GATA2 and Progesterone Receptor Interaction in Endometrial Stromal Cells Undergoing Decidualization

Abstract

The transcription factor GATA2 is important for endometrial stromal cell decidualization in early pregnancy. Progesterone receptor (PGR) is also critical during decidualization but its interaction with GATA2 in regulating genes and pathways necessary for decidualization in human endometrium are unclear. RNA-sequencing (RNA-seq) was performed to compare gene expression profiles (n = 3), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an antibody against GATA2 (n = 2) was performed to examine binding to target genes in human endometrial stromal cells undergoing in vitro decidualization (IVD including estrogen, progestin, and 3',5'-cyclic AMP analogue) or vehicle treatment. We identified 1232 differentially expressed genes (DEGs) in IVD vs vehicle. GATA2 cistrome in IVD-treated cells was enriched with motifs for GATA, ATF, and JUN, and gene ontology analysis of GATA2 cistrome revealed pathways that regulate cholesterol storage, p38 mitogen-activated protein kinase, and the c-Jun N-terminal kinase cascades. Integration of RNA-seq and ChIP-seq data revealed that the PGR motif is highly enriched at GATA2 binding regions surrounding upregulated genes in IVD-treated cells. The integration of a mined public PGR cistrome in IVD-treated human endometrial cells with our GATA2 cistrome showed that GATA2 binding was significantly enhanced at PGR-binding regions in IVD vs vehicle. Interrogating 2 separate ChIP-seq data sets together with RNA-seq revealed integration of GATA2 and PGR action to coregulate biologic processes during decidualization of human endometrial stromal cells, specifically via WNT activation and stem cell differentiation pathways. These findings reveal the key pathways that are coactivated by GATA2 and PGR that may be therapeutic targets for supporting implantation and early pregnancy.

Keywords: JNK; WNT; differentiation; endometrium; p38MAPK; progesterone.

Figure 1.

Differentially expressed genes (DEGs) and pathways in human endometrial cells undergoing in vitro decidualization (IVD) vs vehicle treatment (Veh). A, Heat map of 1232 DEGs in replicates. B, MetaCore-enriched pathways of DEGs and examples of DEGs in those pathways.

Figure 2.

GATA2 binding sites in human endometrial stromal cells during vehicle treatment (Veh) or in vitro decidualization (IVD). A, Venn diagram comparing GATA2 peaks in Veh and IVD cells. B,) Motif analysis of GATA2 peaks in vehicle-treated cells (total target sequences: 10 612; total background sequences: 38 567). C, Motif analysis of GATA2 peaks in IVD cells (total target sequences: 14 597; total background sequences: 34 650). D, Gene ontology analysis of biological processes of genes with GATA2 binding in Veh-treated cells. E, Gene ontology analysis of biological processes of genes with GATA2 binding in IVD-treated cells.

Figure 3.

Analysis of GATA2 binding sites on differentially expressed genes DEGs in human endometrial cells during in vitro decidualization (IVD). A, Motif analyses of peaks corresponding to GATA2-bound downregulated genes (total target sequences: 634; total background sequences: 48 263). B, Motif analyses of peaks corresponding to GATA2-bound upregulated genes (total target sequences: 1007; total background sequences: 48 124). C, GATA2 and progesterone receptor (PGR) binding at genes downregulated during IVD. D, GATA2 and PGR binding at genes upregulated during IVD. Veh, vehicle-treated cells; ****P less than 0.001.

Figure 4.

GATA2 and progesterone receptor (PGR) interaction in human endometrial cells during in vitro decidualization (IVD). A, Immunoprecipitation (IP) of PGR on chromatin followed by immunoblotting for GATA2. B, GATA2 binding centering at all PGR peaks. The x-axis corresponds to base pairs surrounding peak regions from 5’ to 3’. The y axis indicates how many reads (per million mapped reads) located at the specific regions; IVD treatment significantly increased the interaction between GATA2 and PGR binding across the genome. ****P less than 0.001. C, Venn diagram of genes upregulated during IVD that are bound by GATA2 (green) and PGR (pink); the majority of GATA2 and PGR binding sites overlapped (gray) in a statistically significant manner. ****P less than 0.001. D, MetaCore pathway analysis of GATA2- and PGR-bound overlapping genes.