaved PARP alongside decreases in gene expression with particular regard to genes related to the regulation of apoptosis and cell adhesion. They show how these genes are also commonly bound by Miz1, and that Miz1/Myc bind to relatively low-affinity binding sites within the genome. The study also addresses which pathways may be inhibited by Myc/Miz1 interaction, and give data supporting a repression of targets normally regulated and activated by Srf/Mrtf-A. To European Molecular Biology Organization 2

3 this end, the study utilizes a ΔN-MAL construct which, when induced, is a constitutively active form of Mrtf-A and which attenuates the pro-apoptotic processes activated by Myc overexpression. This study overall provides convincing and important insights into a novel element of Myc pathway signaling. If the points below are addressed it would further strengthen the paper. Major Points (1) p.7. Myc and MycVD induce Bim to a similar extent, and the authors state "... elevated Bim levels are tolerated in cells expressing MycVD". Can the authors provide any further explanation for this or expand on this in the Discussion? How would Bim fit into their model in Fig. 7C? A parallel Myc-related pathway not tied to Miz and Srf? (2) p.8. "... genes regulated by Myc and MycVD were similar to previously identified target genes of Myc and confirmed that MycVD was compromised in its ability to repress known Myc target genes". This may be somewhat of an overstatement. The majority of targets for Myc and MycVD are similar to previously identified genes (only 8 clusters total are listed -- how many genes compose each cluster?). Further, for the "compromised in its ability to repress known Myc target genes" statement, this does not seem to match the data overall that well. The normalized expression patterns are very similar between WT and VD, except for the last group, which has a relatively higher False Discovery Rate (>0.05). However, this FDR is not particularly high when compared to other analyses. The methods section does not list the chosen FDR cutoff. Therefore, the extent of "compromised" function for MycVD may not be particularly extensive. (3) p.14., Figure 7B. While significant, and an n = 4 number of samples/experiments analyzed, the effect does not seem very pronounced -- were the p-values significantly different between the -Gln, +4-OHT vehicle vs. Dox. What were the p values and is this experiment sufficiently powered? Additional experiments could be helpful to determine the strength of the effect. Minor Points (1) Are the symbols used for p values as defined in Fig. 1B defined in the same way in subsequent uses such as in Fig. 2D and elsewhere? (2) p.3. "GO-term analyses show that differences... These findings suggest that oncogenic levels of Myc "invade" low affinity binding sites and thereby regulate genes that are functionally distinct from those regulated by physiological Myc levels". Written as-is, this statement is not entirely accurate; based off the previous statements, Myc occupies genes with similar function and overexpression of Myc results in an affinity for genes not normally bound by Myc at physiologic levels. The statement presented here suggest that Myc invades low affinity binding sites that are functionally distinct from those regulated by Myc normally -- contrasting to the statements previously reported by the authors. This needs rephrasing. (3) p.6., Fig. 1F. For the colony assays, what are they stained with? This should be listed in the Figure legend. (4) p.6. It would be helpful for the authors to explain the importance of using Myc-ER instead of regular/total Myc protein expression. Also, the paper in which MycER was first obtained/described should be cited. For example, Alberto Gandarillas and Fiona Watt, 1997 Genes and Development, or another paper. (5) Figure 2B. This could be moved to the Supplemental material section. Figure E2A-C. These are more important and could be moved to the main figures. They could take the place of Fig. 2B. (6) p.11. "... invades low-affinity binding sites in core promoters in a complex with Miz1, leading to transcriptional repression". Although the authors present previous studies demonstrating that Miz1/Myc may invade low-affinity binding sites, the data cited here does not definitively state this. The statement needs revising. As the high-affinity binding sites measured here refer to E-boxes, this does not mean that the Myc/Miz1 interaction invade low-affinity Myc binding sites per se, but rather invade regions that are E-box deficient. These could be low-affinity Myc binding sites or have some alternative form of Myc affinity (perhaps another cofactor). (7) p.31., Figure 2C. This is a little unclear -- are the p-values between Myc-ER and MycVD-ER at a given time point? Also, how are the apoptotic sums calculated? Was there some form of normalization (e.g. were equal amounts of cells sorted)? (8) p.32., Figure 2F. If what is pictured is a representative experiment, how do repeated experiments respond to the same experimental conditions? The error bars for technical triplicates is fine, but it would be better to increase the n >2, normalize data, and report the changes together. As is, the data is somewhat limited and unconvincing. European Molecular Biology Organization 3

