MYSM1 co-activates ERα action via histone and non-histone deubiquitination to confer antiestrogen resistance in breast cancer

  1. Ruina Luan
  2. Ge Sun
  3. Baosheng Zhou
  4. Manlin Wang
  5. Yu Bai
  6. Chunyu Wang
  7. Shengli Wang
  8. Kai Zeng
  9. Jianwei Feng
  10. Mingcong He
  11. Lin Lin
  12. Yuntao Wei
  13. Qiang Zhang
  14. Yue Zhao

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Abstract

Endocrine resistance is a crucial challenge in estrogen receptor alpha (ERα)-positive breast cancer (BCa) therapy. Aberrant alteration in modulation of E2/ERα signaling pathway has emerged as the putative contributor for endocrine resistance in BCa. Thus, identification the efficient ERα cofactor remains necessary for finding a potential therapeutic target for endocrine resistance. Herein, we have demonstrated that Myb like, SWIRM and MPN domains 1 (MYSM1) as a histone deubiquitinase is a novel ERα co-activator with established Drosophila experimental model. Our results showed that MYSM1 participated in up-regulation of ERα action via histone and non-histone deubiquitination. We provided the evidence to show that MYSM1 was involved in maintenance of ERα stability via ERα deubiquitination. Furthermore, silencing MYSM1 induced enhancement of histone H2A ubiquitination as well as reduction of histone H3K4me3 and H3Ac levels at cis regulatory elements on promoter of ERα-regulated gene. In addition, MYSM1 depletion attenuated cell proliferation/growth in BCa-derived cell lines and xenograft models. Knockdown of MYSM1 increased the sensitivity of antiestrogen agents in BCa cells. MYSM1 was highly expressed in clinical BCa samples, especially in aromatase inhibitor (AI) non-responsive tissues. These findings clarify the molecular mechanism of MYSM1 as an epigenetic modifier in regulation of ERα action and provide a potential therapeutic target for endocrine resistance in BCa.

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    • Reviewer #1 (Evidence, reproducibility and clarity (Required))*

    *Summary: ** ER+ breast cancer is the most common form of cancer. Targeting ER-alpha transcriptional cofactors present one potential method to target the disease. The authors demonstrate that MYSM1 is a histone deubiquitinase and a novel ER cofactor, functioning by up-regulating ER action via histone deubiquitination. Loss of MYSM1 attenuated cell growth and increase breast cancer cell lines' sensitivity to anti-estrogens. The authors, therefore, propose MYSM1 as a potential therapeutic target for endocrine resistance in Breast cancer. * *

    *Major Comments: *

    The data as presented is convincing, and the evidence for the role of MYSM1 as a co-activator of ER-alpha is extensive. Given the amount of data, I do not believe any additional experiments are needed. I could not find any description of ethics for the patient samples used.

    Response: Appreciate for the positive response from the reviewer. According to the important suggestions, the ethics approval for the patient specimens have been included in the “Materials and methods” part.

    Minor Comments:

    The data as presented is convincing, and the evidence for the role of MYSM1 as a co-activator of ER-alpha is extensive. Given the amount of data, I do not believe any additional experiments are needed. I could not find any description of ethics for the patient samples used.

    • Response: Appreciate for the positive response from the reviewer. According to the important suggestions, the ethics approval for the patient specimens have been included in the “Materials and methods” part.

    (1)- Figure 4C - is the increase of binding in response to Estrogen significant? It is an important control to show for MCF7 as Fig 4B is in T47D.

    Response: According to the comments from reviewers, we conducted statistical difference analysis in Figure 4C, our results have shown that the recruitment of MYSM1 or ERa on c-Myc ERE region is significantly increased upon E2 treatment in MCF-7 cells.

    *(2)- Figure 6 - Can we clarify that B = Before, A = After *

    Response: Apologize for the unclear description in Figure 6. As clarified by the reviewer, “B” represents before AI treatment, “A” represents after AI treatment. We have included the description in the “Figure legends” section.

