Disruption of Smarce1, a component of the SWI/SNF chromatin remodeling complex, decreases nucleosome stability in mouse embryonic stem cells and impairs differentiation

  1. Katsunobu Kashiwagi
  2. Junko Yoshida
  3. Hiroshi Kimura
  4. Kyoji Horie

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Abstract

The SWI/SNF chromatin remodeling complex consists of more than 10 component proteins that form a large protein complex of > 1 MDa. The catalytic proteins Smarca4 or Smarca2 work in concert with the component proteins to form a chromatin platform suitable for transcriptional regulation. However, the mechanism by which each component protein works synergistically with the catalytic proteins remains largely unknown. Here, we report on the function of Smarce1, a component of the SWI/SNF complex, through the phenotypic analysis of homozygous mutant embryonic stem (ES) cells. Disruption of Smarce1 induced the dissociation of other complex components from the SWI/SNF complex. Histone binding to DNA was loosened in homozygous mutant ES cells, indicating that disruption of Smarce1 decreased nucleosome stability. Sucrose gradient sedimentation analysis suggested an ectopic genomic distribution of the SWI/SNF complex, accounting for the misregulation of chromatin conformations. Unstable nucleosomes remained during ES cell differentiation, impairing the heterochromatin formation that is characteristic of the differentiation process. These results suggest that Smarce1 guides the SWI/SNF complex to the appropriate genomic regions to generate chromatin structures adequate for transcriptional regulation.

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    Reply to the Reviewers

    We appreciate the time and effort that the reviewers dedicated to providing feedback on our manuscript and are grateful for the insightful comments on and valuable improvements to our paper.

    Here is a point-by-point response to the reviewers’ comments and concerns.

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

    __Summary: __

    In this manuscript, Kashiwagi and colleagues examine the role of the BAF complex subunit Smarce1 in mouse ESC. They utilize a gene trap methodology to generate a Smarce1-null cell line (m/m) as well as a Smarce1-rescue cell line (r/r) in which the gene trap is excised. The Smarce1-null cell line exhibited abnormal colony morphology and elevated Nanog expression.

    The authors use several approaches to examine the effects of Smarce1 loss on the chromatin characteristics of mouse ESC. Using salt extraction, they show that depletion of Smarce1 causes nucleosome instability, as marked by enhanced solubility of Histone H3. Using immunoprecipitation and sucrose gradient experiments, they suggest that decreased nucleosome stability is due to loss of Arid1a from the BAF complex and improper targeting of the complex to heterochromatin. This improper targeting of the BAF complex to heterochromatin causes a decompaction of heterochromatin foci, potentially due to disruption or displacement of the PRC2 complex. The changes alter the differentiation potential of the m/m mESC, which exhibit abnormal mesoderm differentiation and enhanced neural differentiation. Taken together, these data suggest that Smarce1 is a critical component of the BAF complex that is required for proper targeting of the complex within the chromatin environment.

    __Major Comments: __*

    • Throughout the manuscript, the authors make conclusive statements about differences in the intensity or pattern of bands in western blots or DNA gels. However, these statements are purely qualitative, and the authors fail to provide any information about the number of replicates performed. As such, there is no way to judge the statistical significance of the observed changes and it is difficult to have confidence in any of the conclusions drawn from these experiments. *

    Responses

    We thank the reviewer for the insightful comments. Many of the results presented in this paper were validated multiple times prior to the initial submission. We plan to compile these data, conduct further experiments, and perform quantitative analyses to demonstrate the reproducibility of our findings.

    *1. Figure 2b: requires validation through multiple replicates. Ideally, the bands would be quantified and data from multiple replicates can be used for statistical analysis. *

    Responses

    As suggested by the reviewer, we plan to present quantified data from multiple replicates.

    *2. Figure 2d: as depicted, it is impossible to draw any conclusions. To assess the stability or "looseness" of nucleosomes, the authors should perform lane densitometry to compare the relative intensity of the bands within the lanes as well as the spacing of the bands. *

    Responses

    We appreciate the reviewer’s critical comments. Because the nucleosome repeat length changes due to chromatin structure alteration, it is extremely important to compare the spacing of the bands between the wt, r/r, and m/m cells. As suggested by the reviewer, we plan to perform a quantitative analysis of the band intensity and band spacing.

    *3. Figure 3: the authors claim that Arid1a pulldown is decreased in the m/m cells whereas there is no change in BRD9 pulldown between the cell lines. However, there is a decrease in BRD9 pulldown in the r/r cell line that appears equal to the decrease of Arid1a in the m/m cells. Multiple replicates and quantification should be provided to validate the findings, otherwise the reported differences seem to just be judgement calls. *

    Responses

    As suggested by the reviewer, we plan to present quantitative data from multiple replicates to confirm the observations of the Arid1a and Brd9 pulldown assays.

    *4. Figure 3: the immunoprecipitations appear to be poorly normalized. For instance, there seems to be more Smarca4 in the m/m input but less Smarca4 in the pulldown. This could suggest that the immunoprecipitation in the m/m cells was less efficient than in the other two cell lines. Again, additional replicates of the experiment seem necessary. *

    Responses

    As suggested by the reviewer, we plan to present quantitative data from multiple replicates to normalize the efficiency of immunoprecipitation.

    5. Figure 4: Validation of the differences observed between the WT and m/m cell line requires multiple replicates of the experiment. Ideally, the bands can be quantified and the data could be presented as line plots or histograms.

    Responses

    As suggested by the reviewer, we plan to perform a quantitative analysis of the band intensity and present the results in a graph.