4 (9) p.33., Figure 4D. Please standardize nomenclature; in the rest of the text, MycVD is referred to as MycVD-ER and not Myc-ERVD. Referee #3: This manuscript from the Eilers lab continues to examine repression by a Myc-Miz complex and its role in apoptosis. They establish conditional expression for Myc and Myc-VD, which cannot bind Myc, and conclude that interaction between Myc and Myc is required for Myc-dependent apoptosis (already established in other papers). This data is quite convincing. The authors then go on to examine the genome wide occupancy of Myc and Miz. Much of this data is convincing, but see #4 below. Further, the authors argue that the VD mutant does not recruit Miz based on a comparison of figures 4 C and D, but it appears that MycVD binds promoters less well than WT making this comparison a challenge. The authors next connect Myc-Miz repressed targets to SRF activated genes. Again quite convincing and figure 6 provides nice evidence to support the physiological relevance of the regulatory mechanism they have proposed. Finally, the authors show that Myc activation suppresses AKT and this can be blocked by constitutive activation of SRF. The western blot in Fig 7A is quite convincing. The effect of the AKT inhibitor shown in figure 7B is less so and it is not clear whether the effect of MK-2206 in the + Myc/-glutamine/+ N-Mal is significant. A more sophisticated statistical analysis of this experiment should be done and an AKT knockdown should be used to validate this pharmacological result. In summary, this is an interesting manuscript. Much of the data is convincing with the mentioned caveats spelled out above and in the other points below. The functional connection and the overall model is less strong than it could be because the AKT inhibitor only subtly, if at all, sensitizes cells to Myc-dependent apoptosis under stress conditions. One also has concerns over the final model presented in Fig 7C because N-Mal does not increase AKT P-S473. Other points: 1. In figure 3A, the authors show a IHC experiments showing that a cells in a tumor section that express Miz also express Myc. This is a single tumor slice and the authors should determine whether Miz expression correlates with Myc expression or Myc-gene signature using one of several available breast cancer expression datasets. 2. Also regarding figure 3, the authors state on page 9 that "expressing high levels of Myc/Mizrepressed genes" I'm confused what this actually means. That the Myc/Miz genes are not suppressed in the lower grade tumors? If so this would seem to contradict their hypothesis that Myc-Miz repressed targets have a tumor suppressive function. Please clarify. 3. The comparison of datasets from U20S and MCF10A is difficult to interpret because cells are clearly differentially dependent on Miz1 for their survival. 4. The concept that high-myc and Miz invade low affinity sites is over stated. Joint binding sites are not enriched in CACGTG sites, but do have a number of non-canonical sites. Older papers show that these non-canonical sites bind Myc:Max with similar affinity to CACGTG. Are non-canonical sites really low affinity? 5. The authors state the "most" of the Myc-Miz bound sites are independent from the SRF sites. They show two examples of this in fig 5F, one with shared and one independent binding. The authors should provide more evidence that "most" co-occupied targets have independent binding sites. 1st Revision - authors' response 08 March 2015 Response to the referees' comments We would like to thank all three reviewers for their efforts in reviewing this manuscript and their helpful comments. European Molecular Biology Organization 4

