{"paper_id":"1736890d-32a7-4baa-8917-7c1521a931b1","body_text":"1 \n \nIncreased chromatin accessibility following 1α,25-dihydroxyvitamin D3 treatment in human \nendometrial stromal cells \n \nMyeongJin Yi a, Hamed Bostan b, and Francesco J. DeMayo a,* \na Reproductive and Developmental Biology Laboratory, National Institute of Environmental Health \nSciences, National Institutes of Health, Research Triangle Park, North Carolina, 27709, United States \nb Integrative Bioinformatics Supportive Group, National Institute of Environmental Health Sciences, \nNational Institutes of Health, Research Triangle Park, North Carolina, 27709, United States \n \nAuthor ORCIDs \nMyeongJin Yi https://orcid.org/0000-0002-6561-3620; Hamed Bostan https://orcid.org/0000-0003-4102-\n807X; Francesco J. DeMayo https://orcid.org/0000-0002-9480-7336  \n \n*Corresponding authors \nCorrespondence to: National Institute of Environmental Health Sciences, National Institutes of Health, \nResearch Triangle Park, North Carolina, 27709, United States.  \nE-mail addresses: francesco.demayo@nih.gov (F.J. DeMayo)  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n2 \n \nHighlights \n• First evidence suggesting the direct impact of active vitamin D, 1α,25-dihydroxyvitamin D3, 1,25(OH)2D3, \nenhanced the signal intensity of chromatin accessibility in human endometrial stromal cells \n• Most accessible chromatin regions were shared between vehicle and ligand-treated human endometrial \nstromal cells \n• 1,25(OH)2D3-responsive transcription occurs largely within pre -accessible chromatin in human \nendometrial stromal cells \n• Assay for transposase-accessible chromatin sequencing (ATAC -seq) defines a chromatin -level \npharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n3 \n \nAbstract \nVitamin D signaling has recognized roles in female reproductive physiology, but its effects at the \nchromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form \nof vitamin D, 1α,25 -dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of \nhuman endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-\nseq) was performed on T -HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment \nincreased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only \nminor changes in the total number of called peaks. Peak annotation revealed that accessible regions \nwere spread across both promoter -proximal and distal genomic areas. Integrating this data with \nCUT&RUN and RNA sequencing showed that most vitamin D -responsive cistromic modifications and \ntranscripts were linked to nearby open chromatin, though fewer were associated with regions that wer e \nsignificantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription \nmainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence \nthat active vitamin D influences the epigenomic landscape of human endometrial stromal cells, \nestablishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, \nrelevant to stromal differentiation and preparation for decidualization. \n \nKeywords \nVitamin D, calcitriol, chromatin accessibility, endometrial stromal cells, assay for transposase-accessible \nchromatin using sequencing (ATAC-seq) \n  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n4 \n \n1. Introduction \nVitamin D is known for its role in calcium homeostasis and skeletal health, but accumulating evidence \nindicates that it also contributes to reproductive physiology. In female reproductive tissues, vitamin D \nsignaling has been implicated in endometrial receptivity, implantation, and decidual function  (Yi et al., \n2026), suggesting that this pathway influences the cellular environment required for pregnancy. Among \nits biologically active forms, 1α,25-dihydroxyvitamin D3, 1,25(OH)2D3, acts through the vitamin D receptor \n(VDR) (Haussler et al., 1968; Holick et al., 1971; Lawson et al., 1971), a ligand-activated nuclear receptor \nthat regulates transcriptional programs involved in differentiation, inflammation, and hormone-responsive \ncellular processes (Haussler and Norman, 1969). As a chemically defined bioactive vitamin D metabolite \nand pharmacologic VDR ligand, 1,25(OH)2D3 provides an important model for understanding how nuclear \nreceptor activation translates chemical exposure into molecular and cellular responses. Despite \nincreasing interest in vitamin D action in the uterus, the chromatin -level effects of ligand exposure in \nhuman endometrial stromal cells remain insufficiently defined. \n \nEndometrial stromal cells are highly dynamic and undergo functional reprogramming in response to \nsteroid hormones and local signaling cues  (Dunn et al. , 2003) . This plasticity requires coordinated \nregulation of gene expression, which is strongly shaped by chromatin state (Vrljicak et al., 2018; Vrljicak \net al., 2023). Chromatin accessibility determines whether transcription factors and cofactors can engage \nregulatory elements such as promoters and enhancers (Thurman et al., 2012; Eustermann et al., 2024). \nTherefore, defining accessible chromatin landscapes provides mechanistic insight into how hormonal \nand metabolic signals are translated into