Affinity-tagged SMAD1 and SMAD5 mouse lines reveal transcriptional reprogramming mechanisms during early pregnancy

Reviewer #1 (Public Review):

Summary
Liao et al leveraged two powerful genomics techniques-CUT&RUN and RNA sequencing-to identify genomic regions bound by and activated or inactivated by SMAD1, SMAD5, and the progesterone receptor during endometrial stromal cell decidualization. Additionally, the authors generated novel knock-in HA-SMAD1 and PA-SMAD5 tagged mice to combat antibody issues facing the field, generating a novel model to advance the study of BMP signaling in the female reproductive tract. During decidualization in a murine model, SMAD1/5 are bound to many genomic sites of genes important in decidualization and pregnancy and coregulated responses with progesterone receptor signaling.

Strengths
The authors utilized powerful next generation sequencing and identified important transcriptional mechanisms of SMAD1/5 and PGR during decidualization in vivo.

Weaknesses
None.

Overall, the manuscript and study are well structured and provide critical mechanistic updates on the roles of SMAD1/5 in decidualization and preparation of the maternal endometrium for pregnancy.

Reviewer #2 (Public Review):

Summary:
Liao and colleagues generated tagged SMAD1 and SMAD5 mouse models and identified genome occupancy of these two factors in the uterus of these mice using the CUT&RUN assay. The authors used integrative bioinformatic approaches to identify putative SMAD1/5 direct downstream target genes and to catalog the SMAD1/5 and PGR genome co-localization pattern. The role of SMAD1/5 on stromal decidualization was assayed in vitro on primary human endometrial stromal cells. The new mouse models offer opportunities to further dissect SMAD1 and SMAD5 functions without the limitation from SMAD antibodies, which is significant. The CUT&RUN data further support the usefulness of these mouse models for this purpose.

Strengths:
The strength of this study is the novelty of new mouse models and the valuable cistromic data derived from these mice. Overall the present manuscript is an excellent resource paper for the field of reproductive biology.

Weaknesses:
The weakness of the present version of the manuscript includes the self-limited data analysis approaches such as the proximal promoter based bioinformatic filter and an outdated method on inferring the cell type composition. Evidence was provided for potential associations between SMAD1/5 and other major transcription factors. However, causal effects of SMAD1/5 on the genome occupancy of other major uterine transcription factors were discussed but not experimentally examined in the present manuscript, which is understandable.

For data in Figure 2B, the current manuscript fails to elaborate the common and distinct features between clusters 1 and 3 as well as the biological significance of having two separate clusters for SMAD1. In addition, Figure S1A shows overlapping genome occupancy between SMAD1 and SMAD5, which is not clearly demonstrated in Figure 2B.

For data in Figure 5A, the result description does not provide adequate information to guide readers to full understanding of the data. The biological meaning behind the three PR clusters is not stated nor speculated. Moreover, Figure 5A and Figure S1B are inherently connected but fail to be adequately described in the main text.

Reviewer #3 (Public Review):

Summary:
As SMAD1/5 activities have previously been indistinguishable, these studies provide a new mouse model to finally understand unique downstream activation of SMAD1/5 target genes, a model useful for many scientific fields. Using CUT&RUN analyses with gene overlap comparisons and signaling pathway analyses, specific targets for SMAD1 versus SMAD5 were compared, identified, and interpreted. These data validate previous findings showing strong evidence that SMADs directly govern critical genes required for endometrial receptivity and decidualization, including cell adhesion and vascular development. Further, SMAD targets were overlapped with progesterone receptor binding sites to identify regions of potential synergistic regulation of implantation. The authors report strong correlations between progesterone receptor and SMAD1/5 direct targets to cooperatively promote embryo implantation. Finally, the authors validated SMAD1/5 gene regulation in primary human endometrial stromal cells. These studies provide a data-rich survey of SMAD family transcription, defining its role as a governor of early pregnancy.

Strengths:
This manuscript provides a valuable survey of SMAD1/5 direct transcriptional events at the time of receptivity. As embryo implantation is controlled by extensive epithelial to stromal molecular crosstalk and hormonal regulation in space and time, the authors state a strong, descriptive narrative defining how SMAD1/5 plays a central role at the site of this molecular orchestration. The implementation of cutting-edge techniques and models and simple comparative analyses provide a straightforward, yet elegant manuscript.