5 Referee #1 Major points Figure 1: To confirm that high expression levels of wildtype Myc are counterselected more rapidly than the mutant variant of Myc, i.e. MycVD, it is recommended to quantify protein levels of Myc, e.g. by densitometric analysis, and to provide also a quantitative analysis of protein expression with data derived from several independent experiments. To address this comment, we have now replaced Figure 1, panel E, by an analysis that is based on three independent biological experiments in MCF10A cells. The panel shows a representative immunoblot and a quantitation of three experiments with corresponding p-values. The immunoblot of HMLE cells, including a densitometric analysis, has been moved to Extended Figure 1 D. Figure 2: Data shown in panel C of figure 2 demonstrate that expression of Myc-ER results in induction of primarily Annexin V single-positive cells. By comparison, expression of wildtype Myc or MycVD leads to an increase in both Annexin V single-positive as well as Annexin V/propidium iodide double-positive cells. Therefore, the question arises whether the different variants of Myc differ in their ability to induce apoptosis compared to other forms of cell death. In our hands, the relative proportion of Annexin V single-positive ( early apoptotic ) to AnnexinV/propidium iodide double-positive ( late apoptotic ) cells varies between different experiments. However, we do not see a consistent difference between constitutive and inducible variants of MYC. We provide two experiments to illustrate this point as a "Figure 1 for the Reviewers" at the end of these comments (see induction of both Annexin V single positive as well as Annexin V / propidium iodide double-positive cells after MYC-ER induction in the right panel). Another example is provided in Figure 6G. We have inserted a sentence in the text (page 7) alerting readers to this issue. Figure 6: It is unclear why the authors used different ways to present data on caspase activation. For example, in panel D they present the percentage of cleaved caspase-3 cells, whereas they show fold increase in caspase-3/-7 cleavage in panels E, F and H. To allow a better comparison of the different sets of experiments, it is recommended to consistently use similar parameters to assess caspase activation. Furthermore, it is recommended to determine cell death induction in addition to caspase activation for the different types of apoptotic stimuli that were used, i.e. doxorubicin, TRAIL, glutamine depletion of glucose depletion. Panel 6 D represents the quantification of the immunofluorescence analysis in panel C, where we used an antibody specific for activated (cleaved) caspase 3 to determine the percentage of apoptotic cells. In contrast, panels E, F and H are the result of Caspase-Glo 3/7 luminescence-based assays, which measure caspase 3 and 7 activities by means of substrate cleavage. Hence, the use of two different ways of plotting is necessary, since the caspase activity assays used in panels E, F and H do not immediately translate into numbers or percentages of apoptotic cells. To make this difference more clear, we changed the axis labels in panels 6E, F and H, as well as Expanded View Item 8A from caspase 3/7 cleavage to caspase 3/7 activity. In addition, Figure 6G and Expanded View Item 8B are FACS assays illustrating cell death in response to doxorubicin, glutamine deprivation and TRAIL, as measured by Annexin V / propidium iodide staining. Figure 7: While data presented in panel B demonstrate that Akt inhibition using the pharmacological inhibitor MK-2206 diminishes the ability of ΔN-MAL to alleviate Myc-induced apoptosis in glutaminedeprived cells. This effect is however only partial. It is therefore suggested to further investigate the role of Akt in mediated survival by using a genetic approach to the pharmacological approach. European Molecular Biology Organization 5

6 To address this comment, we have now considerably expanded Figure 7. First, we have generated new cell pools and repeated experiments using MK-2206 to retest the strength of the effect and improve the statistical power. The results are now shown for three new independent experiments (7B). Second, we show that sirnas targeting Akt1 and Akt2 abolish the ability of SRF to protect against apoptosis in this setting (7C). Please note that the time course of these experiments is slightly longer than the inhibitor experiment and that sirna-mediated depletion of Akt1/2 also reduces proliferation and growth; these effects are stronger after this longer time than in 7B. Therefore, the total caspase activity shown here, which is normalized to cell number at the start of the experiment, slightly underestimates the apoptotic activity in wells treated with siakt1/2. However, the neutralizing effect of sirna to deltan-mal-dependent protection from apoptosis is clear and consistent with the pharmacological experiments in Fig. 7B. Third, we show that expression of constitutively active Akt (v-akt) blocks Myc-induced apoptosis in this system (7D). This is consistent with previous work, which we have quoted. Collectively, the data clearly establish regulation of Akt as one downstream mediator of