    6. Figure 6: same issues as Figure 2b. Additionally, the results for Ezh2, HDAC1, and Kap1 are quite different from those depicted in Figure 2b despite the experiment appearing to be identical. This further emphasizes the need for replication of these experiments.

    Responses

    We thank the reviewer for highlighting this important point. Regarding the data presented in Figure 2b, we analyzed undifferentiated ES cells, and Figure 6e depicts the results of an analysis of differentiated ES cells. It is known that heterochromatin formation is promoted during the differentiation of ES cells. Therefore, we believe that the heterochromatin components such as Ezh2, HDAC1, and Kap1 are more unstable in Figure 6e compared to Figure 2b. In the full revision, we plan to present the difference in heterochromatin formation between the undifferentiated ES cells and differentiated cells using DAPI-staining or an MNase sensitivity assay. Additionally, we observed by immunostaining that the co-localization of Kap1 to heterochromatin is inhibited in the differentiated m/m cells, which is consistent with the observation that Kap1 is more unstable in Figure 6e compared to Figure 2. We plan to present the Kap1 localization data in the full revision. As suggested by the reviewer, we plan to quantify the immunoblot data from multiple replicates and present the reproducibility of the findings illustrated in Figure 6e.

    *Additionally, the interpretation of the differentiation experiments in figure 5 is somewhat confusing and raises several questions:

    1. In C-E, the m/m embryoid bodies appear to have much denser outgrowths than the WT and r/r embryoid bodies. While panels A and B demonstrate that the m/m embryoid bodies are smaller, the images in C-E seem to suggest that there is much more proliferation in the m/m outgrowths. This could be due to the maintenance of stem cell characteristics suggested by the presence of more Nanog-positive cells. The authors should comment on this phenomenon. *

    Responses

    We thank the reviewer for this insightful comment. As highlighted by the reviewer, the maintenance of stem cell characteristics in the m/m cells, which is suggested by more Nanog-positive cells, may influence the proliferation of EB outgrowth. Conversely, we believe that the images in C-E alone are insufficient to assess the proliferation of EB outgrowth, and more observation fields must be analyzed. We plan to address this point by evaluating all areas of the EB outgrowth.

    *2. The authors should consider additional experiments to test the persistence of pluripotent cells in these assays. For instance, these outgrowths could be dissociated and replated in ESC growth conditions to examine the ability of the cells to form ESC-like colonies (which would indicate retention of pluripotency). *

    Responses

    We thank the reviewer for this valuable suggestion. We will examine the retention of pluripotency by investigating the proliferation of dissociated EB-outgrowth in the ESC growth condition. We also plan to evaluate the persistence of several pluripotency markers by qRT-PCR or immunostaining.

    3. The authors describe the m/m cells as having impaired mesodermal differentiation based on SMA staining and the morphology of SMA-positive cells. While the morphology of SMA-positive cells does look altered in the m/m cells, there is extensive SMA staining. Rather than "impaired" (which suggests that mesodermal differentiation is blocked), the authors should consider describing mesodermal differentiation as "abnormal" or "altered." Examination of alternative mesodermal markers would also be informative.

    Responses

    We thank the reviewer for the careful evaluation of the staining data. As suggested, we believe that the SMA staining in the m/m cells is “altered” rather than “impaired”. We will revise the manuscript accordingly during the full revision. We also plan to analyze the expression levels of other mesodermal markers by qRT-PCR to further assess the mesodermal differentiation of the m/m cells.

    *4. Are there m/m cells in this assay that are double-positive for SMA and BIII tubulin? This would be compelling evidence demonstrating that loss of Smarce1 disrupts normal differentiation pathways. *

    Responses

    We thank the reviewer for proposing an attractive model for a novel role of Smarce1 in the regulation of cell differentiation. We have carefully reevaluated our staining data. Although we did not conduct double staining of the m/m cells with anti-SMA and BIII tubulin antibodies, the morphologies of the SMA-positive cells and BIII tubulin-positive cells in a single staining are quite different. This suggests that double staining of the m/m cells with anti-SMA and BIII antibodies is unlikely. However, we believe that the present analysis is insufficient to evaluate the effect of the loss of Smarce1 on normal differentiation pathways. scRNA-seq will provide an overall picture of the effect of Smarce1 loss, which we plan to discuss in the full revision.

    *Minor Comments: *

    1. Sox2 expression levels should be added to figure 1d.

    Responses

    As suggested by the reviewers, we plan to add Sox2 expression levels to Figure 1d.

    *2. Please define the regions being targeted in the Oct4, Nanog, and Sox2 chip-pcrs. Are these the promoters, enhancers, gene bodies, etc…? *

    Responses

    We appreciate the reviewer’s comment. The promoter regions were analyzed in all the ChIP-PCRs. We will elaborate on this in the full revision.

    *3. The ChIP data in figure1e-k are difficult to read as presented. It would be helpful to group the data by target rather than cell line so that adjacent data points are directly comparable. *

    Responses

    We appreciate the reviewer’s comment. As suggested, we will group the ChIP data by the target regions in the full revision.

    4. On lines 378-379, the authors state there is minimal to no histone acetylation at IAP and LINE1. This clearly contradicts the data shown in Figure 1.

    Responses

    We appreciate the reviewer’s comment. Our description was misleading. We intended to say that the difference in histone acetylation at IAP and LINE1 was minimal (if detected at all) between the m/m and wt or r/r cells. We will clarify this point in the full revision.

    *5. Are other BAF subunits disrupted in the m/m cells? It would be useful to look at Smarcc1/2, BAF180, Smarcd1/2, in figure 3. *

    Responses

    We appreciate the reviewer’s comment. We believe that the investigation of other BAF subunits is important to substantiate our conclusion of the role of Smarce1 in BAF complex assembly. We plan to add the data for other BAF subunits to Figure 3 in the full revision.

