(186) Identification of Underlying Repressive Role of VDR in Epigenomic Network in Endometrial Decidualization

MyeongJin Yi¹; Tianyuan Wang²; Abdull J. Massri²; Anne Marie Z. Jukic³; Francesco J. DeMayo¹
1. Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health Sciences, NIH, RTP, NC, USA

2. Biostatistics and Computational Biology Branch, National Institute of Environmental Health Sciences, NIH, RTP, NC, USA

3. Epidemiology Branch, National Institute of Environmental Health Sciences, NIH, RTP, NC, USA

Abstract Text:

Epidemiologic studies link vitamin D levels to menstrual cycles and fertility outcomes, with vitamin D signaling mediated by 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) via the vitamin D receptor (VDR). Preliminary findings showed impaired decidualization in mice on a vitamin D-deficient diet. In human endometrial stromal cells (THESCs), VDR knockdown and 1,25(OH)₂D₃ treatment increased PRL and IGFBP1 expression. We hypothesize that vitamin D deficiency impairs uterine decidualization, while VDR acts as a suppressor of stromal cell differentiation, therefore, this study investigates molecular pathways influenced by 1,25(OH)₂D₃ and VDR in endometrial stromal cells. RNA-seq was conducted on THESCs transfected with non-targeting (siNT) or VDR siRNA (siVDR), followed by treatment with 2 nM 1,25(OH)₂D₃ or vehicle. Differentially expressed genes (DEGs) were analyzed using Ingenuity Pathway Analysis (IPA). Chromatin accessibility and histone modifications (H3K4me3, H3K27ac, and H3K27me3) were assessed via ATAC-seq and CUT&RUN in THESCs transfected with siNT or siVDR. Additionally, VDR activation was analyzed using lentiviral transduction and 1,25(OH)₂D₃ treatment. RNA-seq revealed 626 ligand-regulated and 1,499 receptor-regulated DEGs (FDR-adjusted p-value < 0.05, fold change ≥ |1.4|). VDR knockdown regulated 56 genes in the presence of 1,25(OH)₂D₃, demonstrating receptor-dependent gene regulation. ATAC-seq analysis indicated increased chromatin accessibility following VDR knockdown. Control cells (siNT) exhibited 45,341 open chromatin peaks, whereas knockdown cells (siVDR) had 73,340 peaks, revealing a significant increase in accessible regions. Of the total 79,099 peaks observed, 39,382 peaks were shared between siNT and siVDR, representing 87.3% of siNT peaks and 53.97% of siVDR peaks. Peak intensity was also higher in siVDR cells, underscoring VDR’s role as a chromatin regulator that limits accessibility. Histone modification analysis using CUT&RUN revealed significant changes in open chromatin regions following VDR knockdown. Active histone marks, H3K4me3 and H3K27ac, were increased in siVDR cells, indicating enhanced transcriptional activity, while the repressive mark H3K27me3 decreased, suggesting reduced transcriptional repression. These results highlight the role of VDR in chromatin regulation, as its depletion enhances chromatin accessibility and shifts histone modification dynamics to a more transcriptionally active state. Taken together, these findings suggest that vitamin D deficiency adversely affects mouse decidualization and stromal cell differentiation demonstrating that vitamin D signaling, which is mediated by VDR, modulates endometrial stromal cell differentiation by regulating chromatin accessibility and transcriptional activity. Vitamin D deficiency disrupts these processes, potentially contributing to impaired reproductive performance, thus, understanding the mechanisms provides insights into the role of vitamin D in reproductive health and offers avenues for therapeutic intervention.