SRF. Referee #2 Major Points (1) p.7. Myc and MycVD induce Bim to a similar extent, and the authors state "... elevated Bim levels are tolerated in cells expressing MycVD". Can the authors provide any further explanation for this or expand on this in the Discussion? How would Bim fit into their model in Fig. 7C? A parallel Myc-related pathway not tied to Miz and Srf? We have now added a section to the discussion addressing this important point (page 18). Previous work had clearly established that Myc induces a change in the balance of pro- to anti-apoptotic BH3 proteins at the mitochondrial membrane. Since Akt phosphorylates and thereby inactivates Bad, we believe that this framework can explain our findings. The simplest model is that the combined levels of active Bad and Bim contribute to apoptosis of Myc-expressing cells, and higher levels of Bim are tolerated in cells when Srf/Akt is active. (2) p.8. "... genes regulated by Myc and MycVD were similar to previously identified target genes of Myc and confirmed that MycVD was compromised in its ability to repress known Myc target genes". This may be somewhat of an overstatement. The majority of targets for Myc and MycVD are similar to previously identified genes (only 8 clusters total are listed -- how many genes compose each cluster?). Further, for the "compromised in its ability to repress known Myc target genes" statement, this does not seem to match the data overall that well. The normalized expression patterns are very similar between WT and VD, except for the last group, which has a relatively higher False Discovery Rate (>0.05). However, this FDR is not particularly high when compared to other analyses. The methods section does not list the chosen FDR cutoff. Therefore, the extent of "compromised" function for MycVD may not be particularly extensive. To address this comment, we have now included a GSEA that shows all gene sets that are significantly regulated by Myc (FDR cutoff <=0.25), plotting the normalized enrichment score for each gene set both for Myc-ER and MycVD-ER. This plot is now included as Figure E4D. In this panel, we have labeled in red the 28 gene sets that are annotated as sets of Myc-regulated genes. This plots confirms that sets of Myc-activated genes are induced to the same extent by Myc-ER and MycVD-ER, whereas MycVD-ER is compromised in repression of multiple gene sets. The plot also illustrates that this effect is consistent (and significant), but clearly MycVD is not a black-and-white tool. In addition, we have added the number of genes in each gene set to the table in Figure E4C. (3) p.14., Figure 7B. While significant, and an n = 4 number of samples/experiments analyzed, the effect does not seem very pronounced -- were the p-values significantly different between the -Gln, +4-OHT vehicle vs. Dox. What were the p values and is this experiment sufficiently powered? Additional experiments could be helpful to determine the strength of the effect. European Molecular Biology Organization 6

7 (Please also see the similar comment concerning Figure 7 by reviewer 1.) To address this comment, we have now considerably expanded Figure 7. First, we have generated new cell pools and repeated experiments using MK-2206 to retest the strength of the effect and improve the statistical power. The results are now shown for three new independent experiments (7B). Second, we show that sirnas targeting Akt1 and Akt2 abolish the ability of SRF to protect against apoptosis in this setting (7C). Please note that the time course of these experiments is slightly longer than the inhibitor experiment and that sirna-mediated depletion of Akt1/2 also reduces proliferation and growth; these effects are stronger after this longer time than in 7B. Therefore, the total caspase activity shown here, which is normalized to cell number at the start of the experiment, slightly underestimates the apoptotic activity in wells treated with siakt1/2. However, the neutralizing effect of sirna to deltan-mal-dependent protection from apoptosis is clear and consistent with the pharmacological experiments in Fig. 7B. Third, we show that expression of constitutively active Akt (v-akt) blocks Myc-induced apoptosis in this system (7D). This is consistent with previous work, which we have quoted. Collectively, the data clearly establish regulation of Akt as one downstream mediator of SRF. Minor Points (1) Are the symbols used for p values as defined in Fig. 1B defined in the same way in subsequent uses such as in Fig. 2D and elsewhere? Yes. To avoid confusion, we have now added the definition of p-values to all subsequent figure legends. (2) p.3. "GO-term analyses show that differences... These findings suggest that oncogenic levels of