    *6. Similarly, the authors mention in the discussion that impaired REST interaction may explain the enhanced neuronal differentiation observed in the m/m cells. In this case, the authors should consider including REST or Sin3a in figures 3 and 4. *

    Responses

    We thank the reviewer for the insightful comment. We plan to analyze the interaction between Rest and the BAF complex and Rest and chromatin and will present the results in the full revision. We believe that these experiments would be better performed on differentiated cells, as the Rest function would be more pronounced during neural differentiation.

    *7. Additional assays/markers for heterochromatin would strengthen the authors' conclusions about impaired heterochromatin formation. For instance, are overall H3K9me3 or H4K20me3 levels different in the m/m cells? What about HP1? *

    Responses

    As suggested by the reviewer, we plan to analyze the overall levels of heterochromatin markers and present the results in the full revision.

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

    This work could provide intriguing conceptual advances in the understanding of BAF complex function in mouse ESC. The authors provide sufficient context for this work in their introduction and discussion sections. As referenced in the manuscript, work from several labs has demonstrated the requirement for canonical BAF complexes in mouse ESC. Recent work has also demonstrated the existence of a non-canonical BAF complex that also functions in the maintenance of mouse ESC. SMARCE1 is specific to the canonical BAF complexes, and this work presented here potentially demonstrates the functional requirement for SMARCE1 in canonical BAF complex function. As such, this work is likely to influence an audience with interest in the molecular biology of the BAF complex and chromatin remodeling.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

    This manuscript is a follow-up on a Nature Methods from 2011 (reference 34) describing an astute method to rapidly generate homozygous mutant mouse ES cells. During this study, the authors noticed that inactivation of Smarce1/BAF57, a subunit of the SWI/SNF complex containing an HMG domain, resulted in abnormal morphology of the ES cells. Here, they have extended the characterization of this mutant, and shown that inactivation of BAF57 leaves essentially unaffected the expression of the pluripotency genes Oct3/4, Nanog, and Sox2, while also apparently preserving the MNase digestion patterns, and H3K9ac and H3K9me accumulation at repeats of the L1 or IAP families. In contrast, the mutation causes increased extractability of the Baf250/Arid1a SWI/SNF subunit, of histone H3, and of the transcriptional co-repressor KAP1. Finally, mutant cells were shown to differentiate into smaller embryoid bodies and failed to differentiate properly into mesodermal lineages. The differentiated cells were also found to contain pericentromeric heterochromatin foci of more elongated shape than in the wild types. Based on these observations, the authors propose that inactivation of Smarce1 decreaseb nucleosome stability and impaired heterochromatin formation during differentiation. Overall, the paper is technically sound. Yet, there is also a clear trend towards overinterpretation of the data, and the arguments in favor of a genome-wide impact on chromatin remain week. On the other hand, the impact of the Baf57 mutation on ES cell differentiation and on the extractability of Baf250 and KAP1 are convincing. *

    __Specific comments: __*

    Figure 1: There seems to be a contradiction between the conclusion from this figure (Increased H3K9ac levels at the Yamanaka gene promoters but not at repeats, suggesting a local impact on chromatin) and the overall conclusion from the paper, proposing a global decrease in stability of the nucleosomes. An ATAC-seq experiment would probably yield a more definitive conclusion. *

    Responses

    We thank the reviewer for the important comment. As described below, we believe that our explanation was insufficient. In the full revision, we plan to clarify our explanation as follows and conduct additional experiments to substantiate our findings.

    We proposed nucleosome instability of the m/m cells based on biochemical analysis. We should have emphasized here that at a salt concentration of 75 mM, at which chromatin is considered intact (Thoma F et al, J Cell Biol 1979, 83 : 403-427, Allan J et al, J Cell Biol 1981, 90 : 279-288), no difference was observed in the nucleosome stability between the wt and m/m cells either in the salt extraction assay (Figure 2B) or the MNase assay (Figure 2D). The difference in nucleosome instability was observed when the nucleosomes were artificially destabilized by increasing the salt concentration. From this observation, we can speculate that it may be difficult to detect the difference in the genomic status between the wt and m/m cells by ATAC-seq because ATAC-seq is performed under the condition where nucleosomes are intact.

    However, as the reviewer highlighted, we believe that further experiments are required to assess the extent of the genome-wide effect of the Smarce1 mutation. It is unclear from the current data whether nucleosome instability occurs globally or only in restricted regions of the genome. Additionally, it has not been proven whether nucleosome instability occurs in live cells. Therefore, we plan to investigate nucleosome instability by fluorescence recovery after photobleaching (FRAP) analysis using H2B-mCherry. If we observe increased incorporation of H2B-mCherry into chromatin in FRAP, we can say that nucleosome instability occurs globally in m/m cells. This would also prove that nucleosomes are indeed unstable in live cells. We have already established stable cell lines that express H2B-mCherry in the wt, m/m, and r/r cells and are ready to begin FRAP analysis.

    Figure 2: The increased extractability of Kap1 is difficult to see on panel 2B, while it is clear in the sucrose gradient experiments and in the differentiated cells. These sets of experiments (including ARID1a and histone H3) would therefore be much more ro*bust if the different species were quantified on biological replicates (to allow calculation of a p value). Also, the increased extractability of histone H3 is intriguing, pointing toward a global effect on chromatin, while the MNase experiment showing no impact on the nucleosome ladder argues against such an effect (same issue as for Figure 1). It may be worth exploring whether the extracted H3 is nucleosomal (or alternatively not yet incorporated into chromatin). This could eventually be done by a simple Coomassie staining of the different fractions, that would allow tracking of all the 4 histones simultaneously. *

    Responses

    We thank the reviewer for the important comment. As suggested, we plan to present quantitative data from multiple replicates. We also plan to conduct Coomassie staining to track all four histones in different fractions to examine whether extracted histones are incorporated or not yet incorporated into chromatin.