    (3)- The use of Fig EV was confusing to me, I assume it means supplementary?

    Response: Thank you for your question. Since our priority affiliate journal is belong to EMBO Press, this manuscript was written according to the relevant requirements and “EV” is the abbreviation of “Expanded View”, which is the same as that of the supplementary figures.

    *Reviewer #1 (Significance (Required)): **

    • Discovery science to understand the regulation of the ER is critical in discovering new opportunities to target breast cancer. As far as I can tell this is the first study where MYSM1 is a co-regulator of the ER.
    • The significance would be greatly increased if the manuscript identified opportuinities to target the ER via this pathway using existing compounds. However, it is reasonable to consider this is beyond the scope of this study.*

    Response: According to your valuable suggestion, we thus turned to screen the commercially-available compound in ZINC database to find the compounds that could spatially interact with MYSM1 protein, thereby inhibiting the activity of MYSM1. We plan to perform the additional biological function experiments to explore the effect of MYSM1-targeting compounds on the sensitivity of breast cell lines to anti-estrogen treatment.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    *Below I outline a few suggestions that can help clarify specific aspects of the study. *

    Fig. 2: Ideally a rescue study with a wild-type and catalytically mutant MYSM1 should be performed.

    Response: Thank you for your suggestion. To address this point, we will perform a rescue study with a wild-type and catalytically mutant MYSM1 in the breast cancer cells with stable knocked down of MYSM1 to examine the corresponding protein expression of ERa target genes.

    What is the ERa interactome in the presence and absence of MYSM1? Proteomics studies upon shMYSM1 should be performed. Alternatively, a better characterization of ERa-containing complexes upon shMYSM1 should be performed.

    Response: We agree with the reviewer’s suggestion to functionally address the influence of MYSM1 on ERa interactome. In breast cancer cells with the presence or absence of MYSM1, Co-IP experiments will be conducted to examine the influence of MYSM1 on the interaction between ERa and KAT2B, EP300 and CREBBP complex, which are predicted from String database.

    *Fig. 3: Does MYSM1 control its own protein via deubiquitination? *

    Response: We thank the reviewer for this suggestion and it provides us with a novel perspective upon MYSM1 investigation of whether MYSM1 is the deubiqutination substrate of itself. We would first transfect MYSM1-FL or MYSM1-ΔMPN plasmids and detect whether the endogenous MYSM1 expression changes. Next step, ubiquitination assays will be performed to determine whether MYSM1 control its own protein via deubiquitination.

    *Fig. 4: I propose that the authors perform MYSM1 ChIP-Seq to better show the MYSM1 distribution and overlap with ERa distribution. *

    Response: Appreciate for the reviewer for the valuable and important comments. ChIP-seq will be additionally performed in MCF-7 cells with MYSM1 antibody to examine the MYSM1 occupation on global chromatin in response to E2 and to show its overlap with ERa distribution.

    Fig. 7. Is there a correlation between MYSM1 mRNA and protein levels in cancer and physiological samples? How is the MYSM1 transcriptionally regulated in physiological and cancer cells?

    Response: We thank the reviewer for raising this issue. We will detect MYSM1 mRNA and protein levels in breast cancer and physiological samples, along with physiological and breast cancer cells. Statistics for MYSM1 transcriptional level will be further displayed.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    *Luan et al performed a detailed analysis on the potential coactivator MYSM1 and its role in regulating the expression of ER and ER-dependent genes by being a deubiquitinase of ER as well the repressive mark, H2Aub1. This study has demonstrated an excellent work on the biochemistry aspect of the story with meticulous work on the role of specific domains of MYSM1 and ER and how they interact and how the deubiquitination process is regulated. This was identified initially in Drosophila models, but eventually and promptly explored in multiple breast cancer cell lines and patient samples. *