Myc "invade" low affinity binding sites and thereby regulate genes that are functionally distinct from those regulated by physiological Myc levels". Written as-is, this statement is not entirely accurate; based off the previous statements, Myc occupies genes with similar function and overexpression of Myc results in an affinity for genes not normally bound by Myc at physiologic levels. The statement presented here suggest that Myc invades low affinity binding sites that are functionally distinct from those regulated by Myc normally -- contrasting to the statements previously reported by the authors. This needs rephrasing. We agree and have re-phrased the description accordingly (see page 3). We hope we understood the comment correctly. (3) p.6., Fig. 1F. For the colony assays, what are they stained with? This should be listed in the Figure legend. The colony assays were stained with crystal violet and we have added that information to the figure legend. (4) p.6. It would be helpful for the authors to explain the importance of using Myc-ER instead of regular/total Myc protein expression. Also, the paper in which MycER was first obtained/described should be cited. For example, Alberto Gandarillas and Fiona Watt, 1997 Genes and Development, or another paper. We now quote the correct references on page 6. (5) Figure 2B. This could be moved to the Supplemental material section. Figure E2A-C. These are more important and could be moved to the main figures. They could take the place of Fig. 2B. We agree and have changed Figure 2B into Figure E2A. The old Figure E2A is now Figure 2 C. E2B,C are confirmatory (in a second cell line) and- also for space reasons, we have not moved these two panels. European Molecular Biology Organization 7

8 (6) p.11. "... invades low-affinity binding sites in core promoters in a complex with Miz1, leading to transcriptional repression". Although the authors present previous studies demonstrating that Miz1/Myc may invade low-affinity binding sites, the data cited here does not definitively state this. The statement needs revising. As the high-affinity binding sites measured here refer to E-boxes, this does not mean that the Myc/Miz1 interaction invade low-affinity Myc binding sites per se, but rather invade regions that are E-box deficient. These could be low-affinity Myc binding sites or have some alternative form of Myc affinity (perhaps another cofactor). We agree and have revised the statement to: "invades sites that lack consensus E-Box sequences in complex with Miz1." We have also changed the discussion of affinity at several places in response to a comment made by referee #3. (7) p.31., Figure 2C. This is a little unclear -- are the p-values between Myc-ER and MycVD-ER at a given time point? Also, how are the apoptotic sums calculated? Was there some form of normalization (e.g. were equal amounts of cells sorted)? We have altered the Figure legend to explain better what the p-values refer to. "apoptotic sums" refer to the percentage of total cells, so the normalization is to total cell number. (8) p.32., Figure 2F. If what is pictured is a representative experiment, how do repeated experiments respond to the same experimental conditions? The error bars for technical triplicates is fine, but it would be better to increase the n >2, normalize data, and report the changes together. As is, the data is somewhat limited and unconvincing. We have repeated the experiment several times. For each gene, the number of independent biological replicates is now either n=5 and n=6 and shown. p-values are also shown. In this analysis, one of the four original genes turned out to be more variable than expected, so we have deleted it from the figure. (9) p.33., Figure 4D. Please standardize nomenclature; in the rest of the text, MycVD is referred to as MycVD-ER and not Myc-ERVD. We have changed the Figure legend accordingly. Referee #3 "Further, the authors argue that the VD mutant does not recruit Miz based on a comparison of figures 4 C and D, but it appears that MycVD binds promoters less well than WT making this comparison a challenge. " Association with Miz1 stabilizes Myc on chromatin and protects it from proteasomal degradation. Some of the evidence for this is published (1). To illustrate the effect, we include an analysis of the effect of Miz1 depletion on Myc levels in several human tumor cell lines as Figure 2 for the reviewer at the end of this document. While the effects are not as strong for Myc versus MycV394D in the MCF10A analyzed here, they are consistent with the observed differences in chromatin binding and argue that MycVD binds promoters less well since it does not recruit Miz1. The western blot in Fig 7A is quite convincing. The effect of the AKT inhibitor shown in figure 7B is less so and it is not clear whether the effect of MK-2206 in the + Myc/-glutamine/+ N-Mal is significant. A more sophisticated statistical analysis of this experiment should be done and an AKT knockdown should be used to validate this pharmacological result. European Molecular Biology Organization 8