Although the progesterone receptor exists as a major regulator of early pregnancy, the authors have demonstrated clear evidence that progesterone receptor with SMAD1/5 work in concert to molecularly regulate targets such as Sox17, Id2, Tgfbr2, Runx1, Foxo1 and more at embryo implantation. Additionally, the authors pinpoint other critical transcription factor motifs that work with SMADs and the progesterone receptor to promote early pregnancy transcriptional paradigms.

Weaknesses:

Although a wonderful new tool to ascertain SMAD1 versus SMAD5 downstream signaling, the importance of these factors in governing early pregnancy is not novel. Furthermore, functional validation studies are needed to confirm interactions at promoter regions. Additionally, the authors presume that all overlapped genes are shared between progesterone receptor and SMAD1/5, yet some peak representations do not overlap. Although, transcriptional activation can occur at the same time, they may not occur in the same complex. Thus, further confirmation of these transcriptional events is warranted.

Since whole murine uterus was used for these studies, the specific functions of SMAD1/5 in the stroma versus the epithelium (versus the myometrium) remain unknown. Further work is needed to delineate binding and transcriptional activation of SMAD1/5 and the progesterone receptor in the uterine compartments.

There are asynchronous gene responses in the SMAD1/5 ablated mouse model compared to the siRNA-treated human endometrial stromal cells. These differences can be confounding. Further investigation is needed to understand the meaning of these differences and as they relate to the entire SMAD transcriptome.

Author Response

We thank you for your careful review of our manuscript and helpful comments and suggestions. We have carefully considered each point and have addressed them by adding changes to the manuscript and figures. The text below detailed our responses and edits.

Reviewer #1 (Public Review):

Summary:

Liao et al leveraged two powerful genomics techniques-CUT&RUN and RNA sequencing-to identify genomic regions bound by and activated or inactivated by SMAD1, SMAD5, and the progesterone receptor during endometrial stromal cell decidualization.

Strengths:

The authors utilized powerful next generation sequencing and identified important transcriptional mechanisms of SMAD1/5 and PGR during decidualization in vivo.

Weaknesses:

Overall, the manuscript and study are well structured and provide critical mechanistic updates on the roles of SMAD1/5 in decidualization and preparation of the maternal endometrium for pregnancy. Please consider the following to improve the manuscript:

• Figure 4: A and C show bar graphs, not histograms. Please alter this phrasing.

Figure legends were adjusted as suggested.

• What post hoc test was performed on qPCR analyses? (Figure 6). It is evident that any assumptions of equal variance need to be negated due to the wide dispersion in experimental response invalidating the assumptions of a one-way ANOVA.

Yes, a Tukey’s post hoc test was performed on the qPCR analyses. To address the reviewer’s question regarding equal variance, normality of the dataset was examined by D’agostino & Pearson test in GraphPad Prism. The data demonstrated a normal distribution pattern, thus justifying the one-way ANOVA test.

• Figure 6: what data points are plotted? Are these technical replicates from individual wells or qPCR technical replicates?

The dataset represents three technical and three biological data points.

• Figure 6: Consider changing graph colors to increase visibility of error bars and data points.

Thank you for this suggestion. The colors of the error bars in Figure 6 have been changed to increase visibility. Additionally, different shapes have been utilized to distinguish between different groups.

• Figure 6 legend: no histograms are shown in this figure. Refer to all gene names utilizing proper nomenclature and conventions (gene names should be italicized).

The legend was adjusted as suggested with the correct nomenclature implemented.

• qPCR analyses: qPCR normalization should be done to at least two internal control genes, preferably three according to the MIQE guidelines (PMID: 19246619).

As suggested, we have performed additional qPCR analysis with normalization done to three internal controls.

• Supplement figure 2: graphs are bar graphs, not histograms.

The legends have been changed as suggested.

