Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s

The transcriptional mechanisms for Satb1 gene expression are ill-defined. This study identifies a novel enhancer for Satb1 expression in TH2 cells and to suppress IL-5 production, and for Satb1 expression in activated ILC2s.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** In this paper, Nomura et al exploited a SATB1-Venus reporter mouse model and precisely evaluated the SATB1 expression levels of T-lineage cells. By using genome editing methodology, the authors have determined one cis-regulatory enhancer for Satb1 expression in CD4 TH2 cells. Then, the authors have proved that this enhancer, named as Satb1-Eth2 (enhancer for TH2 cells) is indispensable to up-regulate and maintain Satb1 expression in CD4 TH2 cells and group 2 innate lymphoid cells (ILC2s). Information from public data base suggested the interaction of Stat6 with this enhancer. The results of in vitro experiments have clearly shown that Satb1-Eth2 is essential for repressing IL-5 expression in CD4 TH2 cells though it remains elusive how this enhancer and Satb1 are coordinately involved in physiological regulation of IL5 and allergic reaction in vivo.
Although this paper includes quite a few negative data, all the experiments seem to have been well conducted and precisely executed. Those negative results also have some values. This reviewer has no major concern on this meticulous study, but wants to make a couple of minor comments.
----This is perhaps an overstatement because this paper provides no data directly linking TCR stimulation and SATB1 expression.

