Mouse models with global HA tagged SMAD1 and PA tagged SMAD5 proteins.

A-B) Schematic approaches for generating Smad1HA/HA and Smad5PA/PA knock-in mouse lines. Sanger sequencing of the genotyping results are included as validation of knock-in sequence. Black and blue boxes indicate untranslated and coding regions, respectively. C-D) Immunoblot (IB) analysis of the immunoprecipitation (IP) of HA tagged SMAD1 and PA tagged SMAD5 proteins from different tissues of the tagged mouse lines. Wild type (WT) mice were used as negative controls. Antibodies used for IB and IP are as labeled. Targeted bands of SMAD1 and SMAD5 are indicated by red arrows.

Genomic profiling of SMAD1 and SMAD5 binding sites during decidualization in vivo.

A) Diagram outlining experimental approaches for tissue collection, processing, and CUT&RUN. B) Heatmaps and summary plots showing the enrichment of SMAD1 and SMAD5 binding peaks from one exemplary replicate. Clustering was conducted using k-means algorithm. The colors in the summary plots correspond to clusters labeled in the heatmap below. C-D) Feature distribution of the annotated peaks for the SMAD1 (C) binding sites and SMAD5 (D) binding sites.

SMAD1 and SMAD5 show unique direct target genes during early pregnancy.

A-B) Venn diagrams showing the shared and unique direct up-target genes (A) and down-target genes (B) of SMAD1, SMAD5 Numbers indicate genes numbers. C-D) Motif enrichment analysis from the up-targets and down-targets for SMAD1 (C) and SMAD5 (D). E-F) Dot plot showing Gene Ontology enrichment analysis of shared direct target genes of SMAD1/5 from the up-targets (E) and the down-targets (F), respectively. Dot size represents the gene ratio in the enriched categories compared to background genes, dot colors reflect P-value.

Direct target genes of SMAD1/5 mediate uterine homeostasis.

A) Bar graph of normalized Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of downregulated transcripts in the Control and SMAD1/5 cKO groups as indicated by the label. Histograms represent average +/− SEM of experiments uteri from Control mice (N=3) and SMAD1/5 cKO mice (N=4). Analyzed by an unpaired t-test. B) Integrative Genomics Viewer (IGV) track view of SMAD1, SMAD5 binding activities. Gene loci are as indicated in the figure, genomic coordinates are annotated in mm10. C) Bar graph of FPKM of up regulated transcripts in the Control and SMAD1/5 cKO groups as indicated by the label. D) IGV track view of SMAD1, SMAD5 binding activities. Gene loci are as indicated in the figure, genomic coordinates are annotated in mm10. E) Dot plot showing the gene expression pattern of the key SMAD1/5 direct target genes in different cell types from published human endometrium single-cell RNA-seq dataset.

SMAD1 and SMAD5 co-regulate PR target genes.

A) Heatmaps and summary plots showing the enrichment comparison between SMAD1, SMAD5 and PR binding peaks from one exemplary replicate. Clustering was conducted using k-means algorithm. The colors in the summary plots correspond to clusters labeled in the heatmap below. B) Dot plot showing KEGG pathway enrichment analysis for shared genes bound by SMAD1, SMAD5, and PR. C) IGV track view of SMAD1, SMAD5 and PR binding activities. Gene loci are as indicated in the figure, genomic coordinates are annotated in mm10. D) Table of motif analysis results for shared peaks between SMAD1, SMAD5 and PR, with P-value and motif annotation specified for each motif.

SMAD1 and SMAD5 are required for PR responses during decidualization of human endometrial stromal cells.

A) Schematic approach and timeline outlining in vitro decidualization for endometrial stromal cells (EnSCs). B-C) RT-qPCR results showing mRNA levels of PRL, IGFBP1, FOXO1, RORB and KLF15 after SMAD1/5 perturbation using siRNAs. Data are normalized to siCTL-Veh for visualization. Bar graphs represent average +/− SEM of experiments on cells from three different individuals with technical triplicates. Analyzed by a One-Way ANOVA test with post hoc Tukey test.

Gene numbers with SMAD1/5 promoter binding activities and motif analysis of SMAD1/5 peaks.

A) Venn diagrams showing the shared and unique genes bound by SMAD1 or SMAD5 in the +/− 3kb region of the promoter regions. B) Cell type compositions in the deconvoluted RNA-seq from control mice (Control 1-3) and SMAD1/5 PR-Cre mice (KO1-4). Numbers plotted in the table are absolute values that reflects the absolute proportion of each cell type in the given sample. A larger number indicates a higher proportion of that cell type in the mixture. C) Venn diagrams showing the shared and unique genes bound by SMAD1, SMAD5 or PR in the +/− 3kb of the promoter regions. D-E) Table of motif analysis results for unique peaks for SMAD1(D) and SMAD5 (E), with P-value and motif annotation specified for each motif. F) Venn diagrams showing the shared and unique genes bound by SMAD1, SMAD5 or SOX17 in the +/− 25kb regions near transcription start site. G) Venn diagrams showing the shared and unique genes bound by SMAD1, SMAD5 or NR2F2 in the +/− 25kb regions near transcription start site.

Knockdown effect validation of SMAD1/5 perturbation.

A-B) RT-qPCR results showing mRNA levels of SMAD1(A) and SMAD5 (B) after siRNA treatments in both Veh and EPC conditions. Data are normalized to siCTL for visualization. Bar graphs represent average +/− SEM of experiments on cells from three different individuals with technical triplicates. Analyzed by a One-Way ANOVA test with post hoc Tukey test.

Genotype of the knock-in mouse lines.

A) Schematic design of the genotype primers for Smad1HA/HA and Smad5PA/PA mouse lines. B) Exemplary gel electrophoresis of PCR products derived from homozygous knock-in mice, heterozygous mice, and WT mice using genotyping primers.