    *Figure **3: It is not clear why the authors connect the sedimentation to a link with heterochromatin, as there is no correlation between distribution of the SWI/SNF subunits and that of histone H3 (while, in contrast, this experiment clearly documents some dissociation of Smarcc1 and the Arid proteins from the rest of the SWI/SNF complex). This should be discussed. Also, the lack of an effect of the BAF57 mutation on histone H3 sedimentation would be in favor of nucleosome remaining intact. *

    Responses

    (Note: Figure 3 in the reviewer's comment is most likely Figure 4.) Regarding the connection of the sedimentation to a link with heterochromatin, we believe our explanation was insufficient. At a salt concentration of 75 mM as shown, in Figure 4A, a higher-order chromatin structure is maintained (Thoma F et al, J Cell Biol 1979, 83 : 403-427, Allan J et al, J Cell Biol 1981, 90 : 279-288). Thus, the heterochromatin components would tend to distribute in the bottom fractions, while the euchromatin components and free proteins unbound to chromatin would tend to distribute in the top fractions. Since a portion of the esBAF components, such as Smarca4, Arid1a, and Smarcc1, migrated to the bottom fractions in the m/m cells (as evident in fraction 22 in Figure 4A), we interpreted this result as ectopic binding of the BAF complex to heterochromatin. In support on this interpretation, we possess an immunofluorescence data that shows an ectopic co-localization of Smarca4 with heterochromatin markers such as DAPI foci and H3K9me3 in differentiated m/m cells. Smarca4 is normally distributed throughout the nucleoplasm, not at heterochromatin. We plan to present this data in the full revision. A shift in the PRC2 components Ezh2 and Suz12 to the top fractions in the m/m cells is also consistent with the idea that ectopic heterochromatin localization of the BAF complex evicted PRC2 from the heterochromatin. However, we cannot discount the possibility that the migration of BAF components to the bottom fractions was caused by other factors, such as the binding of the Smarce1 (BAF57)-deficient BAF complex with a large protein complex. We will discuss this point in the full revision.

    Concerning the lack of effect of the Smarce1 mutation on histone H3 sedimentation, we thank the reviewer for highlighting this. A lack of histone H3 sedimentation at a salt concentration of 75 mM is expected because chromatin is generally considered to be intact under this condition, as mentioned above. Conversely, histone H3 is expected to shift toward the top fractions in the m/m cells at a salt concentration of 300 mM, given that the dissociation of histone H3 increased with increasing salt concentration (as shown in Figure 2B). The band images of histone H3 in Figure. 4B are partially saturated and were not appropriate for quantification. We plan to present adequate images and quantify the density of the histone bands in the results of replicated experiments.

    *Figure 6: The modified shape of the pericentromeric foci is remarkable. Their characterization would be greatly improved by 3D reconstruction on their confocal microscope. It will also be important to verify that the effect is not due to an impact of the Baf57 mutation on the cell cycle. A FACS analysis would probably be the best approach, staining with an anti-S10p antibody may also be informative. *

    Responses

    As suggested by the reviewer, we plan to conduct a 3D reconstruction of the microscopic data and cell cycle analysis.

    *Minor point: *

    *The information contained in Supplementary Figure 1 is essentially identical to that provided by Wettler et al, Genomics, 1999, one of the original cloning papers of the murine Smarce1. *

    Responses

    Yeast HNP6A and HNP6B are not presented in Wattler et al. (Genomics, 1999), whereas they were included in the similarity analysis in Supplementary Figure 1. We plan to modify Supplementary Figure 1 to clarify this difference in the full revision.

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

    **This study utilizes mouse embryonic stem cells with homozygous disruption of Smarce1 and those with reversion of Smarce1 to determine the mechanistic function of Smarce1 on cBAF complex assembly, chromatin structure, pluripotency gene expression, and differentiation. The findings indicate that expression of the pluripotency gene, SOX2, increases, and that chromatin structure on Sox2, Nanog, and Oct3/4 becomes more permissible (as evidenced by histone modifications) upon Smarce1 disruption. Other studies suggest that loss of Smarce1 destabilizes nucleosomes and association of chromatin proteins with chromatin. Furthermore, there is disruption of heterochromatin structure and abnormalities in differentiation. *

    __Major Concerns: __*

    The key conclusion that Smarce1 deficiency impacts upon embryonic stem cell morphology, alters cBAF composition and results in changes in chromatin structure are convincing. However, additional experiments are needed to make many of the specific claims. *

    1. Fig. 2B indicates that Smarce1 deficiency destabilizes nucleosomes, yet Fig. 2D shows no change in nucleosome positioning. It is suggested that an ATAC-seq experiment be conducted to better determine changes in nucleosome positioning. *

    Responses

    The nucleosome instability shown in Fig. 2B and no change in nucleosome positioning shown in Fig. 2D seem to be contradictory, but this is due to a lack of an explanation on our part. In the full revision, we plan to include the following explanations and experiments.

