Research resource: Genome-wide profiling of progesterone receptor binding in the mouse uterus

Abstract

Progesterone (P4) signaling through its nuclear transcription factor, the progesterone receptor (PR), is essential for normal uterine function. Although deregulation of PR-mediated signaling is known to underscore uterine dysfunction and a number of endometrial pathologies, the early molecular mechanisms of this deregulation are unclear. To address this issue, we have defined the genome-wide PR cistrome in the murine uterus using chromatin immunoprecipitation (ChIP) followed by massively parallel sequencing (ChIP-seq). In uteri of ovariectomized mice, we identified 6367 PR-binding sites in the absence of P4 ligand; however, this number increased at nearly 3-fold (18,432) after acute P4 exposure. Sequence analysis revealed that approximately 73% of these binding sites contain a progesterone response element or a half-site motif recognized by the PR. Many previously identified P4 target genes known to regulate uterine function were found to contain PR-binding sites, confirming the validity of our methodology. Interestingly, when the ChIP-seq data were coupled with our microarray expression data, we identified a novel regulatory role for uterine P4 in circadian rhythm gene expression, thereby uncovering a hitherto unexpected new circadian biology for P4 in this tissue. Further mining of the ChIP-seq data revealed Sox17 as a direct transcriptional PR target gene in the uterus. As a member of the Sox transcription factor family, Sox17 represents a potentially novel mediator of PR action in the murine uterus. Collectively, our first line of analysis of the uterine PR cistrome provides the first insights into the early molecular mechanisms that underpin normal uterine responsiveness to acute P4 exposure. Future analysis promises to reveal the PR interactome and, in turn, potential therapeutic targets for the diagnosis and/or treatment of endometrial dysfunction.

Fig. 1.

Identification of PR binding locations with vehicle or P4 on whole-mouse uterine tissue and epithelial-isolated uterine tissue. BED files containing chromosome annotation and start-stop positions of the ChIP-seq sites are provided as Supplemental Material SM1. A, ChIP-seq PR-binding site counts and proximity relationships. Venn diagrams of the intersection of multiple intervals files between vehicle vs. P4-treated and whole vs. epithelial. B, Sequence conservation analysis. PR ChIP intervals were aligned at their centers and uniformly expanded 1500 bp in each direction, and phastCons scores were retrieved and averaged at each position. C, Distribution of genome-wide PR-binding locations throughout the murine uterus. PR-binding sites were analyzed using the CEAS module at Cistrome. Promoter region was defined in increments of 2500 bp to 10 kb. When the PR-binding site is within a gene, it is further defined as within the 5′-untranslated region (UTR), 3′-UTR, coding exon or intron. Intergenic region is defined as more than 10 kb from gene boundaries. D, In the P4-treated dataset, the frequency of PR-binding sites located within 10 kb upstream of the transcriptional start site were analyzed. E, Gene counts. Venn diagrams for nonredundant genes with PR-binding locations within ± 25 kb and those genes that are regulated by P4 after 6 h treatment by microarray (>1.2-fold) are presented. P4-regulated nonredundant genes are further grouped underneath the diagram as a list presenting the gene counts that are either up- or down-regulated in the absence or presence of P4. F, HRE motifs were found enriched in PR-binding sites in the P4-treated group using the SeqPos tool at Cistrome.

Fig. 2.

ChIP validation of P4 target genes. Validation of PR-binding sites by ChIP-RT-qPCR with the PR H-190 antibody (Santa Cruz Biotechnology) on uteri isolated from ovariectomized C57 female mice treated 1 h with P4. Negative control (Untr) is a gene-deficient region. Data are represented as fold enrichment of PR binding of PR-location sites over that of the negative control region.

Fig. 3.