9 (Please also see the similar comment concerning Figure 7 by reviewer 1.) To address this comment, we have now considerably expanded Figure 7. First, we have generated new cell pools and repeated experiments using MK-2206 to retest the strength of the effect and improve the statistical power. The results are now shown for three new independent experiments (7B). Second, we show that sirnas targeting Akt1 and Akt2 abolish the ability of SRF to protect against apoptosis in this setting (7C). Please note that the time course of these experiments is slightly longer than the inhibitor experiment and that sirna-mediated depletion of Akt1/2 also reduces proliferation and growth; these effects are stronger after this longer time than in 7B. Therefore, the total caspase activity shown here, which is normalized to cell number at the start of the experiment, slightly underestimates the apoptotic activity in wells treated with siakt1/2. However, the neutralizing effect of sirna to deltan-mal-dependent protection from apoptosis is clear and consistent with the pharmacological experiments in Fig. 7B. Third, we show that expression of constitutively active Akt (v-akt) blocks Myc-induced apoptosis in this system (7D). This is consistent with previous work, which we have quoted. Collectively, the data clearly establish regulation of Akt as one downstream mediator of SRF. One also has concerns over the final model presented in Fig 7C because N-Mal does not increase AKT P-S473. We are not certain we understand this comment. Our model predicts that N-Mal enhances AKT phosphorylation at P-S473 in the presence of active Myc, when SRF activity is suppressed by Myc/Miz1. When Myc levels are low, SRF would be active and expression of N-Mal has no effect on Akt activity. We have added a sentence to better explain this model to the figure legend. Other points 1. In figure 3A, the authors show a IHC experiments showing that a cells in a tumor section that express Miz also express Myc. This is a single tumor slice and the authors should determine whether Miz expression correlates with Myc expression or Myc-gene signature using one of several available breast cancer expression datasets. We have stained a total of 15 tumors. All ten high-grade (triple-negative) tumors show strong expression of both Myc and Miz1. In low-grade tumors, expression of Miz1 is high, but expression of Myc is lower. As a result, Myc-repressed genes are expressed at higher levels in low-grade tumors, consistent with the analysis shown in Figure 3B. The information is now included in Figure 3A. 2. Also regarding figure 3, the authors state on page 9 that "expressing high levels of Myc/Mizrepressed genes" I'm confused what this actually means. That the Myc/Miz genes are not suppressed in the lower grade tumors? If so this would seem to contradict their hypothesis that Myc-Miz repressed targets have a tumor suppressive function. Please clarify. We agree with this analysis. Myc/Miz1-repressed targets have a tumor-suppressive function in the context of wild-type p53, but not in the absence of p53 (see Figure E3). Figure 3, panel B, shows that Myc/Miz1-mediated repression clearly correlates with mutation of p53 in human mammary tumors. A Kaplan-Meier analysis shows that Myc/Miz1-mediated repression is indicative of poor prognosis, so it is possible repression by Myc/Miz1 is oncogenic in a p53-mutant situation. For example, Myc/Miz1 represses Cdk inhibitors and this is oncogenic in lymphomas (2). We did not include these data, since there are many confounding factors for the analysis: for example, Myc/Miz1-dependent repression of course inversely correlates with Myc-dependent activation, so the enhanced Myc-dependent activation could be oncogenic and repression neutral in a p53-mutant situation. 3. The comparison of datasets from U20S and MCF10A is difficult to interpret because cells are clearly differentially dependent on Miz1 for their survival. European Molecular Biology Organization 9