Reviewer #2 (Public Review):

Summary:

Liao and colleagues generated tagged SMAD1 and SMAD5 mouse models and identified genome occupancy of these two factors in the uterus of these mice using the CUT&RUN assay. The authors used integrative bioinformatic approaches to identify putative SMAD1/5 direct downstream target genes and to catalog the SMAD1/5 and PGR genome co-localization pattern. The role of SMAD1/5 on stromal decidualization was assayed in vitro on primary human endometrial stromal cells. The new mouse models offer opportunities to further dissect SMAD1 and SMAD5 functions without the limitation from SMAD antibodies, which is significant. The CUT&RUN data further support the usefulness of these mouse models for this purpose.

Strengths:

The strength of this study is the novelty of new mouse models and the valuable cistromic data derived from these mice.

Weaknesses:

The weakness of the present version of the manuscript includes the self-limited data analysis approaches such as the proximal promoter based bioinformatic filter and a missed opportunity to investigate the role of SMAD1/5 on determining the genome occupancy of major uterine transcription regulators.

Thank you for the comments. We addressed the limitation of the promoter-based analysis in the discussion and pointed out the possibility of analyzing additional genomics features (Lines 548551). Based on the suggestions, we also included an analysis in which we compared SMAD1/5 binding activities in this study to known major uterine transcription regulators’ binding activities (namely, SOX17 and NR2F2) using published ChIP-seq data in the mouse uterus. Results from this analysis are discussed in Lines 426-436. Content from the adjusted manuscript is copied below.

Lines 548-551:

“From pathway enrichment analysis, we demonstrate that genes with SMAD1/5 and PR bound at the promoter regions are enriched for key pathways in directing the decidualization process, such as WNT and relaxin signaling pathways. Future studies can benefit from analyzing binding events beyond the promoter regions.”

Lines 426-436:

“To further evaluate the key roles of SMAD1/5 as major uterine transcription regulators, we cross-compared the genomic binding sites of SMAD1/5 with known key transcription factors, namely aforementioned SOX17 (Supplement Figure 1E), as well as NR2F2 (Supplement Figure 1F), an essential regulator of hormonal response, using our CUT&RUN data sets and published mouse uterine SOX17 and NR2F2 ChIP-seq data sets (GSE118328, GSE232583). Among the annotated genes, 5402 genes are shared between SMAD1/5 and SOX17, and 1922 genes are shared between SMAD1/5 and NR2F2. Such observations indicate a potential co-regulatory mechanism between SMAD1/5 and other key uterine transcription factors in maintaining appropriate uterine functions. Overall, our analyses demonstrate that the transcriptional activity of SMAD1, SMAD5, and PR coordinate the expression of key genes required for endometrial receptivity and decidualization.”

Reviewer #3 (Public Review):

Summary:

As SMAD1/5 activities have previously been indistinguishable, these studies provide a new mouse model to finally understand unique downstream activation of SMAD1/5 target genes, a model useful for many scientific fields. Using CUT&RUN analyses with gene overlap comparisons and signaling pathway analyses, specific targets for SMAD1 versus SMAD5 were compared, identified, and interpreted. These data validate previous findings showing strong evidence that SMADs directly govern critical genes required for endometrial receptivity and decidualization, including cell adhesion and vascular development. Further, SMAD targets were overlapped with progesterone receptor binding sites to identify regions of potential synergistic regulation of implantation. The authors report strong correlations between progesterone receptor and SMAD1/5 direct targets to cooperatively promote embryo implantation. Finally, the authors validated SMAD1/5 gene regulation in primary human endometrial stromal cells. These studies provide a data-rich survey of SMAD family transcription, defining its role as a governor of early pregnancy.

Strengths:

This manuscript provides a valuable survey of SMAD1/5 direct transcriptional events at the time of receptivity. As embryo implantation is controlled by extensive epithelial to stromal molecular crosstalk and hormonal regulation in space and time, the authors state a strong, descriptive narrative defining how SMAD1/5 plays a central role at the site of this molecular orchestration. The implementation of cutting-edge techniques and models and simple comparative analyses provide a straightforward, yet elegant manuscript.