    *Major Comments: *

      • It is really exciting to see how MYSM1 regulates ER activity and it looks like expression of ER is the first event of regulation by MYSM1's. However, H3Ac would be the very intermediate event of ER activity. This brings a question of whether ER complex itself is affected by MYSM1 - for example, does MYSM1 affect p300, SWI/SNF and other ER-associated coactivator binding? Does it affect chromatin accessibility? Which exact histone mark of H3Ac is affected, as different proteins are involved in the acetylation of histones. *

    Response: Appreciate the reviewer for the valuable questions. The Co-IP and ChIP experiments will be conducted respectively to assess the influence of MYSM1 on the binding of ERa with its associated co-activators and their recruitments on EREs upon MYSM1 knockdown. In addition, ChIP assays will also be performed to determine the effect of MYSM1 on histone modification levels (H3K9ac, H3K27ac, et al). MNase assay will be further performed to examine the function of MYSM1 on chromatin accessibility.

    • The regulation of MYSM1 is mainly shown on promoters of ER regulated genes. However, ER primarily bind to enhancers. Is there any general effect on enhancers? *

    Response: Thank you for your comments. We will perform ChIP assays to detect the regulation of MYSM1 on ERa binding to enhancers of ERa regulated genes in breast cancer cells.

    3. MYSM1 is not the complex usually cells prefer to deubiquitinate H2Aub, but BAP1. What is the role of BAP1 here? Are they redundant or any cross-talk?

    Response: Concerning this interesting question, it has been reported that BAP1 co-activator function correlated with increased H3K4me3 and concomitant deubiquitination of H2Aub at target genes. However, BAP1 has not been reported as an ERa co-regulator so far. Moreover, the interaction between ERa and BAP1 cannot be predicted using the STRING database. Whether BAP1 plays a similar role as MYSM1 in breast cancer and how MYSM1 cooperates with the other DUBs to regulate the genome-wide landscape of histone H2A ubiquitination and the gene expression profiles of different mammalian cell types remains to be elusive. It would be necessary to further study in the future.

    • Effect of MYSM1 on histone marks on the EREs - only one ERE is shown. Multiple EREs should be validated by qPCR. Enhancers should also be focused. Does it affect H3K27ac or H3K4me1?*

    Response: Thank you for your suggestions. ChIP experiments will be conducted to examine the effect of MYSM1 on histone marks on multiple EREs of ERa target genes. Furthermore, we will focus on the effect on MYSM1 on hitone marks (H3K27ac and H3K4me1 levels, et al) on enhancers of ERa target genes.

    • It is clear that MYSM1 is required for the response to antiestrogen therapies. However, the link to resistance is not completely clear. This should be investigated with multiple Tamoxifen resistant cell lines. There is one cell line used, but it is responding to tamoxifen even at lower concentrations in Crystal violet assays. MYSM1 overexpression in nonresponders doesn't mean that their activity is also more. Binding analyses should be analysed in proper Tamoxifen-resistant cell lines. Usually, Tamoxifen is used or works at concentrations from 100 nM - 1 uM in vitro to see the transcriptional effects. However, the authors claim that these are very high concentrations, but actually they aren't the concentrations which promote toxicity.*

    Response: We thank reviewer for the valuable comments. According to your suggestion, we will construct Tamoxifen-resistant MCF-7 or T47D cell lines carrying stable knockdown of MYSM1 to perform the biological function experiments with appropriate Tamoxifen concentrations to further confirm the effect of MYSM1 on the sensitivity of cells to anti-estrogen. In addition, we will examine the expression of MYSM1 and ERa target genes or histone H2Aub levels in nonresponders samples to preliminarily determine the activity of MYSM1 in AI-resistant samples.