10 We agree that this is a caveat. However, this panel provides an - in our view necessary - argument that the transcriptional phenotype of the VD mutant is due to its reduced interaction with Miz1. 4. The concept that high-myc and Miz invade low affinity sites is over stated. Joint binding sites are not enriched in CACGTG sites, but do have a number of non-canonical sites. Older papers show that these non-canonical sites bind Myc:Max with similar affinity to CACGTG. Are non-canonical sites really low affinity? (Please see the reply to a similar comment (6) made by Referee#2.) We have replaced the statement by the more precise statement "invades sites deficient of consensus E-Box sequences in complex with Miz1." The issue of how much Myc/Max binding affinity discriminates canonical from non-canonical sites is still vigorously debated. Biophysical measurements with intact DNA binding domains clearly show discrimination with a difference in K d values of about 200fold (for Max homodimers), in vivo the situation is less clear (3, 4). We discuss these analyses now in the text and have also deleted a couple of references to affinity where they are not necessary. 5. The authors state the "most" of the Myc-Miz bound sites are independent from the SRF sites. They show two examples of this in fig 5F, one with shared and one independent binding. The authors should provide more evidence that "most" co-occupied targets have independent binding sites. In order to address this issue, we have added a plot of the distance between Myc and Miz1 and Myc and SRF binding sites for all jointly bound repressed genes as a new panel to Figure 5F. The plot shows that Myc and Miz1 binding sites co-localize precisely, whereas Myc and SRF binding sites show a wide range of distances from each other. The example genes have now been moved to Figure E7C. References 1. Salghetti SE, Kim SY, & Tansey WP (1999) Destruction of Myc by ubiquitin-mediated proteolysis: cancer-associated and transforming mutations stabilize Myc. The EMBO journal 18(3): van Riggelen J, et al. (2010) The interaction between Myc and Miz1 is required to antagonize TGFbeta-dependent autocrine signaling during lymphoma formation and maintenance. Genes & development 24(12): Guo J, et al. (2014) Sequence specificity incompletely defines the genome-wide occupancy of Myc. Genome Biol 15(10): Sauve S, Naud JF, & Lavigne P (2007) The mechanism of discrimination between cognate and non-specific DNA by dimeric b/hlh/lz transcription factors. J Mol Biol 365(4): European Molecular Biology Organization 10

11 Figures for the Reviewer Figure % of all cels % of all cells ctr ctr ctr MYC-ER MYCVD-ER MYC-ER MYCVD-ER Annexin V positive Annexin V/PI double-positive The panel shows the percentage of annexin-v positive (early apoptotic) and annexinv/propidium iodide (PI) double-positive (late apoptotic) cells from two experiments, in which MycER and MycVD-ER are activated in MCF10A cells. MycVD is clearly compromised in both experiments; there are variations in the relative percentage in late versus early apoptotic cells. Figure 2 A. kda kda 100 B plko shmiz1 #1 shmyc U2OS shluc shmiz1 #1 shmiz1 #2 -Miz1 -Myc -Actin relative mrna expression HCT shmyc shluc shmiz1 #1 shmiz1 #2 shmyc Myc Miz1 plko shmiz1 #1 shmyc shluc HaCat shmiz1 #1 shmiz1 #2 shmyc relative protein levels plko Miz1 Myc shmiz1 #1 shmyc 100 -Miz1 55 -Myc 40 -Actin Miz1 Myc Miz1 stabilizes Myc in human tumor cells. Panel A shows relative Myc and Miz1 protein and mrna levels of Myc and Miz1 in HeLa cells. Quantification is from three independent biological replicas. Panel B shows similar experiments using two distinct shrnas in three different human European Molecular Biology Organization 11

12 tumor cell lines. We concluded that a fraction of Myc is stabilized by Miz1 in human tumor cells; fractionation experiments show that this is bound to chromatin (Not shown). We believe, therefore, that the reduced binding of MycVD to chromatin in MCF10A cells is due to its reduced binding to Miz1. These are yet unpublished data. 2nd Editorial Decision 25 March 2015 Thank you for submitting your revised manuscript for consideration by the EMBO Journal. It has now been seen by the referees again, whose comments are enclosed. As you will see, the referees are broadly in favor of publication, pending satisfactory minor revision. I would thus like to ask you to address the remaining concerns raised by referee #3 in a final version of your manuscript. I am therefore formally returning the manuscript to you for a final round of minor revision. Once we should have received the revised version, we should then be able to swiftly proceed with formal acceptance and production of the manuscript! Referee #2: All my previous concerns have been adequately addressed in the revision. Referee #3: This revision answers most of my main criticisms. However, I have several remain concerns that should be addressed. Most importantly, the connection to AKT as a downstream effector of MRTF:SRF is not convincing. This really needs to be addressed or the paper reworked without this data. I think this could easily be accomplished without reducing the impact of their overall findings. Points: 1) Can the authors really conclude that MYC:VD drives less apoptosis in the presence of doxorubicin or the absence of glutamine? The control cells with Myc:VD are less apoptotic on their own. Please clarify. 