The test includes a few grammatical mistakes.
Reviewer #2 (Comments to the Authors (Required)): In this paper, these authors study how SATB1 expression is regulated. The authors identify an enhancer for Satb1 expression in Th2 cells and ILC2. While the deletion of this enhancer decreases the Satb1 expression, the authors not find functional relevancy of SATB1 decrease in Th2 cells or ILC2, in vivo. Only an increase in IL5 expression in vitro differentiated Th2 cells are observed.
In the abstract, the authors do not describe about all the conclusions they can draw from their results. They only talk about the results in favour of their title. For example, they do not say that the enhancer deletion has no functional relevance in vivo.
Throughout the article (in particular in introduction), they talk about Satb1 regulates/controls without saying in which sense activation/inhibition, expression/repression. It is difficult for the reader to follow the different studies.
The authors characterized the Satb1-venus fusion reporter mouse in thymus and periphery. The authors describe the venus expression by cytometry by analyzing the MFI only. the authors do not report the % of cells that came positive, only the decrease of venus expression on all population (positive + negative). For example in Figure 1E only a part of effector memory cells are positive for Venus. Same in Figure EV1F. Why are these cells divided into Venus positive and negative cells? What is the functional relevance? What is the functional relevance of these different levels of Venus (Satb1 expression) in thymus and in periphery ? Figure 2B line 202 "significantly reduced": MFI 2573 to 2044; What is the functional relevance ? what is the % of cells that express venus ? Why the deletion of enhancer do not decrease the expression in all the cells ? Figure 2C: It is important to increase the number of experiments to do statistical. Figure EV2D: the quantification of the western blot is necessary.
Using public data, the authors show that Sabt1-A region as a genomic enhancer via STAT6. The authors need to demonstrate this mechanism, by performing an ChIP anti stat6 in wt and deleted Sabt1-a region mice.
In Satb1 deltaEth2/ deltaEth2, the authors do not analyse the development of T cells in periphery, while the enhancer is important in effector CD4 T cells.
The authors need to analyse the IL4, IL5 and IL13 production after PMA/iono, but also after TCR engagement.
In vivo, using Alternaria Alternate model, the authors show no physiological relevance of the absence of the Sabt1. In others models as asthma (house dust mite or ovabulmin models) ???
In general manner, in different figures the statistics are no present. The paper need a functional relevancy.
Reviewer #3 (Comments to the Authors (Required)): The authors constructed a SATB1-Venus expressing reporter mouse and investigated SATB1 expression in various stages of T cell development and multiple subpopulations of T cells. Using this reporter system, the authors investigated the roles of two candidates of distal enhancers in SATB1 expression regulation in T cells. The authors found that the Satb1-a, one of these two sequences can regulate the expression of SATB1 in Th2 and ILC2 cells. The authors further analyzed the mechanism of regulation of SATB1 expression by this regulatory sequence in Th2 cells. The authors also analyzed the effect of this regulatory sequence on the Th2 immune response. The study contributes to the understanding of the complex regulatory mechanism of SATB1 in T cells. But there are still some concerns with the current manuscript. Major concerns: 1. This study needs to be very cautious about the extent to which the SATB1-Venus reporter system constructed by the author can truly reflect the protein expression level of SATB1. Indumathi Patta et al. used Western blot to observe that the level of SATB1 protein was higher in CD4 SP cells than in DP cells (Nucleic Acids Research, 2020, Vol. 48, No. 11 5873-5890), which is inconsistent with the observation here using SATB1-Venus. Also, I noticed that the Th1 MFI of Satb1+/Venus mice was higher than Th2 ( Fig 2C), but Western Blot showed that SATB1 protein was higher in Th2 than in Th1 ( Figure EV2D). This inconsistency made me suspect that SATB1-Venus could not truly reflect SATB1 protein levels. Therefore, the author needs to use Western Blot or other alternative methods to confirm that the protein level expression of SATB1 is consistent with that of SATB1-Venus. 2. Fig EV1E. The altered ratio of CD8SP in Satb1 Venus/Venus mice is evident and it can not be ignored. The authors need to explain this in the manuscript. 3. On page 7, line 150, the author says "The SATB1-Venus hi IEL population expressed CD62L, with their CD4 and CD8alpha expression profiles similar to that in splenic T cells". The authors should provide data and analysis. 4. The authors used ATAC-seq data to search enhancers, which is somewhat limited. CTCF binding sites and silencers are also accessible regions. While the histone modification H3K27 acetylation is a better enhancer marker, it would be better to combine the ChIP-seq data of H3K27 acetylation to analyze the candidate enhancers of SATB1. ChIP-seq of H3K27 acetylation can be added to Fig 2A to help understand enhancer characteristics. 5. The resolution of the Fig 3B Hi-C heatmap is too low, and the black line is too thick, obscuring the details of the SATB1 locus in the heatmap. Suggest a better presentation of the Hi-C data. In addition, the heat map of Hi-C could not provide the specific interaction between Satb1-a and the SATB1 promoter. It is recommended to perform a 4C assay or similar techniques to confirm the interaction between the enhancer and the promoter. 6. The mice information in this manuscript is confusing. What is the difference between SATB1 Venus-Δa, SATB1 ΔEth2, and SATB1 Venus-ΔEth2? The authors should state this clearly in the manuscript. Since the CRISPR-Cas9 system excision is not perfect, there will be some variation in deletion regions. What is the difference between the deletion sequences in these mice? The authors should provide sequencing validation results for each mouse line. Minor: 1. Cd4-cre (Line114, Page5) or CD4-cre ( Figure EV1E)? Need to be consistent. 2. Figure 1C, 1D, 1E, and 1G have missing x-axis numbers. Although this paper includes quite a few negative data, all the experiments seem to have been well conducted and precisely executed. Those negative results also have some values. This reviewer has no major concern on this meticulous study but wants to make a couple of minor comments.
We are very thankful for Reviewer #1`s positive evaluation on our study.
----This is perhaps an overstatement because this paper provides no data directly linking TCR stimulation and SATB1 expression.
We thank Reviewer #1 for this suggestion. We agree that we did not provide any evidence showing a direct link of TCR stimulation with SATB1 upregulation in thymocytes. Therefore, we rewrote this statement to point out this possibility in the revised manuscript below in line 128, on page 6.
"which may suggest that Satb1 expression is positively regulated during positive selection signals."

The test includes a few grammatical mistakes.
We appreciate errors pointed out by this reviewer. We have now corrected the grammatical errors as much as possible.
Reviewer #2 (Comments to the Authors (Required)): In this paper, these authors study how SATB1 expression is regulated. For example, they do not say that the enhancer deletion has no functional relevance in vivo.
First, we thank Reviewer #2 for his/her constructive comments. In the title and abstract, we wanted to avoid overstating our results: we mainly focused on describing the key findings derived from our study. In terms of "functional relevance of enhancer deletion in vivo", we have confirmed that, in the response to lung inflammation, the Satb1-Eth2 enhancer is required to maintain SATB1 expression in both in vivo Th2 cells and activated ILC2s. Thus, we believe that this enhancer, which we identified, has a functional relevance in regulating SATB1 expression in vivo. The problem is that we have yet to understand how this enhancer and SATB1 are co-ordinately involved in physiological regulation of IL5 to finely regulate allergic reaction in vivo. We are afraid that the Reviewer #2 is possibly misdirected by the results that showed no significant effects of enhancer deletion in the Th2 immune responses. As we pointed out in the discussion section, these negative results do not exclude the possible roles of this enhancer/low Satb1 expression in Th2/ILC2 in regulating Th2 immune responses under other experimental settings, which again would require additional intensive studies. Therefore, at this moment, we were afraid to make a strong statement on this point in the abstract to prevent misleading to our readers.