transcriptional outputs. In the endometrium, where stromal cells \nundergo cyclical changes in proliferation, differentiation, and immune adaptation, chromatin accessibility \nis especially relevant for understanding how regulatory programs are established and modified (Gellersen \nand Brosens, 2014; Vrljicak et al., 2018; Vrljicak et al., 2023). \n \nAssay for transposase-accessible chromatin using sequencing (ATAC -seq) is a powerful approach for \ninterrogating genome-wide chromatin accessibility using relatively limited input material  (Buenrostro et \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n5 \n \nal., 2015; Miskimen  et al. , 2017) . Compared with transcriptomic analysis alone, ATAC -seq provides \ninformation on the regulatory architecture that permits, constrains, or primes gene expression responses. \nThis is particularly important in hormonally responsive cells, where transcriptional changes arise either \nthrough broad chromatin remodeling or through selective use of regulatory elements that are accessible \n(Wiench et al., 2011; Grontved and Hager, 2012; Hoffman et al., 2022; Gomez Acuna et al., 2024). From \na pharmacologic perspective, defining chromatin accessibility provides a molecular mechanism for \nunderstanding how ligand exposure alters regulatory competence before or alongside measurable \ntranscriptional output. Thus, ATAC-seq is well-suited to determine whether ligand treatment induces \naccessibility changes or instead acts within a pre-existing permissive chromatin landscape. \n \nT-HESCs, an immortalized human endometrial stromal cell line, provide a useful model for investigating \nhormone-responsive signaling and chromatin regulation in the stromal compartment (Krikun et al., 2004a; \nKrikun et al., 2004b; Li et al., 2022). Because stromal cell differentiation is central to endometrial function \nand preparation for decidualization (Li et al., 2022), this system offers a biologically relevant context for \nexamining how vitamin D influences the epigenomic architecture of uterine biology. Although previous \nstudies have described transcriptional and functional effects of vitamin D in reproductive tissues, much \nless is known about whether 1,25(OH) 2D3 alters chromatin accessibility in human endometrial stromal \ncells and how such changes relate to downstream gene expression. \n \nIn the present study, we used ATAC -seq to define the chromatin accessibility landscape of T -HESCs \nfollowing treatment with 1,25(OH) 2D3. We asked whether ligand exposure broadly remodels accessible \nchromatin or instead reinforces accessibility within an open regulatory environment. We further integrated \nchromatin accessibility profiles with transcriptomic data to assess the relationship b etween open \nchromatin regions and vitamin D -responsive genes. By focusing on the epigenomic consequences of \nexposure to a chemically defined VDR ligand, this study provides a concise pharmacologic scheme for \nunderstanding how active vitamin D influences regulatory architecture in human endometrial stromal cells. \n  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n6 \n \n2. Materials and methods \n2.1. Materials and software \nhTERT-immortalized endometrial stromal cells (T -HESCs) were purchased from the American Type \nCulture Collection (ATCC;  Cat. CRL -4003, VA, USA). Dulbecco's Modified Eagle Medium/Nutrient \nMixture F-12 (DMEM/F12; Cat. 11330032), fetal bovine serum (FBS; Cat. 10082147), trypsin-EDTA (Cat. \n25200056), optimized minimum essential medium (Opti -MEM; Cat. 31985070), and charcoal st ripped \nFBS (Cat. 12676029) were purchased from Gibco (MA, USA). 1α,25-dihydroxy vitamin D3 (1,25(OH)2D3, \ncalcitriol; Cat. 71820) was manufactured by Cayman (MI, USA). Penicillin-streptomycin was from Sigma-\nAldrich (Cat. P0781, MO, USA). Qubit double -stranded (ds) DNA high sensitivity (HS) assay kit (Cat. \nQ32854), Qubit RNA high sensitivity (HS) assay kit (Cat. Q32852), TRIzol (Cat. 15596018), and \nUltraPure DEPC-treated water (Cat. 750023) were purchased from Invitrogen (CA, USA). Tagment DNA \nTDE1 Enzyme and Buffer Kits (Cat . 20034198; Illumina, USA) were used to prepare the ATAC -seq \nlibraries. MinElute PCR purification kit was from Qiagen (Cat. 28004; Germany).  Ingenuity Pathway \nAnalysis was developed by Qiagen (CA, USA). RStudio-2025.05.0-496 and R 4.5.0 were from Posit PBC \n(MA, USA). The Integrative Genomics Viewer (IGV; University of California and Broad Institute, \nhttps://igv.org/app/) was used to load the peak track and visualize the signal.  \n \n2.2. Cell culture of human telomerase reverse transcriptase ( hTERT)-immortalized human \nendometrial stromal cell line (T-HESC) and exposure of 1,25(OH)2D3 in vitro assays \nThe cells were cultured and maintained in Dulbecco’s Modified Eagle Medium /Nutrient Mixture F -12 \n(DMEM/F12), which was supplemented with 10% (v/v) fetal bovine serum (FBS), 1 mM sodium pyruvate, \n100 units/mL penicillin, and 100 µg/mL streptomycin at 37˚C , containing 5% CO 2 in a humidified \natmosphere (Michalski et al., 2018; Li et al., 2022; Maurya et al., 2022; Montague Redecke et al., 2025; \nMontague Redecke et al., 2026; Yi et al., 2026). To evaluate the impact of ligand, T-HESCs were treated \nwith 2 nM 1,25(OH)2D3. The ligand was diluted in Opti -MEM supplemented with 2% (v/v) charcoal -\nstripped FBS, 1 mM sodium pyruvate, 100 units/mL penicillin, and 100 µg/mL streptomycin to minimize \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n7 \n \nbackground hormonal effects from standard FBS. Vehicle -treated cells were prepared under the same \nculture conditions and used as controls for comparison with 1,25(OH)2D3-treated cells. \n \n2.3. ATAC-seq library preparation \nATAC-seq libraries were prepared as previously described with minor modifications  (Buenrostro et al., \n2013; Buenrostro et al., 2015). Three technical replicates were generated for each treatment condition, \nresulting in a total of six samples. Cells were collected and washed with cold PBS, followed by lysis in \nGreenleaf buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl₂, and 0.1% IGEPAL CA-630). Nuclei were \nisolated by incubation on ice for 5 min and centrifugation at 500 × g for 5 min at 4 °C. Nuclei were counted \nusing a hemocytometer, and 50,000 nuclei per sample were used for transposition. For the transposition \nreaction, nuclei were resuspended in 50 µL of transposase reaction mix containing 25 µL TD buffer, 2.5 \nµL Tn5 transposase (Illumina Nextera DNA Library Preparation Kit), and 22.5 µL nuclease -free water. \nSamples were incubated at 37 °C for 15 min. Following tran sposition, DNA was purified using the \nMinElute PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. Purified DNA was \namplified by PCR using 10 µL of eluted DNA, indexed adapters, and PCR master mix. Libraries were \namplified for a tot al of 15 cycles. Amplified libraries were purified using 150 µL SPRI beads (Beckman \nCoulter). Beads were incubated at room temperature for 15 min, followed by magnetic separation for 5 \nmin. The supernatant was removed, and the beads were washed twice with 80% ethanol. After air-drying, \nDNA was eluted in 50 µL elution buffer (10 mM Tris -HCl, pH 8.0). Final libraries were quantified and \nprepared for high-throughput sequencing according to standard protocols. \n \n2.4. Evaluation of chromatin accessibility by 1,25(OH)2D3 in T-HESC using ATAC-seq \nSequencing was performed on a NovaSeq (Illumina). Raw reads (50 bp, paired -end) processed by \nadaptor trimming and quality filtering using Trim Galore version 6.7, with reads retained when the average \nquality score was greater than 20. Filtered reads were aligned to the human reference genome hg38 \nusing Bowtie2 with unique mapping and up to two mismatches permitted for each read (-m 1 -v 2). Reads \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n8 \n \nmapped to mitochondrial DNA and duplicated reads were removed before downstream analysis. \nUniquely mapped, non-duplicated reads from each sample were normalized by down -sampling to 100 \nmillion reads. The first 9 bp of each read were used for downstream accessibility analysis. \nOpen chromatin regions were identified using MACS 3 with a Q -value cutoff of 0.0001, followed by \nmerging genomic intervals within 100 bp of each other (Zhang et al., 2008; Liu, 2014). Peaks identified \nfrom vehicle- and 1,25(OH)2D3-treated samples were used to evaluate global chromatin accessibility \npatterns, treatment -associated changes in ATAC -seq signal intensity, and differentially accessible \nregions (DARs). DARs were defined by comparing the normalized ATAC-seq signal between vehicle - \nand 1,25(OH)2D3-treated cells across merged peak intervals. (Zhang et al., 2008; Liu, 2014). \n \n2.5. Annotation of the nearest gene and motif analysis from ATAC-seq called peaks \nThe gene associated with each ATAC -seq peak was predicted by searching for transcription start sites \n(TSSs) of nearby genes within a 100 kb range using the “annotatePeaks.pl function ” of the \nHypergeometric Optimization of Motif Enrichment (HOMER) motif discovery tool (Heinz et al., 2010; \nRoberson, 2018) . Peak locations were annotated relative to genomic features, including promoter -\nproximal regions, untranslated regions, exons, introns, and intergenic regions.  Promoter-associated \npeaks were defined based on their proximity to the TSS. HOMER’s findMotifsGenome.pl function was \nused to perform motif enrichment analysis for selected peak sets (Genomes Project et al., 2012; Khurana \net al., 2013; Tuoresmaki et al., 2014; Wen et al., 2014) \nFor genomic feature classes, we assigned using transcript annotation and prioritized as promoter, 5′UTR, \n3′UTR, exon, intron, or intergenic region, with promoter defined as within 2 kb of the nearest TSS.  \nReference gene annotation files were generated using the hg38 reference genome annotation. The \nhg38.refGene.gtf file was used for genomic feature annotation  and downloaded from UCSC Genome \nBrowser. “ genePredToGtf -utr hg38 refGene hg38.RefGene.gtf” was used to generate transcript \nannotation files containing untranslated region information for downstream peak annotation. \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n9 \n \n2.6. Overlay of analyses of chromatin accessible peaks with cistromic and transcriptomic \ndatasets  \nTo compare accessible chromatin regions between vehicle - and 1,25(OH)2D3-treated T-HESCs, direct \noverlap analyses were performed using summit-centered ATAC-seq peak coordinates. Peak summit files \nfor each condition were represented as 1 bp genomic positions, and each summit was extended by ±100 \nbp to generate fixed 200 bp wind ows centered on the peak summit. Overlap between vehicle and \n1,25(OH)2D3 ATAC-seq peak sets was then assessed based on genomic intersection between these \nsummit-centered windows. Peaks  were classified as shared if a summit -centered window from one \ncondition overlapped a summit-centered window from the other condition, and as condition-specific if no \noverlap was detected. \nTo assess concordance between accessible chromatin and ligand-associated cistromic regions, overlap \nanalyses were also performed between 1,25(OH)2D3 ATAC-seq summit-centered windows and publicly \navailable 1,25(OH)2D3 CUT&RUN peak intervals (GSE306127). ATAC -seq summits were extended by \n±100 bp and intersected with CUT&RUN peak coordinates in hg38. Overlap was summarized as the \nnumber and proportion of ATAC summit windows that intersected CUT&RUN peaks, the number and \nproportion of CUT&RUN peaks that interse cted ATAC summit windows, and the total number of \noverlapping peak pairs. Overlapping CUT&RUN peaks were further annotated to nearby transcripts and \ngenes using the same nearest -TSS framework described above. Because these analyses were based \non genomic pr oximity and coordinate intersection, overlap between datasets was interpreted as \ncolocalization or association rather than direct evidence of regulatory interaction. \nTo relate accessible chromatin regions to transcriptomic responses, summit -centered ATAC-seq peaks \nor merged peak sets were annotated to nearby genes using the hg38 reference transcript annotation. \nThe nearest transcription start site (TSS) was assigned to each peak, and peak-associated genes were \ncompared with the list of differentially expressed genes (DEGs) identified from publicly available RNA-\nseq analysis of 1,25(OH)2D3-treated versus vehicle -treated T -HESCs (GSE254251). Peak -to-gene \nrelationships were summarized both at the peak level and as collapsed lists of unique genes.  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n10 \n \nAll coordinate parsing, overlap classification, nearest -transcript assignment, and summary table \ngeneration were performed computationally in Python  3.13. All transcriptomic regulators and pathways \nwere analyzed bioinformatically in Qiagen IPA. \n \n2.7. Validation of differentially accessible regions in open chromatin by qPCR \nTotal RNAs from T -HESCs were isolated using TRIzol reagent according to the manufacturer’s \ninstructions. All isolated RNAs were quantified and certified using a Qubit RNA High Sensitivity kit \n(Invitrogen). Each cDNA was synthesized with a High-Capacity cDNA Reverse Transcription kit (Applied \nBiosystems), adding 2 µg of total RNA to the reaction mixture, and the reaction mixtures were incubated \nat room temperature for 10 min followed by additional incubation at 37˚C for 2 hr. Each synthesized cDNA \nwas quantified and certified using a Qubit dsDNA High Sensitivity kit (Invitrogen). The primers to amplify \nthe enhancer region of GPAT3  (LOC112997542) (Barakat et al. , 2018) ; forward 5’ -\nGGGTCTTCAATAAACAGCAG-3’, reverse 5’ -GTCACTGAGAACGACGTCTG-3’, and the enhancer \nregions of MAMDC2 (LOC127814909) (Barakat et al., 2018); forward 5’-GAATGGAATCAACTCGAGAG-\n3’, reverse 5’ -GGAAGTCACGTAAATGAATG-3’, respectively. qPCR was performed with the CFX96 \nReal-Time PCR Detection System (Bio-Rad). Each value was derived from the comparative CT method, \nwhich compared the Ct value of one target gene to a reference gene using the 2 -ΔΔCt formula according \nto the manufacturer’s guidelines. ΔCt indicates the differences in threshold cycles for target and reference \n(Ct, target – Ct, reference), and ΔΔC t represents the relative change in these differences between the target \nand reference (ΔC t, target – ΔCt, reference). Therefore, the expression of the target, normalized to a \nhousekeeping gene, was given by 2-ΔΔCt and adjusted as a fold-change. \n \n2.8. Statistical analysis \nAll quantitative data were first tested for normality. When the normality assumption was met, comparisons \namong multiple groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons \ntest, and comparisons between two groups were perfor med using Student’s t -test. For non -normally \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n11 \n \ndistributed data, comparisons among multiple groups were performed using the Kruskal -Wallis test \nfollowed by Dunn’s multiple comparisons test, and comparisons between two groups were performed \nusing the Mann-Whitney test. Statistical significance was defined as p < 0.05 unless otherwise indicated. \nFor sequencing-based analyses, normalized read counts and peak -associated signal intensities were \nused to evaluate differences between vehicle- and 1,25(OH)2D3-treated groups. Differential accessibility \nand transcriptomic overlap analyses were interpreted using adjusted statistical thresholds where \napplicable. Data were summarized as counts, percentages, or normalized signal intensity values, \ndepending on the analysis. \n \n  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n12 \n \n3. Results \n3.1. 