P4 regulates the expression of several genes involved in circadian rhythm regulation in the uterus. A, ChIP-RT-qPCR validation of PR binding near genes involved in regulation of circadian rhythm (Clock, Npas2, Cry1 and Per1, Nr1d2, Rorc, Ppargc1a) with the PR H-190 antibody (Santa Cruz Biotechnology) on uteri isolated from ovariectomized C57 female mice treated 1 h with P4. Data are represented as fold enrichment of PR binding of PR-location sites over that of the negative control region. B, Analysis of several regulators of circadian rhythm. Ovariectomized mice were treated with vehicle or P4 for 6 h, and uterine gene expression was analyzed by RT-qPCR. All circadian genes confirmed to contain PR binding demonstrate P4 regulation. Expression data for each gene were normalized to that of 18s rRNA. Data are based on three independent experiments. Error bars represent sem. *, P < 0.05, **, P < 0.01; ***, P < 0.001.

Fig. 4.

Identification of a potential novel PR transcriptional interaction with Sox17. A, UCSC Genome Browser illustrations of select genes showing distributions of PR binding in vehicle and P4 treatments and potential Sox-binding locations based on motif analysis. Sequence positions and other generic UCSC annotations were removed for clarity. Light blue indicates PR binding in vehicle condition, purple indicates PR binding in P4 condition and yellow indicates location of putative Sox motifs. B, Validation of PR binding on the Sox17 gene by ChIP-RT-qPCR. Two putative locations, within the Sox17 gene and one approximately 17 kb upstream, were validated with the PR H-190 antibody (Santa Cruz Biotechnology) on uteri isolated from ovariectomized C57 female mice treated 1 h with P4. Data are represented as fold enrichment of PR binding of PR-location sites over that of the negative control region. C, Analysis of Sox17 mRNA expression. Analysis performed on RNA isolated from uteri of ovariectomized mice treated with vehicle or P4 for 6 h. Expression data for each gene were normalized to that of 18s rRNA. Data based on three independent experiments. D, Immunofluorescence of SOX17 protein on uterine sections of ovariectomized mice treated with vehicle or P4 for 6 h with SOX17 Ab (AF1924; R&D Systems). E, In vitro analysis of PR-interval region within the Sox17 gene cloned into the pGL4.24 minimal promoter construct cotransfected with PRA and PRB expression vectors (pcDNA3) treated with R5020 for 24 h in the Hec-1a cell line. Results obtained from triplicate experiments. Error bars represent sem. ***, P < 0.001.

Fig. 5.

Analysis of in vivo Sox17 binding by ChIP-on-chip. A, ChIP-on-chip Sox17-binding site counts and proximity relationships. Venn diagrams of the intersection of multiple interval files from vehicle and P4-treated PR-binding sites, and P4-treated Sox17 sites. All locations present on chromosomes 10, 13, and 14. B, Distribution of Sox17-binding locations on chromosomes 10, 13, and 14. Binding locations were analyzed using the CEAS module at Cistrome. Promoter region was defined in increments of 2500 bp up to 10 kb. When the PR-binding site is within a gene, it is further defined as within the 5′-untranslated region (UTR), 3′-UTR, coding exon, or intron. Intergenic region is defined as more than 10 kb from gene boundaries. C, Sox17 binding is enriched along the 10-kb promoter region. The percentage of ChIP regions that reside in a 10-kb region upstream of gene boundaries was calculated and compared with the genome background percentages of the same region. Detailed analysis of calculation can be found in Ref. . D, Gene counts. Venn diagrams for nonredundant genes with PR (vehicle and P4) and Sox17 (P4) binding locations within ± 10 kb. E, UCSC Genome Browser illustration of the ERα gene showing distributions of PR and Sox17 binding in P4-treated uteri. Binding of the two factors demonstrate direct overlap in two interval locations. Sequence positions and other generic UCSC annotations were removed for clarity. Purple indicates PR binding and yellow indicates Sox17 binding. F, Validation of Sox17 binding sites by ChIP-RT-qPCR on uteri isolated from ovariectomized C57 female mice treated 6 h with P4. Negative control (Untr) is a gene-deficient region. Data are represented as fold enrichment of Sox17-binding sites over that of the negative control region. G, Cotransfection of 19-kb enhancer region of Ihh placed into the pGL4.24 minimal promoter construct with PRA, PRB expression vectors (pcDNA3) and a mouse Sox17 expression vector (pcDNA6) treated with R5020 for 24 h in the Hec-1a cell line. Results are obtained from triplicate experiments. Error bars represent sem. ***, P < 0.001.