Throughout the article (in particular in introduction), they talk about Satb1 regulates/controls without saying in which sense activation/inhibition, expression/repression. It is difficult for the reader to follow the different studies.
We thank the Reviewer #2 for this comment. We have revised the text, aiming to increase the clarity and for easy reading. We had the same question raised by Reviewer #2 why there are Satb1-high and -low cells in CD44 + CD62L -CD4 T cells subsets. We wished to address this but could not find good experimental approaches to directly answer this question. If Reviewer #2 can suggest us specific experiments that would address this question, we are willing to perform them.
As such, at this moment, we did not reveal the functional relevance of these heterogenous SATB1-Venus levels within these central and effector memory T cell populations. However, we would ask the Reviewer #2 to appreciate the merit of the Satb1-Venus reporter model.
Without this fluorescent reporter model, it would be impossible to quantify SATB1 protein level in various T cell subpopulations with a single cell resolution. Thus, heterogenous expression of Satb1 protein was clearly shown at the first time by our approach. In addition, to understand how Satb1 expression is regulated in various T cell subpopulations, we utilized the Satb1-Venus reporter mice to validate the physiological function of cis-regulatory candidate regions, and eventually we have identified a novel Th2 specific enhancer regulating Satb1 expression not only in vitro but also in vivo differentiated Th2 cells. We believe that our approach is one of the most reliable ways to address the functional relevance of the different levels of SATB1 by perturbing its physiological regulation. Of course, the Satb1-Eth2 enhancer is not the sole regulatory region for Satb1 expression and comprehensive understanding of the functional relevance of heterogenous SATB1 expression will requires additional genetic approaches and intensive characterization of these SATB1-hi and SATB1-lo central/effector memory T cell populations, which we believe is beyond the scope of this manuscript.

The authors conclude that T cell activation in the periphery induces down-regulation of Satb1 expression. It is not a down regulation in all the cells, but the lost in one population of cells.
Why ? There is a correlation with proliferative/ activated cells ? More experiments with a kinetic on TCR-activated naïve CD4 cells are necessary.
We thank Reviewer #2 for raising these points and apologise for not showing frequencies of SATB1-Venus Hi and SATB1-Venus L cells within naïve, central memory and effector memory T cell subsets. We now provide this data in Figure S1I

The authors indicate that Satb1 expression declines after activation of T cells, but they do not
show the expression of Satb1 in naïve CD4 T cells before the Th differentiation ( Figure 1G).
We thank Reviewer #2 for raising these critical comments, which are related to above criticisms by the same reviewer. We agree that we did not examine all effector cells differentiated in vivo for Satb1-expression, but this is practically very difficult since in general not all effector cells have not been identified. But our data confirmed that all naïve T cell shows uniform and high SATB1-venus expression pattern. Thus, our statement was based on the different SATB11-Venus expression levels in different T cell populations developed in vivo.
As requested, we have repeated the CD4 T-helper cultures to include Satb1-Venus expression before differentiation (sorted naïve CD4 T cells prior to activation/differentiation) and 5 days after Th0/1/2/17iTreg differentiation, all on the same flow cytometric settings, and present the data in Fig 1H of   We thank the Reviewer #2 for raising this question. As we show in Figures 1E and S1I and when compared to non-venus control (wildtype) cells, all (100%) naïve CD4 T cells expressed SATB1-Venus (i.e. they are all SATB1-Venus hi). In Figure 2B, deletion of the Satb1-a enhancer caused a small but significant decrease in SATB11-Venus MFI (this is clearly judged by the shift of SATB1-Venus peak, toward the left-hand side). Thus, the deletion of the Satb1a enhancer promoted a small decreased in SATB1-Venus expression in all naïve CD4 T cells.
We did elaborate on this finding in our discussion on why would there a small decline in SATB1-Venus expression in naïve CD4 T cells (see lines 422-434, page 17).
As mentioned throughout our manuscript, there are STAT-DNA binding sites in the Satb1-a regulatory region, which is indeed occupied by STAT6, but can also be accessible for other STAT members, such as STAT5. Naïve T cells require the IL-7 cytokine for their homeostatic proliferation, and therefore, it is possible that Satb1-a is also occupied by STAT5, to maintain SATB1 expression. But since we only observed a small decrease in SATB1 expression in  We thank Reviewer #2 for raising this point. We have now added additional biological replicates from Satb1 +/Venus and Satb1 +/Venus-Δa mice in Figure 2C to support our conclusion from the statistical viewpoint.
On the other hand, please note that we could not add additional replicates from Satb1 +/Venus-Δb mice, as we have not been breeding them since Sept 2022 after we consistently observed no significant differences in SATB1-Venus expression in in vivo T cell subpopulations in the Satb1 +/Venus-Δb mice as shown in Fig. 2B and two times experiments for in vitro T cell differentiation. Although it was possible to retrieve this mouse strain from the frozen stock, we must consider the strict polices for conducting animal research (3Rs) and judged that additional experiments with this mouse strain would not be essential for this work. We therefore ask this reviewer and the editor to evaluate whether additional replicates from Satb1 +/Venus-Δb mice are necessary for publishing. Figure EV2D: the quantification of the western blot is necessary We thank Reviewer #2 for raising this point. We have now quantified our SATB1 immunoblots in Figure S2D and included a summarized graph with the appropriate statistical analyses.