Although the progesterone receptor exists as a major regulator of early pregnancy, the authors have demonstrated clear evidence that progesterone receptor with SMAD1/5 work in concert to molecularly regulate targets such as Sox17, Id2, Tgfbr2, Runx1, Foxo1 and more at embryo implantation. Additionally, the authors pinpoint other critical transcription factor motifs that work with SMADs and the progesterone receptor to promote early pregnancy transcriptional paradigms.

Weaknesses:

Although a wonderful new tool to ascertain SMAD1 versus SMAD5 downstream signaling, the importance of these factors in governing early pregnancy is not novel. Furthermore, functional validation studies are needed to confirm interactions at promoter regions. Addtionally, the authors presume that all overlapped genes are shared between progesterone receptor and SMAD1/5, yet some peak representations do not overlap. Although, transcriptional activation can occur at the same time, they may not occur in the same complex. Thus, further confirmation of these transcriptional events is warranted.

Thank you for the review; we appreciate these valuable comments. Although we used an overlap approach to investigate the gene regulatory networks between SMAD1/5 and PR at the gene level, we functionally validated the regulatory effect in an in vitro decidualization model using a qPCR approach. We acknowledge that gene activations may not occur at the exact same complex, but functional validation screenings at the promoter level are beyond the scope of the study. However, we added the discussion about the possibility of proposed investigations in Lines 553-558. Our current dataset and validation studies support our conclusions with robust evidence. Content from Lines 553-558 is copied below.

Lines 553-558: “In this study, we determined the overlapped transcriptional control between SMAD1/5 and PR at the gene level, and functionally validated the regulatory effect at the transcript level in a human stromal cell decidualization model. While we observe a subset of peak representations that do not overlap at the base pair level in the promoter regions, future functional screenings at the promoter level, such as luciferase reporter assays to assess transcriptional co-activation by SMAD1/5 and PR, will advance this study.”

• Since whole murine uterus was used for these studies, the specific functions of SMAD1/5 in the stroma versus the epithelium (versus the myometrium) remain unknown. Specific roles for SMAD1/5 in the uterine stroma and epithelial compartments still need to be examined. Also, further work is needed to delineate binding and transcriptional activation of SMAD1/5 and the progesterone receptor in stromal versus epithelial uterine compartments.

Thank you for the comments. Indeed, our study was performed in the whole mouse uterus, which includes stroma, epithelium and myometrium. Our previous data shows that nuclear SMAD1/5 are localized to both the stroma and epithelium in the decidua zone during the decidualization process at 4.5 dpc (PMID:34099644). Published in vivo studies also demonstrate the essential role of SMAD1/5 in the uterine epithelium and stroma compartments, respectively (PMIDs:35383354/27335065/17967875). Although we believe the binding/transcriptional activation of SMAD1/5 and PR occurs in both compartments based on the mouse phenotypic data, opportunities for further compartment-specific analysis were granted and discussion regarding such investigations was added (Lines 501-513). Content from Lines 501-513 is copied below.

Lines 501-513:

“Published studies have shown that nuclear SMAD1/5 localize to the stroma and epithelium during the decidualization process at 4.5dpc during the window of implantation. Conditional deletion of SMAD1/5 exclusively in the uterine epithelium using lactoferrin-icre (Ltf-icre) results in severe subfertility due to impaired implantation and decidual development. Conditional deletion of SMAD1/5/4 exclusively in the cells from mesenchymal lineage (including uterine stroma) using anti-Mullerian hormone type 2 receptor cre (Amhr2-cre) results in infertility with defective decidualization. Given the essential roles of SMAD1/5 in both stroma and epithelium identified by previous studies, we believe that transcriptional co-regulation by SMAD1/5 and PR reported here using the whole uterus validates a relationship between SMAD1/5 and PR in both the stromal and epithelial compartments. However, it does not rule out the potential coregulation of SMAD1/5 and PR in the myometrium, immune cells, and/or endothelium, given that whole uterus was used. The specific transcriptional evaluations of SMAD1/5 in the stroma versus the epithelium would require future single-cell sequencing (i.e., digital cytometry) and/or spatial transcriptomic analysis.”

• There are asynchronous gene responses in the SMAD1/5 ablated mouse model compared to the siRNA-treated human endometrial stromal cells. These differences can be confounding, and more clarity is required in understanding the meaning of these differences and as they relate to the entire SMAD transcriptome.