    It is known that the higher-order structure of chromatin is preserved at a salt concentration of 75 mM (Thoma F et al, J Cell Biol 1979, 83 : 403-427, Allan J et al, J Cell Biol 1981, 90 : 279-288). The nucleosome positioning experiment illustrated in Fig. 2D was performed under this concentration. Conversely, the salt extraction assay depicted in Fig. 2B was performed at concentrations of 75 mM, 150 mM, 300 mM, and 450 mM. It should be emphasized here that under the same 75 mM salt concentration used in the experimental data presented in Fig. 2D, no difference was observed between the wt, m/m, and r/r in Fig. 2B. Therefore, it was assumed that there is little difference in histone-DNA binding between different cell types when looking at static images of chromatin. In contrast, as shown in Fig. 2B, histone dissociation was enhanced by increasing the salt concentration in the m/m cells. This suggests that histones move in and out of chromatin more dynamically in the m/m cells. To examine this dynamic state, we plan to perform FRAP using cells that express H2B-mCherry and quantify the fluidity of histone-DNA binding. We have already established stable cell lines that express H2B-mCherry in the wt, m/m, and r/r cells and are ready to begin FRAP analysis.

    As highlighted by the other reviewer, we did not compare the nucleosome repeat length between the wt, r/r, and m/m cells. Because the nucleosome repeat length changes due to chromatin structure alteration, we plan to measure the nucleosome repeat length of these cells.

    *2. Fig. 4A shows Arid1a but not Smarca4 migrated at fractions 4 and 6, suggesting that Arid1a is dissociated from the BAF complex. However, there was an increase in BAF components, Smarc1/2 and Smarca4 migrating to the bottom of the sucrose gradient despite the absence of Smarce1 and dissociation of Arid1a. The authors took the lack of size reduction in the complex to mean that in the absence of Smarce1 and dissociation of Arid1a, there is inappropriate interaction of the BAF complex with heterochromatin. Although this is a possibility, there are other possibilities that explain the observation. There could be an increase in PBAF association or association with other proteins that cause the shift. Co-localization studies, chromatin immunoprecipitations or cut and run could more clearly show that there are changes in the interaction of BAF components with heterochromatin. Also, the data is not convincing that there is an increase in the migration of PRC2 components to the top of the gradient. *

    Responses

    We thank the reviewer for their insightful comments. Regarding the interaction of the BAF components with heterochromatin in the m/m cells, we have the following supportive data which were not shown in our initial manuscript. Using immunostaining, we found an ectopic co-localization of Smarca4 with heterochromatin markers such as DAPI foci and H3K9me3 in differentiated m/m cells. Smarca4 is normally distributed throughout the nucleoplasm. Therefore, this observation supports an ectopic distribution of the BAF complex to heterochromatic regions in m/m cells. We plan to present this result in the full revision. As suggested by the reviewer, we believe that the possibility of the association of the BAF complex with other proteins remains, regardless of the results of the co-localization study. We plan to discuss this point in the full revision.

    Regarding the migration of the PRC2 components to the top of the gradient, we plan to present quantitative data from multiple replicates. We speculate that the PRC2 components that migrated to the top fractions are evicted from heterochromatic regions and exist in the nucleoplasm as chromatin-unbound proteins. To investigate this possibility, we plan to conduct FRAP analysis using EGFP-Ezh2. The quicker recovery of the fluorescent signal of EGFP-Ezh2 in m/m cells than wt and r/r cells would indicate an unstable association of Ezh2 with chromatin in m/m cells. The result would also support the notion of the ectopic migration of PRC2 to the top fractions.

    *3. Fig. 5 nicely shows changes that Smarce1 disruption causes changes in the ability of the ES cells to differentiate. However, to more convincingly show that Smarce1 compromises endodermal differentiation and enhances ectodermal differentiation, it will be important to look at expression of some lineage specific markers. *

    Responses

    As suggested by the reviewer, we will analyze the expression of several lineage-specific markers by qRT-PCR.

    *4. What is different about Fig. 6A compared to Fig. 2B? In combination, Figs 2B and Fig. 6A indicate that both BAF and PRC2 components and other repressor proteins have looser association with chromatin. How does this result in a shift in BAF components to heterochromatin compartments while PRC2 is lost from these compartments? Additional experiments are needed to support this claim especially since the changes in migration of PRC2 components is very small as shown in Fig. 4A. *

    Responses

    (Note: Fig. 6A in the reviewer's comment is most likely Fig. 6E.)

    We thank the reviewer for the important comment. Regarding the difference between Figure. 6 and Figure. 2B, we believe that our explanation was insufficient. Regarding the data presented in Figure 2B, we analyzed undifferentiated ES cells, and in Figure 6E, we analyzed differentiated ES cells. It is known that heterochromatin formation is promoted during the differentiation of ES cells. For this reason, we believe that heterochromatin components such as Ezh2, HDAC1, and Kap1 are more unstable in Figure 6 compared to Figure 2B. We plan to present the difference in heterochromatin formation between the undifferentiated ES cells and differentiated cells using DAPI-staining or an MNase sensitivity assay in the full revision. Additionally, we observed by immunostaining that co-localization of Kap1 to heterochromatin is inhibited in the differentiated m/m cells, which is consistent with the observation that Kap1 is more unstable in Figure 6E compared to Figure 2. We plan to show the Kap1 localization data in the full revision.

    Regarding the shift in the BAF components to heterochromatin compartments and the loss of PRC2 from these compartments, we believe that additional experiments are required to support this theory. As the reviewer observed, the migration of PRC2 to the top fractions is small in Figure 4A. We plan to conduct FRAP analysis using EGFP-Ezh2 to demonstrate an unstable association of Ezh2 with chromatin in m/m cells as mentioned above. Alternatively, we will perform a salt extraction assay of PRC2 in the differentiated cells to evaluate the strength of the interaction of PRC2 with chromatin in the m/m cells.