    • Discussion about DUB inhibitors - how specific are these? Would they be useful to target MYSM1 activity and thus ER regulation in nonresponders or resistant cell lines? This would add up strongly on the clinical potential of the study. *

    Response: The DUB inhibitors mentioned in discussion are specific to USP14 and UCHL5, but not MYSM1. We thus turned to screen the commercially-available compound in ZINC database to find the compounds that could spatially interact with MYSM1 protein, thereby inhibiting the activity of MYSM1. We plan to perform the additional biological function experiments to explore the effect of MYSM1-targeting compounds on the sensitivity of breast cell lines to anti-estrogen treatment.

    • OPTIONAL: ChIP-seq analyses on the factors would be more informative to look at the unbiased mechanisms including enhancers. *

    Response: We appreciate your important comments. We plan to perform ChIP-seq in MCF-7 cells with MYSM1 antibody to examine the MYSM1 occupation on global chromatin in response to E2 and to show its overlap with ERa distribution.

    • Number of replicates aren't clear in figure legends. Are they biological or technical replicates?*

    Response: We thank for your comments. We have included the number of replicates in the “materials and methods” and “Figure legends” sections.

    *Minor comments: *

    • Please give page numbers and line numbers in the manuscript.*

    Response: We have given page numbers and line numbers in the revised manuscript.

    • Title - "MYSM1 co-activates ER action". "Action" is not needed to be mentioned here.*

    Response: We have modified the title according to the reviewer’s suggestion. The title has been modified as below: “MYSM1 acts as a novel co-activator of ERα via histone and non-histone deubiquitination to confer antiestrogen resistance in breast cancer”.

    • Abstract talks about the work on Drosophila mainly, but apart from the first experiment, everything else is done on mammalian cell culture and also clinically relevant patient samples. *

    Response: Thank you for your important comments. We have modified the abstract contents with breast cancer-derived cell lines instead of Drosophila experimental system.

    • Abstract Line 13 - the work is done many ER regulated genes and not gene.*

    Response: We've modified the text into “ERa-regulated genes” in Abstract section.

    • Pg 6 first paragraph - What/how many mutants were screened here? *

    Response: Thank you for your suggestion. In this study, about 300 fly lines carrying loss of function mutants obtained from Bloomington Stock Center were used for screening.

    • CoIP protocol is not clear. It says followed with manufacturer instructions but no kit information is provided.*

    Response: Apologize for the misrepresentation. Co-IP experiments were performed as that in the previous study. We have corrected the description for CoIP protocol and cited our previous study in the Materials and Methods section.

    • Fig. 1H, etc - can you show a zoomed in or DAPI removed (from merge) picture to show the interactions clearly? It's hard to follow the yellow co-interaction spots as they are hidden behind the blue colour. Any kind of quantification analyses would be wonderful.*

    Response: Thank you for your suggestion, we have merged the red and green colours to precisely show the co-location of MYSM1 and ERa.

    • Fig. EV1H - can you link this with the results from Fig. 1F to discuss if the delta SANT-MYSM1 lost the interaction with ER also in the IF studies? *

    Response: Thank you for your question. Commonly, the fluorescence intensity of confocal results mainly represents the amount of ectopic expression of MYSM1 or ERa, Co-IP experiments more exactly represent the association between proteins. It would be better to pick up the similar cell number in confocal experiments to assess the intensity of protein interaction. We will repeat the confocal again to show the exact fluorescence intensity.

    • Pg 7 - 3-4th line from last - These lines should move above where AF1 and AF2 are introduced. According to Fig. 1G, the interaction of AF2 and MYSM1 is important. Why do we see an effect on AF1 as well in Fig. 2B?*

    Response: Thank you for your comments. The GST ERa-AF1 and GST ERa-AF1 fusion proteins contain 29-180aa and 282-595aa of ERa truncated mutants respectively, while the ERa-AF1 and ERa-AF2 expression plasmids used in luciferase assay in Fig 2B encode 1-282aa and 178-595aa fragments. We can see the ERa-AF1 mutant in Fig 2B contains more amino acid segments than that in GST ERa-AF1 in Fig. 1G. We speculate that MYSM1 may interact with the extra segment (180aa-282aa) to upregulate ERa-AF1 induced transcription. To make it clear, we have included relevant description in the text along with a schematic representation of ERa, ERa-AF1, and ERa-AF2 plasmids used in luciferase reporter assays in Fig EV2B and in materials and methods section.