Thank you for the comments. From the mouse models with SMAD1/5 conditional deletions, we observed phenotypic defects at 4.5 dpc, which is the beginning of decidualization in the mouse. Our study used human endometrial stromal cells as a model to validate our findings functionally, aiming to mimic the specific time point during decidualization. Differences between the two models may arise from the strategy used to perturb SMAD1/5; in the mouse, a complete knockout of SMAD1/5 was used, resulting in failed decidualization, while the human endometrial stromal cells used an siRNA knockdown approach, which decreased the potential for decidualization. As such, this information needs to be considered when evaluating genome-wide effects on the transcriptome. We added a discussion of this point to Lines 564-572. Content from Lines 564-572 is copied below.

Lines 564-572:

“Since mice only undergo decidualization upon embryo implantation whilst human stromal cells undergo cyclic decidualization in each menstrual cycle in response to rising levels of progesterone, asynchronous gene responses may occur in comparison between mouse models and human cells. However, cellular transformation during decidualization is conserved between mice and humans, which makes findings in the mouse models a valuable and transferable resource to be evaluated in human tissues. Accordingly, our functional validation studies were performed using human endometrial stromal cells induced to decidualize in vitro for four days, which models the early phases of decidualization. Additional transcriptomic studies of the SMAD1/5 perturbations in human endometrial stromal cells will be of great resource in understanding the entire SMAD1/5 regulomes in humans.”

Reviewer #1 (Recommendations For The Authors):

• Minor grammatical errors requiring attention such as inserting punctuation at the end of sentences and including figure legends prior to the end of sentence punctuation.

Thanks for the comments. Additional proofreading was conducted for the revision.

Reviewer #2 (Recommendations For The Authors):

1. Between SMAD1 and SMAD5, does losing one SMAD affect the other SMAD's genome occupancy?

Thanks for the comments. Based on the mouse phenotypic data that conditional deletion of SMAD1 in the uterus does not affect female fertility, while conditional deletion of SMAD5 leads to subfertility, and conditional deletion of both SMAD1 and SMAD5 leads to complete infertility. We believe losing one SMAD will affect the other SMAD's genome occupancy. This point is discussed in Lines 514-517, with contents copied below.

Lines 514-517: “Although our studies herein confirm that SMAD1 and SMAD5 proteins have distinct transcriptional regulatory activities, our previous studies demonstrated that while SMAD5 can functionally replace SMAD1, SMAD1 cannot replace SMAD5 in the uterus. How this epistatic relationship is established in a tissue-specific manner still needs to be determined by further biochemical investigations.”

1. In light of SMAD1/5 and PGR co-occupied cis-acting elements and coregulating uterine transcriptome, does loss of SMAD1/5 alter the PGR and ESR1 genome occupancy?

Thanks for the comments. In the SMAD1/5 double conditional knockout mice, we observe the hyposensitivity towards progesterone and unopposed estrogen responses. We hypothesize that loss of SMAD1/5 alters PR genome occupancy and subsequently ER genome occupancy is altered as a secondary effect. To functionally address this question, genomic profiling studies need to be performed in the SMAD1/5 knockout mice, and, ideally, also performed in the PR knockout mice. However, such large-scale studies are beyond the scope of the current study and will not affect our conclusions under physiological conditions. We did include additional discussion regarding this comment in Lines 551-553, with the contents copied below.

Lines 551-553: “Profiling the PR genome occupancy in the SMAD1/5 deficient mice would provide an interesting perspective to reevaluate the major regulatory roles of SMAD1/5 in mediating uterine transcriptomes.”

1. In terms of investigating the impact of SAMD1/5 on cell type composition, perhaps the digital cytometry approach (e.g., PMID: 31061481) could provide unbiased inferences.

Thank you for the comments. We included expression analysis of a subset of SMAD1/5 direct target genes over different uterine compartments (Figure 4E). We also added the discussion of the opportunities for further compartment-specific analysis, including but not limited to the digital cytometry approach in Lines 506-513, with the contents copied below.