1,25(OH)2D3 enhances chromatin accessibility in T-HESCs \nTo determine whether active vitamin D alters chromatin accessibility in human endometrial stromal cells, \nATAC-seq was performed in T -HESCs treated with vehicle or 1,25(OH)2D3. Genome-wide ATAC-seq \nprofiles revealed that ligand treatment enhanced chromatin accessibility, as reflected by increased ATAC-\nseq signal intensity across accessible regions in the maximum signal intensity at the midpoint of identified \npeaks (Fig. 1A) and open regions surrounding the transcriptional start sites (TSS)  (Fig. 1B). Although \nligand treatment enhanced ATAC -seq signal intensity, it did not substantially alter the overall peak \nlandscape. Direct summit-centered overlap analysis revealed that most accessible regions were shared \nbetween vehicle- and 1,25(OH)2D3-treated cells, with 104,503 shared summits, corresponding to 80.85% \nof vehicle peaks and 90.15% of 1,25(OH)2D3 peaks (Fig. 1C and Supplementary Table 1). These findings \nindicate that 1,25(OH)2D3 primarily strengthens the accessibility of regulatory regions rather than inducing \nwidespread chromatin opening. This result suggests that 1,25(OH)2D3 acts primarily within a pre-existing \naccessible chromatin landscape in T -HESCs, increasing the magnitude of accessibility at regulatory \nregions that are open rather than inducing widespread chromatin opening. \n \n3.2. 1,25(OH)2D3 preserves a largely shared accessible chromatin landscape \nAlthough 1,25(OH)2D3 did not broadly increase the total number of accessible regions, comparison of \ncalled peaks between vehicle- and ligand-treated cells revealed condition-associated differences at the \nlevel of individual genomic intervals (Fig. 1D). Vehicle-treated cells contained 24,743 summit regions not \noverlapping the 1,25(OH)2D3 peak set, whereas 1,25(OH)2D3-treated cells contained 11,413 summits not \noverlapping the vehicle peak set (Fig. 1C and Supplementary Table 1) . Thus, ligand treatment was \nassociated with selecti ve differences in peak representation, but these changes occurred within the \ncontext of a largely conserved accessible chromatin.  These findings suggest that 1,25(OH)2D3 \npredominantly strengthens accessibility across a shared set of regulatory regions while introducing a \nmore limited number of condition -associated accessible loci. This pattern supports a model in which \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n13 \n \nligand exposure modulates a permissive chromatin environment rather than establishing an entirely \ndistinct accessibility architecture. \n \n3.3. Distribution of differentially accessible regions (DARs) following ligand treatment \nTo further evaluate heterogeneity within ligand -responsive accessible regions, DARs were clustered \naccording to ATAC-seq signal patterns across treatment conditions. Clustered heatmaps and average \nsignal profiles revealed distinct groups of regions with treatment -associated accessibility changes (Fig. \n2A). Notably, clusters showing higher signal in 1,25(OH)2D3-treated cells displayed stronger accessibility \nintensity compared with the corresponding vehicle condition, supporting the interpretation that ligand \nexposure enhances accessibility at selected chromatin regions. Together, these analyses indicate that \n1,25(OH)2D3 produces a distinct set of DARs, with the most prominent effect reflected by increased signal \nintensity at a subset of ligand-responsive regions rather than by widespread gain of new accessible peaks. \nTo identify genomic regions with statistically significant ligand -dependent changes in accessibility, \ndifferential accessibility analysis was performed between vehicle - and 1,25(OH)2D3-treated T-HESCs. \nUsing an adjusted significance threshold and fold -change cutoff, we identified a set of DARs (Fig. 2B). \nAmong these regions, 110 peaks showed increased accessibility following 1,25(OH)2D3 treatment, \nwhereas 369 peaks showed reduced accessibility (Fig. 2B). Thus, the number of regions classified as \ndecreased DARs was greater than the number of increased DARs. Annotation of DARs across genomic \nfeatures showed that ligand-responsive accessibility changes were distributed across promoter-proximal \nand distal regulatory regions, including intronic and intergenic intervals (Fig. 2 C). Increased DARs \nshowed a relatively higher proportion of promoter -associated regions compared with decreased DARs. \nIntergenic regions represented the largest fraction of both DAR classes. This distribution suggests that \n1,25(OH)2D3-responsive chromatin regulation involves both promoter -proximal and distal regulatory \nelements. \nBecause DAR counts alone do not fully capture the magnitude of accessibility changes, we next \nexamined ATAC-seq signal intensity