    • It's confusing to have HEK and breast cancer cell line datasets swapped inconsistently between main figures and Supplementary figures. It would be nice to keep them consistent. *

    Response: We have reverse the order of Fig 2B and Fig EV2C to maintain the consistency of the cell line datasets.

    • RPMI is spelled wrong in Pg. 19. *

    Response: We have corrected the spelling error of RPMI.

    • How long is the estrogen treatment done in each experiment? What is the concentration? This should be mentioned in the figure legends. 12 or 24 hrs time point is a later stage of estrogen receptor induction. Even 1-3 hrs would be sufficient to promote a stronger effect on RNA transcription than that of these later time points. What you are looking at is all effect on later time points and the effect should be observed on earlier time points to observe dynamic and immediate effects. p-values are required for the comparison on no E2 vs E2 here.*

    Response: We appreciate your valuable comment. We have rephrased the description on estrogen treatment in “Material and methods” and “Discussion” parts to more clearly state that E2 (100nM) was given for 4-6h in the experiments detecting transcriptional levels, while 16-18h in the experiments detecting translation levels. In addition, p-values have included to display the change of MYSM1 and ERa recruitment on ERE region upon E2 treatment.

    • Fig. 2G - effect on c-Myc after MYSM1 knockdown is not clear comparing to the previous WB in 2E.*

    Response: We will replace a clear image in Fig 2G to show the change of c-Myc protein expression after MYSM1 knockdown.

    • Pg. 8 - start of the last paragraph - "Unexpectedly, in Co-IP experiment as shown in Figure 2E and F" - These are not Co-IP experiments. *

    Response: Apologize for the writing error. We have re-written the sentence “Unexpectedly, in western blot experiments as shown in Figure 2E and F” in line 229.

    • Fig. 3C and E - Quantification with comparison needed.*

    Response: Relative ERa levels were semi-quantified by densitometry and normalized by the relative expression of 0 hour to compare the ERa degradation rate in Fig 3C and E.

    • Pg 10 - subtitle - multiple gene promoters have been looked, but the subtitle says "gene". Only ERE for c-MYC is looked at, but it says EREs.*

    Response: We have modified the word “genes” and “ERE” in correct forms in the text.

    • MYSM1 is in the nucleus in IF even before E2 treatment, however it is recruited after estrogen treatment in ChIP assays. Explain why there is a difference seen here. What other targets they might bind to in the nucleus?*

    Response: The aim of ChIP experiments is to examine the recruitment of MYSM1 protein on the DNA in the presence of E2, while IF results represent the MYSM1 subcellular distribution in the nucleus even in the absence of E2. MYSM1 has been reported to bind to promoters of numerous target genes, including Ebf1 in B cell progenitors, Pax5 in naïve B cells, miR150 in B1a cells, Id2 in NK cell progenitors, Flt3 in dendritic cell precursors, and Gfi1 in hematopoietic stem and progenitor cells. In our study, we plan to perform ChIP-seq to further show its potential binding elements on the global genome in ER-positive breast cancer.

    • Pg. 10 last line - the sentence should be combined with comma.*

    Response: Thank you for pointing this out, we have combined a comma in the sentence.

    • Fig. 5H - What about Ki67 which is a proliferative marker for cancer cell growth?*

    Response: We will further perform IHC experiments to compare Ki67 expression in the shCtrl and shMYSM1 group of xenograft tumors from nude mice.