    *Minor concerns: *

    1. The Smarca4 co-IP in Fig. 3 should account for the apparent decrease in Smarca4 immunoprecipitation from the mutant ES cells as well as the variable inputs. It would be better to repeat this experiment to get more consistent inputs between the cell lines and more consistent Smarca4 IPs. Alternatively, quantitation of the IPs relative to inputs would help convince readers that there is a decreased association between Smarca4 and Arid1a but not Brd9. *

    Responses

    As suggested by the reviewer, we plan to present quantitative data from multiple replicates.

    2. In Fig. 4A , it looks like Arid1b also migrates at fractions 4 and 6 in mutant ES cells. There should be discussion on this.

    Responses

    We thank the reviewer for the important remark. As highlighted by the reviewer, Arid1b migrates toward the top fractions in mutant ES cells. We confirmed the reproducibility of this finding. As we mentioned in the Introduction section, Arid1b is not included in the esBAF complex and is thought to be incorporated into the BAF complex during differentiation. ES cells cultured in a serum-containing medium, which was used in this study, are known to fluctuate between undifferentiated and partially differentiated states. We believe that the effect of the loss of Smarce1 on the migration of Arid1b indicates the presence of an Arid1b-containing BAF complex in partially differentiated ES cells. We plan to discuss this point in the full revision.

    3. Throughout the text, it is claimed that GBAF is not affected, yet only BRD9 is interrogated. Without experiments on other GBAF subunits, it is better to conclude that one subunit is not affected rather than the whole GBAF complex.

    Responses

    We agree with the reviewer’s comment that the analysis of Brd9 alone is insufficient to make a conclusion regarding the GBAF complex. We plan to perform an immunoprecipitation assay for other GBAF subunits to clarify the effect of the loss of Smarce1 on the GBAF complex.

    *4. The figure legends should indicate how many independent experiments each dataset represents. *

    Responses

    As suggested by the reviewer, we will indicate the number of independent experiments in the figure legends in the full revision.

    *5. There should be discussion on the findings of this study in context with the recently published manuscript on the relationship between SMARCE1 and BRD9 (PMID: 35681054). *

    Responses

    We thank the reviewer for highlighting this important paper. As mentioned above, we plan to analyze other GBAF subunits to clarify the relationship between Smarce1 and Brd9. Based on those results, we will discuss our findings in light of the above-mentioned paper.

    *Reviewer #3 (Significance (Required)): **

    Significance: This manuscript provides both technical and conceptual advances. The construction of embryonic stem cells with homozygous disruption and reversion of Smarce1 is a technical advance. Although a recent publication ( PMID: 35681054) just showed that Smarce1 disruption destabilizes cBAF complexes, there are other novel conceptual insights provided by this study that are significant. The mechanistic insights into Smarce1 function in embryonic stem cells should be of interest to the chromatin community. Furthermore, since Smarce1 is disrupted in meningiomas and in Coffin-Siros syndrome, it should be of interest to the cancer and developmental biology fields. My expertise is in SWI/SNF chromatin remodeling during cellular differentiation and in cancer. *

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    Referee #3

    Evidence, reproducibility and clarity

    This study utilizes mouse embryonic stem cells with homozygous disruption of Smarce1 and those with reversion of Smarce1 to determine the mechanistic function of Smarce1 on cBAF complex assembly, chromatin structure, pluripotency gene expression, and differentiation. The findings indicate that expression of the pluripotency gene, SOX2, increases, and that chromatin structure on Sox2, Nanog, and Oct3/4 becomes more permissible (as evidenced by histone modifications) upon Smarce1 disruption. Other studies suggest that loss of Smarce1 destabilizes nucleosomes and association of chromatin proteins with chromatin. Furthermore, there is disruption of heterochromatin structure and abnormalities in differentiation.

    Major Concerns:

    The key conclusion that Smarce1 deficiency impacts upon embryonic stem cell morphology, alters cBAF composition and results in changes in chromatin structure are convincing. However, additional experiments are needed to make many of the specific claims.

    1. Fig. 2B indicates that Smarce1 deficiency destabilizes nucleosomes, yet Fig. 2D shows no change in nucleosome positioning. It is suggested that an ATAC-seq experiment be conducted to better determine changes in nucleosome positioning.
    2. Fig. 4A shows Arid1a but not Smarca4 migrated at fractions 4 and 6, suggesting that Arid1a is dissociated from the BAF complex. However, there was an increase in BAF components, Smarc1/2 and Smarca4 migrating to the bottom of the sucrose gradient despite the absence of Smarce1 and dissociation of Arid1a. The authors took the lack of size reduction in the complex to mean that in the absence of Smarce1 and dissociation of Arid1a, there is inappropriate interaction of the BAF complex with heterochromatin. Although this is a possibility, there are other possibilities that explain the observation. There could be an increase in PBAF association or association with other proteins that cause the shift. Co-localization studies, chromatin immunoprecipitations or cut and run could more clearly show that there are changes in the interaction of BAF components with heterochromatin. Also, the data is not convincing that there is an increase in the migration of PRC2 components to the top of the gradient.
    3. Fig. 5 nicely shows changes that Smarce1 disruption causes changes in the ability of the ES cells to differentiate. However, to more convincingly show that Smarce1 compromises endodermal differentiation and enhances ectodermal differentiation, it will be important to look at expression of some lineage specific markers.
    4. What is different about Fig. 6A compared to Fig. 2B? In combination, Figs 2B and Fig. 6A indicate that both BAF and PRC2 components and other repressor proteins have looser association with chromatin. How does this result in a shift in BAF components to heterochromatin compartments while PRC2 is lost from these compartments? Additional experiments are needed to support this claim especially since the changes in migration of PRC2 components is very small as shown in Fig. 4A.