separately across increased and decreased DARs. Regions with \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n14 \n \nincreased accessibility showed a marked elevation of ATAC -seq signal in 1,25(OH)2D3-treated cells \ncompared with vehicle -treated cells, as shown by both the average signal profile and heatmap \nvisualization (Fig. 2D). This result indicates that, although increased DARs were fewer in number, they \nrepresented loci with clear ligand -enhanced accessibility. In contrast, regions classified as decreased \nDARs showed comparatively modest differences between treatment groups in both metaplot and \nheatmap analyses (Fig. 2E). These findings suggest that the accessibility gain induced by 1,25(OH)2D3 \nis concentrated at a subset of regulatory regions but is more pronounced in signal magnitude. \nOverall, although increased DARs were fewer in number than decreased DARs, they showed a more \npronounced gain in ATAC -seq signal intensity, indicating that 1,25(OH)2D3 enhances accessibility at a \nstrongly responsive subset of regulatory regions. These results reinforce the model that active vitamin D \nmodulates the T -HESC chromatin landscape primarily through quantitative strengthening of selected \naccessible regions, while the broader chromatin architecture remains largely pre-accessible. \n \n3.4. Accessible chromatin regions overlap a subset of 1,25(OH)2D3-associated cistromic changes \nTo examine the relationship between accessible chromatin and ligand -associated cistromic changes, \nATAC-seq summit-centered windows from 1,25(OH)2D3-treated T-HESCs were compared with publicly \navailable 1,25(OH)2D3 CUT&RUN peak intervals (GSE306127). A total of 6,092 1,25(OH)2D3-associated \nCUT&RUN peaks from GSE306127 were included in the overlap analysis. Among these, 3,630 peaks \noverlapped accessible chromatin regions, representing 59.59% of the 1,25(OH)2D3-associated cistromic \npeaks (Fig. 3A  and Supplementary Table 2). These findings indicate that a substantial proportion of \nligand-associated cistromic changes occurs within regions of open chromatin. \nTo further characterize these shared regions, de novo motif analysis was performed using HOMER on \nthe 3,630 overlapping peaks (Fig. 3B and Supplementary Table 2). The most significantly enriched motif \ncorresponded to Fra1 (p-value = 1e-163; 17.30%), followed by motifs for VDR, TEAD2, TEAD4, NR1I3 \n(constitutive androstane receptor; CAR), FOXJ3, TEAD3, PU.1, NFκB, SP5, RUNX1, VDR (DR3), and \nZNF692 (Fig. 3B and Supplementary Table 2). The enrichment of VDR-related motifs supports the ligand \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n15 \n \nresponsiveness of these shared regions, whereas the presence of additional transcription factor motifs \nsuggests that 1,25(OH)2D3-associated cistromic regulation may occur in combination with other \nregulatory factors. Together, these results show that a subset of 1,25(OH)2D3-associated cistromic \nchanges is localized within accessible chromatin regions and is enriched for motifs linked to ligand -\nresponsive transcriptional regulation. \n \n3.5. Accessible chromatin regions converge on a shared set of annotated genes \nTo assess whether ligand -associated differences in accessible chromatin altered the gene repertoire \nlinked to open regulatory regions, ATAC-seq peaks were annotated to their nearest genes within 100 kb \nof the transcription start site (Supplementary Table 3). A total of 19,789 and 19,114 genes were \nassociated with open chromatin regions in vehicle- and 1,25(OH)2D3-treated T-HESCs, respectively (Fig. \n3C). The majority of annotated genes were shared between conditions, with 18,322 genes common to \nboth datasets. These shared genes represented 92.62% of vehicle -associated genes and 95.86% of \n1,25(OH)2D3-associated genes.  These findings indicate that vehicle - and 1,25(OH)2D3-treated cells \nmaintain a broadly similar open chromatin -associated gene repertoire, even though accessibility differs \nat selected genomic loci. Thus, ligand -dependent chromatin regulation appears to occur within an \nestablished stromal regulation rather than through wholesale reorganization of the genes associated with \naccessible chromatin. \n \n3.6. Vitamin D -responsive transcripts are primarily associated with pre -existing accessible \nchromatin \nTo determine how chromatin accessibility relates to transcriptional responses to 1,25(OH)2D3, ATAC-seq \npeak-associated genes were integrated with RNA -seq data (GSE254251) generated from vehicle- and \nligand-treated T -HESCs (Fig. 3 D). Among the 626 differentially expressed genes (DEGs), 540 were \nassociated with accessible chromatin regions in vehicle-treated cells, whereas 530 were associated with \naccessible chromatin regions in 1,25(OH)2D3-treated cells (Fig. 3D). A total of 521 DEGs were linked to \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n16 \n \naccessible chromatin in both conditions, indicating that most ligand -responsive transcripts were \nassociated with regulatory regions embedded within a shared accessible chromatin landscape \n(Supplementary Table 3). These findings suggest