Sabt1-a region mice.
We thank the Reviewer #2 for raising this point. We did attempt to provide STAT6 ChIP-qPCR result to confirm STAT6 binding in our in vitro differentiated CD4 Th2 cells during preparation of the original manuscript and we used 2 commercial anti-STAT6 murine antibodies.
Unfortunately, our evaluation of STAT6 ChIP experiment using Gata3 locus (that contains known STAT6 biding region in Th2 cells) indicated that the 2 anti-STAT6 antibodies we used in ChIP did not work well probably due to low quality of antibody. Therefore, at this moment in time, it was very difficult to practically perform and optimise STAT6 ChIP-qPCR in Th0 and TH2 cells. Thus, we used the published STAT6 ChIP-seq data from murine Th2 cells to demonstrate STAT6 binding to our Satb1-a enhancer in the original manuscript.
In addition, please note that performing STAT6-ChIP in Satb1-Δa CD4 Th2 cells is practically impossible, because the STAT6 binding site in Satb1-a region has been deleted in Satb1-Δa mice.

In Satb1 deltaEth2/ deltaEth2, the authors do not analyse the development of T cells in periphery, while the enhancer is important in effector CD4 T cells
We thank the Reviewer #2 for asking this question. We found that T cell development in Satb1 ΔEth2/ΔEth2 mice are normal in a steady state. We show these results for Reviewer #2 in Figure   R1.
In addition, we showed in Figure 5C of our original paper that, after Alternaria induced lung inflammation, CD4 Th2 differentiation was unaffected in Satb1 ΔEth2/ΔEth2 mice, suggesting that the Satb1-Eth2 is dispensable for CD4 TH2 differentiation.
The authors need to analyse the IL4, IL5 and IL13 production after PMA/iono, but also after TCR engagement.
We thank the Reviewer #2 for this suggestion. We measured intracellular IL4 and IL5 production after anti-CD3 treatment and obtained similar results after PMA/iono stimulation.
We present this result in Figure R2 for Reviewer #2 and have included this data in Figure S3C.
Since we didn't not see any significant differences in Il-13 expression ( Figure S3C) from our PMA/Iono treated Th2 cells, we felt that that it was unnecessary to reanalyse Il-13 expression by qPCR in our anti-CD3 treated Th2 cells.

