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.

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.
• 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.
• Figure 6: what data points are plotted? Are these technical replicates from individual wells or qPCR technical replicates?
• Figure 6: Consider changing graph colors to increase visibility of error bars and data points.
• 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).
• qPCR analyses: qPCR normalization should be done to at least two internal control genes, preferably three according to the MIQE guidelines (PMID: 19246619).
• Supplement figure 2: graphs are bar graphs, not histograms.

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.

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.

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.

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.