Impact of vitamin D deficiency on defective endometrial decidualization and the repressive role of VDR in the epigenomic network [CUT&Run]

The identification of the factors regulating female reproduction is critical to understanding how the environment and nutrition impact female fertility and reproductive health. Vitamin D and its cognate receptor, VDR, are recognized for their role in calcium homeostasis; however, their broader impact on female reproduction remains underexplored. Here, we demonstrate that vitamin D and VDR are involved in the hormonal induction of uterine stromal cell differentiation, known as decidualization. Mice fed a vitamin D-deficient diet displayed an impaired hormonally induced decidual response. In a human endometrial stromal cell line, T-HESC, VDR decreases during decidualization. siRNA knockdown of VDR in T-HESC enhanced decidualization, while overexpression of VDR inhibited decidualization. Chromatin accessibility and histone modification analyses revealed that VDR functions as a chromatin regulator, restricting accessibility and maintaining transcriptional repression in specific genomic regions. Transcriptomic analyses confirmed that VDR broadly modulates gene expression, with most ligand-mediated effects dependent on its presence. These findings identify VDR as a key regulator of transcriptional and chromatin landscapes in endometrial stromal cells, offering novel insights into vitamin D signaling in reproduction. This study highlights the potential of targeting vitamin D pathways to treat uterine disorders associated with impaired decidualization. Overall design: Experimental Design: 2 Different Experiments Experiment 1. Identification of cistromic changes in human endometrial stromal cells by the receptor (VDR) and the ligand (1,25(OH)2D3). There are 8 groups, and 24 samples in total. All libraries were prepared from the human endometrial stromal cell line (T-HESC). 1) Control lentiviral GFP vector transduced T-HESCs treated with vehicle and precipitated with VDR antibody, n=3 2) Control lentiviral GFP vector transduced T-HESCs treated with vehicle and precipitated with IgG, n=3 3) Control lentiviral GFP vector transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with VDR antibody, n=3 4) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with IgG, n=3 5) Lentiviral VDR plasmid transduced T-HESCs treated with vehicle and precipitated with VDR antibody, n=3 6) Lentiviral VDR plasmid transduced T-HESCs treated with vehicle and precipitated with IgG, n=3 7) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with VDR antibody, n=3 8) Lentiviral VDR plasmid transduced T-HESCs treated with 1,25(OH)2D3 and precipitated with IgG, n=3 Experiment 2. Impact of VDR and 1,25(OH)2D3 on histone modifications There are 8 groups, and 24 samples in total. All libraries were prepared from the human endometrial stromal cell line (T-HESC). 1) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K4me3, n=3 2) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K27me3, n=3 3) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with H3K27ac, n=3 4) Non-targeting pooled siRNA-transfected T-HESCs and precipitated with IgG, n=3 5) VDR siRNA-transfected T-HESCs and precipitated with H3K4me3, n=3 6) VDR siRNA-transfected T-HESCs and precipitated with H3K27me3, n=3 7) VDR siRNA-transfected T-HESCs and precipitated with H3K27ac, n=3 8) VDR siRNA-transfected T-HESCs and precipitated with IgG, n=3 Data Analysis Strategy: Dr. Austin Bell-Hensley, Dr. Tianyuan Wang, and Dr. Abdull Massri from the informatics group supported Dr. MyeongJin Yi with the analysis. 1) Remove adaptor 2) QC with trim-galore 3) Map the pair-end reads 4) Count reads mapped to chrM 5) Remove duplicated reads 6) Remove unmapped reads 7) Remove reads mapped to chrM 8) Prepare SAM file based on trimmed 9bp reads 9) Random sampling to the same depth 10) Compare between different groups