Line 506-513:

“Given the essential roles of SMAD1/5 in both stroma and epithelium identified by previous studies, we believe that the transcriptional co-regulatory roles of SMAD1/5 and PR reported here using the whole uterus validates a relationship between SMAD1/5 and PR in both the stromal and epithelial compartments. However, it does not rule out potential co-regulatory roles of SMAD1/5 and PR in the myometrium, immune cells, and/or endothelium, given that whole uterus was used. The specific transcriptional evaluations of SMAD1/5 in the stroma versus the epithelium would require future single-cell sequencing (i.e., digital cytometry) and/or spatial transcriptomic analysis.”

1. The limitation of focusing on the promoter occupied SMADs should be discussed.

Additional discussion of the limitation of focusing on the promoter regions was added in Lines 548-551, with contents copied below.

Lines 548-551:

“From pathway enrichment analysis, we demonstrate that genes with SMAD1/5 and PR bound at the promoter regions are enriched for key pathways in directing the decidualization process, such as WNT and relaxin signaling pathways. Future studies can benefit from analyzing binding events beyond the promoter regions.”

1. Methods: The reagent and the condition for PGR CUT&RUN is missing.

Information added in Line 153.

1. Line 260: Please clarify the statement of "suggesting the transcriptional of PR depends on BMP/SMAD1/5 signaling".

Thanks for the suggestion. The sentence was rephrased to (Lines 258-261) “Our previous studies revealed that conditional ablation of SMAD1 and SMAD5 in the uterus decreased P4 response during the peri-implantation period, suggesting that the transcriptional activities of PR depend on BMP/SMAD1/5 signaling.”

1. Line 280-289: This statement belongs to the discussion section.

The statement was moved as suggested.

1. Figure 4E is not cited in the result section.

Figure 4E was cited in the results section in the revised version. (Line 386)

1. Figures 3C, 3D, 3E, 3F, 5B and 5D: please include the full lists in the supplemental data so that labs with limited bioinformatic capabilities could use these findings to facilitate scientific discovery.

Data regarding the aforementioned figures were included in Supplement Tables 3-8 and Supplement Files 1-2.

1. Figure 2B and Figure 5A: the heatmaps without further grouping on common and distinct genome occupancy among assayed factors provided minimum useful information. Please reconsider the presentation format in order to deliver more meaningful results.

Figure 2B and Figure 5A were replotted with clustering using the k-means algorithm. Methods and legends were updated accordingly.

Reviewer #3 (Recommendations For The Authors):

To delineate specific roles for SMAD1/5 in the uterine stroma and epithelial compartments, methods such as single cell sequencing or spatial transcriptomic analysis may be warranted.

The manuscript now includes the discussion of future opportunities in investigating the roles of SMAD1/5 in different uterine compartments using single-cell sequencing and/or spatial transcriptomic analysis (Lines 498-513), with contents copied below.

Lines 498-513:

“Our studies also examined the role of SMAD1/5 in mediating progesterone responses at the genomic and transcription levels. Similarly, our analysis was based on data sets generated from the whole mouse uterus, which contains multiple compartments of the uterine structures, including but not limited to epithelium and stroma. Published studies have shown that nuclear SMAD1/5 localize to the stroma and epithelium during the decidualization process at 4.5 dpc, during the window of implantation. Conditional deletion of SMAD1/5 exclusively in the uterine epithelium using lactoferrin-icre (Ltf-icre) results in severe subfertility due to impaired implantation and decidual development. Conditional deletion of SMAD1/5/4 exclusively in the cells from mesenchymal lineage (including uterine stroma) using anti-Mullerian hormone type 2 receptor cre (Amhr2-cre) results in infertility with defective decidualization. Given the essential roles of SMAD1/5 in both stroma and epithelium identified by previous studies, we believe that the transcriptional co-regulatory roles of SMAD1/5 and PR reported here using the whole uterus validates a relationship between SMAD1/5 and PR in both the stromal and epithelial compartments. However, it does not rule out potential co-regulatory roles of SMAD1/5 and PR in the myometrium, immune cells, and/or endothelium, given that whole uterus was used. The specific transcriptional evaluations of SMAD1/5 in the stroma versus the epithelium would require future single-cell sequencing (i.e., digital cytometry) and/or spatial transcriptomic analysis.”