2) The SRF activation of adhesion genes is proposed to be a critical regulator of AKT signaling. The data showing that FAK and IlK promote AKT-S473P is in the extended figures and they don't actually show that N-Mal elevates FAK and or Ilk expression. 3) The affect of N-Mal on AKT-S473P is very subtle (Fig 7A) Further the affects of AKT inhibitor or AKT1/2 knockdown on survival are subtle and not the same in the absence of glutamine. MK increased survival in -Gln control and in N-Mal expression cells (Fig 7B), yet AKT1/2 knockdown didn't increase survival in -Gln medium (Fig 7C). In both experiments the effects are subtle and makes one wonder whether the effects observed are more a reflection of the error in the assay rather than a reflection of the underlying role of AKT. European Molecular Biology Organization 12

13 2nd Revision - authors' response 28 March 2015 Response to Reviewer`s Comments Reviewer #3 "Can the authors really conclude that MYC:VD drives less apoptosis in the presence of doxorubicin or the absence of glutamine? The control cells with Myc:VD are less apoptotic on their own. Please clarify." MycVD cells are generally "happier" under all circumstances, stressing them by glutamine deprivation or DNA damage brings the difference to cells expressing Myc WT to the forefront. We do not imply a specific role in glutamine or DNA damage signaling. We have rephrased the statement in the text to better reflect our findings (last sentence on page 5). "The SRF activation of adhesion genes is proposed to be a critical regulator of AKT signaling. The data showing that FAK and IlK promote AKT-S473P is in the extended figures and they don't actually show that N-Mal elevates FAK and or Ilk expression." The Ilk/Fak blot is intended to show that integrins are a relevant upstream regulator of AKT phosphorylation, reproducing multiple published data in our experimental setting. We do not suggest that Myc or N-Mal regulate Ilk/Fak expression. "The affect of N-Mal on AKT-S473P is very subtle (Fig 7A) Further the affects of AKT inhibitor or AKT1/2 knockdown on survival are subtle and not the same in the absence of glutamine. MK-2206 increased survival in -Gln control and in N-Mal expression cells (Fig 7B), yet AKT1/2 knockdown didn't increase survival in -Gln medium (Fig 7C). In both experiments the effects are subtle and makes one wonder whether the effects observed are more a reflection of the error in the assay rather than a reflection of the underlying role of AKT." In Figure 7A, we argue that Myc represses AKT activity (compare lane 1 to 3) and that N-Mal restores this (compare lane 3 to 4). For both statements, the readout is both AKT-P-S473 (Modification of AKT indicative of active status) and GSK3-PS9 (downstream target of AKT). We would politely disagree that these are subtle effects. Functionally, we now show three experiments addressing the causal role of AKT. The rescue of apoptosis by vakt (Figure 7D) is complete, so AKT is a relevant player in this experimental setting. We agree that the effects with MK-2206 and siakt are more subtle. However, the experiment shown in Figure 7B has now been performed a total of seven times: of these, three results obtained with one batch of cells are shown here. Careful statistics tell us that the effect is significant. The same was true for the other four results obtained in an independent batch of cells (see 1 st submission). We state in the text that MK-2206 "diminishes" the rescue, so part of the SRF-effects are AKT-dependent. SRF-restored cells are also more adhesive, so they are generally more robust, but this is hard to formalize. The description of the siakt result in the text was very condensed and we have expanded this now (page 15). The critical result (no rescue by N-Mal in the presence of siakt) is the same as obtained with the inhibitor. We have re-checked and realized on the basis of the comments that the discussion lacked a qualifying statement that the effects of AKT depletion/inhibition are only partial, so we have altered a sentence on page 18 ("We therefore propose that one mechanism...") to make this absolutely clear. European Molecular Biology Organization 13

14 Acceptance 30 March 2015 Thank you very much for sending your revised manuscript to us. I appreciate the introduced changes and I am happy to accept your manuscript in principle for publication in The EMBO Journal. European Molecular Biology Organization 14