In vivo, using Alternaria Alternate model, the authors show no physiological relevance of the absence of the Sabt1. In others models as asthma (house dust mite or ovabulmin models) ???
We thank the Reviewer #2 for this thoughtful suggestion. We have also used the OVA-Th2 model to study the function of the Satb1-Eth2. As expected, the deletion of this enhancer did not cause any significant changes in BAL Eosinophils numbers, CD4 Th2 numbers, ILC2 numbers and IL-5 secretion ( Figure R3). We have also included these results in Figures S4D and S4E.
We agree that our two in vivo models could not find differences in Th2 responses and IL5 expression. However, we believe that these results do not formally exclude the involvement of SATB1 in controlling TH2 immune responses in vivo under different experimental settings.
Unfortunately, testing several in vivo models with different settings/conditions will take time.
Using lung inflammation models, we clearly showed that the enhancer is also functional in activated ILC2s, which is an important and a novel finding. As suggested by the Reviewer #1, we too think that presenting and identifying this novel functional enhancer has some value, even without its detailed role in regulating Th2 responses at this point. We hope that Reviewer #2 understands our thoughts on this point. We thank the Reviewer #2 for pointing out our errors. We have checked all figures for missing statistics. We appreciate Reviewer #3 for his/her positive evaluation on our study.   Research, 2020, Vol. 48, No. 11 5873-5890), which is inconsistent with the observation here using SATB1-Venus. Also, I noticed that the Th1 MFI of Satb1+/Venus mice was higher than Th2 (Fig 2C), but Western Blot showed that SATB1 protein was higher in Th2 than in Th1 ( Figure EV2D). This inconsistency made me suspect that SATB1-Venus could not truly reflect SATB1 protein levels. Therefore, the author needs to use Western Blot or other alternative methods to confirm that the protein level expression of SATB1 is consistent with that of SATB1-Venus.

Reviewer #3 (Comments to the Authors (Required)): The authors constructed a SATB1-Venus expressing reporter mouse and investigated SATB1 expression in various stages of T cell development and multiple subpopulations of T cells. Using this reporter system, the authors investigated the roles of two candidates of distal enhancers in SATB1 expression regulation in T cells. The authors found that the
We thank Reviewer #3 for raising this important question.
Firstly, we think there are some technical differences in how we analysed SATB1 expression in CD4 SPs versus those in Patta et al. We noted that the SATB1 immunoblot data by Patta et al, ( Figure 1G) did not specify whether the CD4 SP thymocytes were gated from CD24 lo TCRβ hi thymocytes: it is possible that there is contamination of protein lysates coming from immature CD69 + CD24 + CD4 + SP populations, which express higher SATB-1 levels than mature CD4 SP thymocytes. In our study, we gated for CD24 lo TCRβ hi CD4 + CD8thymocytes (see Figure S1G of our manuscript for gating strategy and Satb1-Venus expression) to quantify SATB1-Venus levels. These gating strategies are also very similar to those used by the IMMGEN team and they too have demonstrated that DPs express higher levels of Satb1-mRNA than mature CD4 SP thymocytes. We have now included the IMMGEN data in Figure 1B concerns about the authenticity of his data and they have now published a "Expression of concern" letter, to caution readers about the study. Moreover, JBC have withdrawn the 2001 paper, due to extreme concerns with immunoblot duplication and the overall authenticity of the data.
Nevertheless, we performed immunoblot analyses from DP and CD4 SP thymocytes from WT and Satb1-Venus mice ( Figure R4) and found that no significant differences between endogenous SATB1 and SATB1-Venus protein in both sorted DP and CD4-SP thymocytes.
Similarly, by using Satb1 +/Venus mice we showed that there was no significant differences between endogenous SATB1 and SATB1-Venus protein in total thymocytes ( Figure S1C). Therefore, we believe that SATB1-Venus does functionally represent endogenous SATB1 protein levels.
We also thank Reviewer #3 for pointing out the inconsistencies in our SATB1-Venus analyses in Th2 versus Th1 cell. We again performed immunoblot analyses of in vitro differentiated Th cell subsets, including Th17 and Treg from Wild-type and Satb1-Venus mice, and observed that Th1, Th2 and Th17 expressed comparably higher levels of SATB1-Venus than Th0 and Tregs ( Figure R5). Our revised flow cytometry data are now included in Figure 1H. These data also show a good correlation of Satb1-venus intensity and endogenous Satb1 protein levels.
Having these new results, we re-checked our previous data and realized that the original data showing a higher SATB1 expression in Th2 than in Th1 shown in Fig S2D was not a representative one, therefore we replaced the old image with more representative image and eliminated exceptional data points from our analyses. We believe that our new analyses preclude concerns raised by Reviewer #3 and support our claim that SATB1-Venus does functionally represent endogenous SATB1 protein levels.