    Minor concerns:

    1. The Smarca4 co-IP in Fig. 3 should account for the apparent decrease in Smarca4 immunoprecipitation from the mutant ES cells as well as the variable inputs. It would be better to repeat this experiment to get more consistent inputs between the cell lines and more consistent Smarca4 IPs. Alternatively, quantitation of the IPs relative to inputs would help convince readers that there is a decreased association between Smarca4 and Arid1a but not Brd9.
    2. In Fig. 4A , it looks like Arid1b also migrates at fractions 4 and 6 in mutant ES cells. There should be discussion on this.
    3. Throughout the text, it is claimed that GBAF is not affected, yet only BRD9 is interrogated. Without experiments on other GBAF subunits, it is better to conclude that one subunit is not affected rather than the whole GBAF complex.
    4. The figure legends should indicate how many independent experiments each dataset represents.
    5. There should be discussion on the findings of this study in context with the recently published manuscript on the relationship between SMARCE1 and BRD9 (PMID: 35681054).

    Significance

    This manuscript provides both technical and conceptual advances. The construction of embryonic stem cells with homozygous disruption and reversion of Smarce1 is a technical advance. Although a recent publication ( PMID: 35681054) just showed that Smarce1 disruption destabilizes cBAF complexes, there are other novel conceptual insights provided by this study that are significant. The mechanistic insights into Smarce1 function in embryonic stem cells should be of interest to the chromatin community. Furthermore, since Smarce1 is disrupted in meningiomas and in Coffin-Siros syndrome, it should be of interest to the cancer and developmental biology fields.

    My expertise is in SWI/SNF chromatin remodeling during cellular differentiation and in cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript is a follow-up on a Nature Methods from 2011 (reference 34) describing an astute method to rapidly generate homozygous mutant mouse ES cells. During this study, the authors noticed that inactivation of Smarce1/BAF57, a subunit of the SWI/SNF complex containing an HMG domain, resulted in abnormal morphology of the ES cells.

    Here, they have extended the characterization of this mutant, and shown that inactivation of BAF57 leaves essentially unaffected the expression of the pluripotency genes Oct3/4, Nanog, and Sox2, while also apparently preserving the MNase digestion patterns, and H3K9ac and H3K9me accumulation at repeats of the L1 or IAP families.

    In contrast, the mutation causes increased extractability of the Baf250/Arid1a SWI/SNF subunit, of histone H3, and of the transcriptional co-repressor KAP1. Finally, mutant cells were shown to differentiate into smaller embryoid bodies and failed to differentiate properly into mesodermal lineages. The differentiated cells were also found to contain pericentromeric heterochromatin foci of more elongated shape than in the wild types.

    Based on these observations, the authors propose that inactivation of Smarce1 decreaseb nucleosome stability and impaired heterochromatin formation during differentiation. Overall, the paper is technically sound. Yet, there is also a clear trend towards overinterpretation of the data, and the arguments in favor of a genome-wide impact on chromatin remain week. On the other hand, the impact of the Baf57 mutation on ES cell differentiation and on the extractability of Baf250 and KAP1 are convincing.

    Specific comments:

    Figure 1: There seems to be a contradiction between the conclusion from this figure (Increased H3K9ac levels at the Yamanaka gene promoters but not at repeats, suggesting a local impact on chromatin) and the overall conclusion from the paper, proposing a global decrease in stability of the nucleosomes. An ATAC-seq experiment would probably yield a more definitive conclusion.

    Figure 2: The increased extractability of Kap1 is difficult to see on panel 2B, while it is clear in the sucrose gradient experiments and in the differentiated cells. These sets of experiments (including ARID1a and histone H3) would therefore be much more robust if the different species were quantified on biological replicates (to allow calculation of a p value). Also, the increased extractability of histone H3 is intriguing, pointing toward a global effect on chromatin, while the MNase experiment showing no impact on the nucleosome ladder argues against such an effect (same issue as for Figure 1). It may be worth exploring whether the extracted H3 is nucleosomal (or alternatively not yet incorporated into chromatin). This could eventually be done by a simple Coomassie staining of the different fractions, that would allow tracking of all the 4 histones simultaneously.

    Figure 3: It is not clear why the authors connect the sedimentation to a link with heterochromatin, as there is no correlation between distribution of the SWI/SNF subunits and that of histone H3 (while, in contrast, this experiment clearly documents some dissociation of Smarcc1 and the Arid proteins from the rest of the SWI/SNF complex). This should be discussed. Also, the lack of an effect of the BAF57 mutation on histone H3 sedimentation would be in favor of nucleosome remaining intact.

    Figure 6: The modified shape of the pericentromeric foci is remarkable. Their characterization would be greatly improved by 3D reconstruction on their confocal microscope. It will also be important to verify that the effect is not due to an impact of the Baf57 mutation on the cell cycle. A FACS analysis would probably be the best approach, staining with an anti-S10p antibody may also be informative.

    Minor point:

    The information contained in Supplementary Figure 1 is essentially identical to that provided by Wettler et al, Genomics, 1999, one of the original cloning papers of the murine Smarce1.

    Significance

    Some interesting observations, but overall, a modest contribution to the field.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Kashiwagi and colleagues examine the role of the BAF complex subunit Smarce1 in mouse ESC. They utilize a gene trap methodology to generate a Smarce1-null cell line (m/m) as well as a Smarce1-rescue cell line (r/r) in which the gene trap is excised. The Smarce1-null cell line exhibited abnormal colony morphology and elevated Nanog expression.