    • Pg 12 - Samples were used from patients treated with AI adjuvant treatment. A small summary of details are needed here including n, arm, details of administration, etc even though mentioned in Methods.*

    Response: We have restated the patients’ condition and administration details in lines 342-250.

    • MYSM1 is upregulated in nonresponders, but it is also downregulated in responders which is ignored. What would this mean mechanistically? Don't patients need MYSM1 for the response or after treatment? Does estrogen inhibition regulate MYSM1 upstream? *

    Response: Appreciate for your important questions. The changes of intracellular environment caused by AI treatment are complicated and varied. The mechanism underlying such a phenomenon is largely unclear. We plan to perform western blot and ubiquitination assays to compare the expression and activity of MYSM1 in endocrine-resistant breast cancer cells treated or untreated with endocrine drugs to identify the effects of estrogen inhibition on MYSM1 expression. Moreover, we will detect whether MYSM1 expression is correlated with cell cycle and cell proliferation states.

    • Pg 13 - Is this data associated with any trial? More details are needed. *

    Response: We appreciate for your helpful comment. We have rearranged the logic of the article in order to clarify our reasoning for presenting this data. The modified contents included in lines 367-371 in the modified version are followed below: The regulation of MYSM1 on ERa action indicates that MYSM1 acts as a novel ERa co-activator, suggesting that MYSM1 may play an important role in breast cancer. We then conducted western blot and IHC experiments to estimate MYSM1 expression and the correlation between MYSM1 expression and clinicopathologic factors of the patients.

    • Last lines of Pg 15 - These were already introduced in the results. *

    Response: We thank reviewer for their highlighting this redundancy in our text. We have simplified the text in lines 442-444.

    • Pg 16 - third last line of the first paragraph - makes typo.*

    Response: Thanks for pointing out this typo. We have corrected the word “make” in line 456.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Luan et al performed a detailed analysis on the potential coactivator MYSM1 and its role in regulating the expression of ER and ER-dependent genes by being a deubiquitinase of ER as well the repressive mark, H2Aub1. This study has demonstrated an excellent work on the biochemistry aspect of the story with meticulous work on the role of specific domains of MYSM1 and ER and how they interact and how the deubiquitination process is regulated. This was identified initially in Drosophila models, but eventually and promptly explored in multiple breast cancer cell lines and patient samples.

    Major Comments:

    1. It is really exciting to see how MYSM1 regulates ER activity and it looks like expression of ER is the first event of regulation by MYSM1's. However, H3Ac would be the very intermediate event of ER activity. This brings a question of whether ER complex itself is affected by MYSM1 - for example, does MYSM1 affect p300, SWI/SNF and other ER-associated coactivator binding? Does it affect chromatin accessibility? Which exact histone mark of H3Ac is affected, as different proteins are involved in the acetylation of histones.
    2. The regulation of MYSM1 is mainly shown on promoters of ER regulated genes. However, ER primarily bind to enhancers. Is there any general effect on enhancers?
    3. MYSM1 is not the complex usually cells prefer to deubiquitinate H2Aub, but BAP1. What is the role of BAP1 here? Are they redundant or any cross-talk?
    4. Effect of MYSM1 on histone marks on the EREs - only one ERE is shown. Multiple EREs should be validated by qPCR. Enhancers should also be focused. Does it affect H3K27ac or H3K4me1?
    5. It is clear that MYSM1 is required for the response to antiestrogen therapies. However, the link to resistance is not completely clear. This should be investigated with multiple Tamoxifen resistant cell lines. There is one cell line used, but it is responding to tamoxifen even at lower concentrations in Crystal violet assays. MYSM1 overexpression in nonresponders doesn't mean that their activity is also more. Binding analyses should be analysed in proper Tamoxifen-resistant cell lines. Usually, Tamoxifen is used or works at concentrations from 100 nM - 1 uM in vitro to see the transcriptional effects. However, the authors claim that these are very high concentrations, but actually they aren't the concentrations which promote toxicity.
    6. Discussion about DUB inhibitors - how specific are these? Would they be useful to target MYSM1 activity and thus ER regulation in nonresponders or resistant cell lines? This would add up strongly on the clinical potential of the study.
    7. OPTIONAL: ChIP-seq analyses on the factors would be more informative to look at the unbiased mechanisms including enhancers.
    8. Number of replicates aren't clear in figure legends. Are they biological or technical replicates?