Fig EV1E. The altered ratio of CD8SP in Satb1 Venus/Venus mice is evident and it can not be ignored. The authors need to explain this in the manuscript
We thank the Reviewer #3 for raising this important question. We agree that the ratio of CD4SP to CD8SP thymocytes appears to be increased, but further analyses of thymocyte numbers revealed no significant alterations in CD4 SP and CD8 SP numbers our Satb1 Venus/Venus mice. We have now adjusted Fig S1E to include CD4 SP and CD8 SP numbers and clarified this normal phenotype in the main text.  We thank the Reviewer #3 for raising this point, we added data from both splenic and IEL derived to show CD4/CD8 expression patterns from CD62L hi versus CD62L lo gated T cells the data in Fig 1H in Figure 2B). Therefore, this strongly support our findings that Satb1-a (or Satb1-Eth2) functions as an enhancer for SATB1 in CD4 T cells.

The resolution of the Fig 3B Hi-C heatmap is too low, and the black line is too thick, obscuring
the details of the SATB1 locus in the heatmap. Suggest a better presentation of the Hi-C data. In addition, the heat map of Hi-C could not provide the specific interaction between Satb1-a and the SATB1 promoter. It is recommended to perform a 4C assay or similar techniques to confirm the interaction between the enhancer and the promoter.
We apologise for poorly presenting the Hi-C data and thank Reviewer #3 for these suggestions. We have now added one additional Hi-C data of thymocytes and have provided Hi-C figures with increased quality/resolution (See Figure 3B). We have also provided 3C experimental data to show the interaction of Satb1-a enhancer with the Satb1 promoter.

The mice information in this manuscript is confusing. What is the difference between SATB1
Venus-Δa, SATB1 ΔEth2, and SATB1 Venus-ΔEth2? The authors should state this clearly in the manuscript. Since the CRISPR-Cas9 system excision is not perfect, there will be some variation in deletion regions. What is the difference between the deletion sequences in these mice? The authors should provide sequencing validation results for each mouse line.
We thank Reviewer #3 for this comment and apologise for the confusion. We modified the text to increase clarity.
Satb1-Δa and Satb1-ΔEth2 are the same; we just changed the name from Δa to ΔEth2 after confirming its role as an enhancer for Th2 cells (Eth2).
Likewise, Satb1 Venus-Δa and Satb1 Venus-ΔEth2 are the same, but they both express SATB1-Venus fusion protein.
We provided sequences data for Satb1 Venus-Δa and Satb1 Venus-Δb mice in Fig EV2 in the original manuscript and added information of Satb1-ΔEth2 mice on the B6 background.
We thank Reviewer #3 for pointing out these minor issues of our manuscript and have attempted to correct all the above. Thank you for submitting your revised manuscript entitled "Identification of a novel enhancer essential for Satb1 expression in TH2 cells and activated ILC2s.". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. The authors identified a new enhancer that promotes Satb1 expression in Th2 cells. First the authors characterized the expression of Satb1 in thymocytes and spleen cells using a satb1-venus fusion reporter. The authors correlate the expression of Venus with mRNA data and WB against Satb1. Secondly, the authors identified an enhancer that is necessary for the full expression of Satb1. The reduction of Satb1 expression is associated with an increase of IL5 in Th2 cells. Although the role of this enhancer in vivo is not described, the enhancer has a role in satb1 expression in ILC2 and Th2 cells. The authors have enriched the manuscript with data following the comments of the referees.
Minors concerns : Figure 1B : It might be better to say that there is an increase in the number of cells expressing Satb1 rather than that Satb1 expression is increased from DN to DP.
Page 9 line 223 : « Satb1-a is essential ». May be say that the enhancer allow to maintain Satb1 expression at high level.
In general manner, it is necessary to clarify what is meant by 3 independent experiences. It would be nice to say in the materials and methods, how many mice are done per group and per independent experiments.
Reviewer #3 (Comments to the Authors (Required)): In this manuscript, the authors use the constructed SATB1-Venus reporter mouse model to study the regulation mechanism of SATB1. The authors identified an enhancer regulating SATB1 in CD4+ Th2 cells, which can maintain the expression of SATB1 in CD4 Th2 cells and ILC2s. However, the authors did not observe the functional relevancy of the decrease of SATB1 expression caused by the loss of this enhancer on T cell function. Experiments in this study were well conducted, the conclusion is reliable, and it is valuable for understanding the transcriptional regulation of SATB1. All issues arising in the previous version have been resolved in this revision. A small error in Fig. 2A. It should be 300kb instead of 300b.