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

Zian Liao
Suni Tang
Kaori Nozawa
Keisuke Shimada
Masahito Ikawa
Diana Monsivais author has email address
Martin M. Matzuk author has email address

Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, 77030, USA
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA
Graduate Program of Genetics and Genomics, Baylor College of Medicine, Houston, TX, 77030, USA
Center for Drug Discovery, Baylor College of Medicine, Houston, TX, 77030, USA
Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan

https://doi.org/10.7554/eLife.91434.1

Open access
Copyright information

Figures and data

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. 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) Histogram 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 a 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) Histogram 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 of PR binding peaks from one exemplary replicate. 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. Histograms represent average +/- SEM of experiments on cells from three different individuals with technical triplicates. Analyzed by a One-Way ANOVA 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) Venn diagrams showing the shared and unique genes bound by SMAD1, SMAD5 or PR in the +/- 3kb of the promoter regions. C-D) Table of motif analysis results for unique peaks for SMAD1(C) and SMAD5 (D), with P-value and motif annotation specified for each motif.

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 the both Veh and EPC conditions. Data are normalized to siCTL for visualization. Histograms represent average +/- SEM of experiments on cells from three different individuals with technical triplicates. Analyzed by a One-Way ANOVA 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.

Uncropped western Blots for figure 1

Sign up for email alerts