    Minor comments:

    1. Please give page numbers and line numbers in the manuscript.
    2. Title - "MYSM1 co-activates ER action". "Action" is not needed to be mentioned here.
    3. Abstract talks about the work on Drosophila mainly, but apart from the first experiment, everything else is done on mammalian cell culture and also clinically relevant patient samples.
    4. Abstract Line 13 - the work is done many ER regulated genes and not gene.
    5. Pg 6 first paragraph - What/how many mutants were screened here?
    6. CoIP protocol is not clear. It says followed with manufacturer instructions but no kit information is provided.
    7. Fig. 1H, etc - can you show a zoomed in or DAPI removed (from merge) picture to show the interactions clearly? It's hard to follow the yellow co-interaction spots as they are hidden behind the blue colour. Any kind of quantification analyses would be wonderful.
    8. Fig. EV1H - can you link this with the results from Fig. 1F to discuss if the delta SANT-MYSM1 lost the interaction with ER also in the IF studies?
    9. Pg 7 - 3-4th line from last - These lines should move above where AF1 and AF2 are introduced. According to Fig. 1G, the interaction of AF2 and MYSM1 is important. Why do we see an effect on AF1 as well in Fig. 2B?
    10. It's confusing to have HEK and breast cancer cell line datasets swapped inconsistently between main figures and Supplementary figures. It would be nice to keep them consistent.
    11. RPMI is spelled wrong in Pg. 19.
    12. How long is the estrogen treatment done in each experiment? What is the concentration? This should be mentioned in the figure legends. 12 or 24 hrs time point is a later stage of estrogen receptor induction. Even 1-3 hrs would be sufficient to promote a stronger effect on RNA transcription than that of these later time points. What you are looking at is all effect on later time points and the effect should be observed on earlier time points to observe dynamic and immediate effects. p-values are required for the comparison on no E2 vs E2 here.
    13. Fig. 2G - effect on c-Myc after MYSM1 knockdown is not clear comparing to the previous WB in 2E.
    14. Pg. 8 - start of the last paragraph - "Unexpectedly, in Co-IP experiment as shown in Figure 2E and F" - These are not Co-IP experiments.
    15. Fig. 3C and E - Quantification with comparison needed.
    16. Pg 10 - subtitle - multiple gene promoters have been looked, but the subtitle says "gene". Only ERE for c-MYC is looked at, but it says EREs.
    17. MYSM1 is in the nucleus in IF even before E2 treatment, however it is recruited after estrogen treatment in ChIP assays. Explain why there is a difference seen here. What other targets they might bind to in the nucleus?
    18. Pg. 10 last line - the sentence should be combined with comma.
    19. Fig. 5H - What about Ki67 which is a proliferative marker for cancer cell growth?
    20. Pg 12 - Samples were used from patients treated with AI adjuvant treatment. A small summary of details are needed here including n, arm, details of administration, etc even though mentioned in Methods.
    21. MYSM1 is upregulated in nonresponders, but it is also downregulated in responders which is ignored. What would this mean mechanistically? Don't patients need MYSM1 for the response or after treatment? Does estrogen inhibition regulate MYSM1 upstream?
    22. Pg 13 - Is this data associated with any trial? More details are needed.
    23. Last lines of Pg 15 - These were already introduced in the results.
    24. Pg 16 - third last line of the first paragraph - makes typo.