    The authors use several approaches to examine the effects of Smarce1 loss on the chromatin characteristics of mouse ESC. Using salt extraction, they show that depletion of Smarce1 causes nucleosome instability, as marked by enhanced solubility of Histone H3. Using immunoprecipitation and sucrose gradient experiments, they suggest that decreased nucleosome stability is due to loss of Arid1a from the BAF complex and improper targeting of the complex to heterochromatin. This improper targeting of the BAF complex to heterochromatin causes a decompaction of heterochromatin foci, potentially due to disruption or displacement of the PRC2 complex. The changes alter the differentiation potential of the m/m mESC, which exhibit abnormal mesoderm differentiation and enhanced neural differentiation. Taken together, these data suggest that Smarce1 is a critical component of the BAF complex that is required for proper targeting of the complex within the chromatin environment.

    Major Comments:

    Throughout the manuscript, the authors make conclusive statements about differences in the intensity or pattern of bands in western blots or DNA gels. However, these statements are purely qualitative, and the authors fail to provide any information about the number of replicates performed. As such, there is no way to judge the statistical significance of the observed changes and it is difficult to have confidence in any of the conclusions drawn from these experiments.

    1. Figure 2b: requires validation through multiple replicates. Ideally, the bands would be quantified and data from multiple replicates can be used for statistical analysis.
    2. Figure 2d: as depicted, it is impossible to draw any conclusions. To assess the stability or "looseness" of nucleosomes, the authors should perform lane densitometry to compare the relative intensity of the bands within the lanes as well as the spacing of the bands.
    3. Figure 3: the authors claim that Arid1a pulldown is decreased in the m/m cells whereas there is no change in BRD9 pulldown between the cell lines. However, there is a decrease in BRD9 pulldown in the r/r cell line that appears equal to the decrease of Arid1a in the m/m cells. Multiple replicates and quantification should be provided to validate the findings, otherwise the reported differences seem to just be judgement calls.
    4. Figure 3: the immunoprecipitations appear to be poorly normalized. For instance, there seems to be more Smarca4 in the m/m input but less Smarca4 in the pulldown. This could suggest that the immunoprecipitation in the m/m cells was less efficient than in the other two cell lines. Again, additional replicates of the experiment seem necessary.
    5. Figure 4: Validation of the differences observed between the WT and m/m cell line requires multiple replicates of the experiment. Ideally, the bands can be quantified and the data could be presented as line plots or histograms.
    6. Figure 6: same issues as Figure 2b. Additionally, the results for Ezh2, HDAC1, and Kap1 are quite different from those depicted in Figure 2b despite the experiment appearing to be identical. This further emphasizes the need for replication of these experiments

    Additionally, the interpretation of the differentiation experiments in figure 5 is somewhat confusing and raises several questions:

    1. In C-E, the m/m embryoid bodies appear to have much denser outgrowths than the WT and r/r embryoid bodies. While panels A and B demonstrate that the m/m embryoid bodies are smaller, the images in C-E seem to suggest that there is much more proliferation in the m/m outgrowths. This could be due to the maintenance of stem cell characteristics suggested by the presence of more Nanog-positive cells. The authors should comment on this phenomenon.
    2. The authors should consider additional experiments to test the persistence of pluripotent cells in these assays. For instance, these outgrowths could be dissociated and replated in ESC growth conditions to examine the ability of the cells to form ESC-like colonies (which would indicate retention of pluripotency).
    3. The authors describe the m/m cells as having impaired mesodermal differentiation based on SMA staining and the morphology of SMA-positive cells. While the morphology of SMA-positive cells does look altered in the m/m cells, there is extensive SMA staining. Rather than "impaired" (which suggests that mesodermal differentiation is blocked), the authors should consider describing mesodermal differentiation as "abnormal" or "altered." Examination of alternative mesodermal markers would also be informative.
    4. Are there m/m cells in this assay that are double-positive for SMA and BIII tubulin? This would be compelling evidence demonstrating that loss of Smarce1 disrupts normal differentiation pathways.

    Minor Comments:

    1. Sox2 expression levels should be added to figure 1d
    2. Please define the regions being targeted in the Oct4, Nanog, and Sox2 chip-pcrs. Are these the promoters, enhancers, gene bodies, etc...?
    3. The ChIP data in figure1e-k are difficult to read as presented. It would be helpful to group the data by target rather than cell line so that adjacent data points are directly comparable.
    4. On lines 378-379, the authors state there is minimal to no histone acetylation at IAP and LINE1. This clearly contradicts the data shown in Figure 1.
    5. Are other BAF subunits disrupted in the m/m cells? It would be useful to look at Smarcc1/2, BAF180, Smarcd1/2, in figure 3.
    6. Similarly, the authors mention in the discussion that impaired REST interaction may explain the enhanced neuronal differentiation observed in the m/m cells. In this case, the authors should consider including REST or Sin3a in figures 3 and 4.
    7. Additional assays/markers for heterochromatin would strengthen the authors' conclusions about impaired heterochromatin formation. For instance, are overall H3K9me3 or H4K20me3 levels different in the m/m cells? What about HP1?

    Significance

    This work could provide intriguing conceptual advances in the understanding of BAF complex function in mouse ESC. The authors provide sufficient context for this work in their introduction and discussion sections. As referenced in the manuscript, work from several labs has demonstrated the requirement for canonical BAF complexes in mouse ESC. Recent work has also demonstrated the existence of a non-canonical BAF complex that also functions in the maintenance of mouse ESC. SMARCE1 is specific to the canonical BAF complexes, and this work presented here potentially demonstrates the functional requirement for SMARCE1 in canonical BAF complex function. As such, this work is likely to influence an audience with interest in the molecular biology of the BAF complex and chromatin remodeling.

  5. Version published to 10.1101/2022.05.18.492397v1 on bioRxiv