    Significance

    • The study seems to be novel as MYSM1 is never studied before as a coactivator for ER. This expands the wealth of knowledge we have on coactivators which can be explored for its potential targeting to treat advanced breast cancers. The study seems to be support the biochemical aspects of ER interaction, but vaguely uncovers the functional or epigenetic mechanisms.
    • Studies on coactivators/coregulators of ER is very important, as modulating ER alone is not efficient enough to solve the puzzle of antiestrogen resistance. The expression/activity levels of the coregulators are very important as these can be modulated in cancers due to epigenetic reprogramming during resistance and mutations on these genes dominate. They can also serve as potential targets especially when cells don't respond to classical ER targeting therapies.
    • Strength - Biochemical analyses of the interactions and detailed mechanistic information
    • Limitation - Studies are very much limited to the biochemical regulation on ER and not on the molecular or epigenetic mechanisms. Association of MYSM1 in resistance mechanisms isn't clear.
    • Audience - this can be interesting for both basic research and clinical audience. Biochemical knowledge would help people to understand how a nonclassical deubiquitinase can promote nuclear receptor associated transcription by targeting genomic and nongenomic targets simultaneously. Clinically this study would be relevant if the MYSM1-ER interaction can be targeted using DUB inhibitors, as requested.
    • Area of expertise of the reviewer - breast cancer, nuclear receptors, estrogen receptor biology, epigenetics, bioinformatics
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    Referee #2

    Evidence, reproducibility and clarity

    Below I outline a few suggestions that can help clarify specific aspects of the study.

    Fig. 2: Ideally a rescue study with a wild-type and catalytically mutant MYSM1 should be performed. What is the ERa interactome in the presence and absence of MYSM1? Proteomics studies upon shMYSM1 should be performed. Alternatively, a better characterization of ERa-containing complexes upon shMYSM1 should be performed.

    Fig. 3: Does MYSM1 control its own protein via deubiquitination?

    Fig. 4: I propose that the authors perform MYSM1 ChIP-Seq to better show the MYSM1 distribution and overlap with ERa distribution.

    Fig. 7. Is there a correlation between MYSM1 mRNA and protein levels in cancer and physiological samples? How is the MYSM1 transcriptionally regulated in physiological and cancer cells?

    Significance

    This is a very comprehensive study characterizing the role of MYSM1 deubiquitinase in ERa transcriptional programs in breast cancer systems. Breast cancer therapy is an unmet need and the role of deubiquitinases warrants further investigation. This accounts for the high significance of the story.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    ER+ breast cancer is the most common form of cancer. Targeting ER-alpha transcriptional cofactors present one potential method to target the disease. The authors demonstrate that MYSM1 is a histone deubiquitinase and a novel ER cofactor, functioning by up-regulating ER action via histone deubiquitination.

    Loss of MYSM1 attenuated cell growth and increase breast cancer cell lines' sensitivity to anti-estrogens. The authors, therefore, propose MYSM1 as a potential therapeutic target for endocrine resistance in Breast cancer.

    Major Comments:

    • The data as presented is convincing, and the evidence for the role of MYSM1 as a co-activator of ER-alpha is extensive.
    • Given the amount of data, I do not believe any additional experiments are needed.
    • I could not find any description of ethics for the patient samples used.

    Minor

    • Figure 4C - is the increase of binding in response to Estrogen significant? It is an important control to show for MCF7 as Fig 4B is in T47D.
    • Figure 6 - Can we clarify that B = Before, A = After
    • The use of Fig EV was confusing to me, I assume it means supplementary?

    Significance

    • Discovery science to understand the regulation of the ER is critical in discovering new opportunities to target breast cancer. As far as I can tell this is the first study where MYSM1 is a co-regulator of the ER.
    • The significance would be greatly increased if the manuscript identified opportuinities to target the ER via this pathway using existing compounds. However, it is reasonable to consider this is beyond the scope of this study.
  5. Version published to 10.1101/2022.12.23.521780 on bioRxiv