that transcriptional  responses to 1,25(OH)2D3 occur \npredominantly within a permissive chromatin environment that is already accessible before ligand \nexposure, rather than through widespread establishment of newly accessible regions. Consistent with \nthis interpretation, only a small subset of DEGs showed condition -specific associations with accessible \nchromatin regions. Specifically, 19 genes: COL10A1, CSF2RB, FABP4, FCMR, GLYAT, HCN2, KCNS2, \nLIF, LINC01081, LINC01123, MEST, MKX, PLEKHA7, PURG, RASSF2, STX1B, TGFB2, TNFRSF1B, \nand TRIM58 were uniquely associated with vehicle -specific accessible regions, whereas 9 genes: \nARHGAP9, CCDC121, CYP3A5, CYTH4, NECAB1, NSDHL, PRRT4, SHANK1, and TUBE1 were \nuniquely associated with 1,25(OH)2D3-specific accessible regions (Supplementary Tabl e 3). Thus, \ncondition-specific chromatin associations accounted for only a minor fraction of ligand -responsive \ntranscripts. \nTo further characterize the 1,25(OH)2D3-responsive transcripts associated with accessible chromatin, the \n530 genes linked to open chromatin regions in 1,25(OH)2D3-treated cells were subjected to Ingenuity \nPathway Analysis (IPA) (Fig. 3E and Supplementary Table 3). In causal network analysis, VDR was \nidentified as the most significant predicted activated master regulator. Other highly ranked activating \ncausal regulators included astemizole, calcitriol (1,25(OH)2D3), GTF2B, RXR/VDR/vitamin D 3 complex, \nand ETV4 (Fig. 3E and Supplementary Table 3). In upstream regulator analysis, calcitriol, KAT5, KAT2A, \nBMP10, and budesonide were among the most strongly predicted activating regulators (Fig. 3E and \nSupplementary Table 3). Canonical pathway analysis showed enrichment of transcriptional and immune-\nrelated pathways, along with Molecular Mechanisms of Cancer, Osteoarthritis Pathway, Idiopathic \nPulmonary Fibrosis Signaling Pathway, RAR Activation, Role of Macrophages, Fibroblasts and \nEndothelial Cells in Rheumatoid Arthritis, VDR/RXR Activation, Tumor Microenvironment Pathway, Role \nof Osteoblasts in Rheumatoid Arthritis Signaling Pathway, RHO GTPase Cycle, and Myocardin Signaling \nPathway (Fig. 3E and Supplementary Table 3). These analyses indicate that transcripts associated with \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n17 \n \naccessible chromatin in ligand-treated cells are enriched for regulatory networks centered on VDR and \nrelated transcriptional programs. \nOverall, these findings indicate that 1,25(OH)2D3-responsive transcription in T -HESCs is largely \nassociated with an established accessible chromatin framework. Rather than being accompanied by \nextensive generation of new open chromatin regions, ligand treatment appears to act mainly within pre-\nexisting accessible regulatory regions linked to vitamin D-responsive gene expression. \n \n3.7. Representative genomic loci support selective ligand-dependent chromatin remodeling \nTo further evaluate the relationship between ligand-responsive chromatin accessibility and transcriptional \nregulation, representative loci were selected from accessible regions overlapping 1,25(OH)2D3-\nresponsive DEGs ( Fig. 4A). Aggregate ATAC -seq profiles across ligand DEG -overlapping accessible \nregions further indicated that 1,25(OH)2D3-associated gain and loss peaks displayed distinct accessibility \npatterns between treatment conditions. The GPAT3-associated accessible region was derived from the \nupregulated accessible peak set overlapping ligand -responsive DEGs and showed increased \naccessibility after 1,25(OH)2D3 treatment in the ATAC-seq analysis (Fig. 4B). By contrast, the MAMDC2-\nassociated accessib le region was derived from the downregulated accessible peak set overlapping \nligand-responsive DEGs and showed reduced accessibility following ligand treatment  (Fig. 4 B). \nQuantitative PCR analysis using RNA from vehicle- and 1,25(OH)2D3-treated T-HESCs confirmed ligand-\nassociated regulation at these representative loci (Fig. 4C).  \nCollectively, these representative loci reinforce the genome -wide conclusion that active vitamin D acts \nprimarily within a pre -existing accessible chromatin landscape in T -HESCs. Although 1,25(OH)2D3 \ninduces selective changes at specific regulatory regions, the relationship between accessibility and \ntranscription is locus-dependent and cannot always be inferred from the direction of change alone. \n  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n18 \n \n4. Discussion \nThis study provides new insight into how the active form of vitamin D, 1α,25 -dihydroxyvitamin D ₃, \n1,25(OH)2D3, modulates chromatin accessibility in human endometrial stromal cells. From a \npharmacologic perspective, 1,25(OH)2D3 is a chemically defined bioactive metabolite and ligand for VDR \n(Slatopolsky et al., 1984; Tsukamoto et al., 1991; Perez-Mijares et al., 1993), making it an appropriate \nmodel for investigating how nuclear receptor activation reshapes molecular regulatory architecture in \ntarget cells. Although vitamin D signaling has been studied in reproductive physiology, implantation -\nrelated biology, immune regulation, and decidual function (Hosseinirad et al., 2022; Rashidi et al., 2023; \nMetz et al., 2026), its direct impact on the accessible chromatin landscape of human endometrial stromal \ncells remains insufficiently defined. More broadly, chromatin accessibility itself has received relatively \nlimited attention in endometrial stromal cell biology compared with transcriptional and hormonal \nregulation, despite its importance in determining which regulatory regions are available f or signal -\ndependent gene control. By applying ATAC -seq to vehicle - and 1,25(OH)2D3-treated T -HESCs and \nintegrating these data with transcriptomic profiles, we found that ligand exposure increased chromatin \naccessibility primarily at the level of ATAC -seq signal intensity, while most open chromatin regions \nremained shared between treatment conditions. Importantly, both a substantial subset of 1,25(OH)2D3-\nassociated cistromic changes and most ligand-responsive transcripts were associated with regions within \nthis accessible chromatin landscape. These findings indicate that active vitamin D does not broadly \nestablish a new accessible chromatin landscape in this cellular context. Instead, 1,25(OH)2D3 appears to \nact largely within a pre -existing permissive chromatin environment, with selective accessibility changes \nat a smaller subset of loci. \n \nA central observation of this study is that 1,25(OH)2D3 increased the magnitude of chromatin accessibility \nwithout causing a proportional expansion in the number of accessible regions. The most prominent effect \nof ligand treatment was enhanced ATAC -seq signal across regions that were accessible, rather than \nwidespread formation of new peaks. This pattern supports a model in which active vitamin D strengthens \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n19 \n \nthe accessibility of pre -existing regulatory elements, potentially increasing their competence for \ntranscription factor engagement or regulatory activity. This finding is particularly relevant to endometrial \nstromal cells, which must remain responsive to multiple hormonal, metabolic, inflammatory, and paracrine \nsignals. Stromal cell differentiation and preparation for decidualization require extensive transc riptional \ncoordination, yet these changes likely depend on a chromatin landscape that is poised or pe rmissive \nbefore full differentiation occurs (Dunn et al., 2003; Krikun et al., 2004b; Gellersen and Brosens, 2014). \nIn this context, vitamin D signaling functions more as a regulatory input that enhances the responsiveness \nof existing chromatin architecture. Such a mechanism would allow T -HESCs to adjust gene expression \nprograms efficiently without requiring extensive chromatin reorganization  (Li et al. , 2022) . This \ninterpretation is consistent with the concept that stromal cells possess a flexible regulatory architecture \nthat can be shaped by ligand-dependent nuclear receptor signaling (Michalski et al., 2018; Vrljicak et al., \n2018). \n \nThe integration of ATAC-seq with RNA-seq further clarifies the nature of the ligand response in T-HESCs. \nMost 1,25(OH)2D3-responsive transcripts were associated with nearby accessible chromatin regions that \nwere present in vehicle -treated cells, in dicating that vitamin D -dependent transcription occurs largely \nwithin a pre-existing permissive regulatory environment. Similarly, overlap analysis with public CUT&RUN \ndata showed that a substantial proportion of 1,25(OH)2D3-associated cistromic changes also occurred \nwithin open chromatin regions. Together, these observations indicate that both the cistromic and \ntranscriptomic effects of 1,25(OH)2D3 are concentrated within chromatin regions tha t are already \naccessible. Few differentially expressed genes were linked specifically to differentially accessible regions, \nsuggesting that broad transcriptional responsiveness to ligand exposure is not primarily driven by \nwidespread creation of new access ible chromatin sites. Rather, the major contribution of 1,25(OH)2D3 \nmay be to modulate the functional output of accessible regulatory elements. Thus, chromatin accessibility \ndefines the regulatory territory available for ligand -responsive signaling, wherea s ligand exposure \ninfluences how that territory is used. When peaks were annotated to nearby genes, the majority of \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n20 \n \nassociated genes were shared between conditions.  This suggests that distinct accessible regions can \nconverge on a broadly similar gene repertoire (Salamone et al., 2025). Gene regulation is rarely controlled \nby a single regulatory element ; i nstead, promoters, enhancers, and distal regulatory regions often \nfunction as networks, with multiple elements contributing to the regulation of the same gene (Doane and \nElemento, 2017; Vitale et al., 2021; Nazarova and Sexton, 2026). Therefore, a change in the accessibility \nof individual peaks does not necessarily imply a complete change in the associated gene program. The \nhigh degree of gene-level overlap observed in this study suggests that 1,25(OH)2D3 fine-tunes regulatory \nelement usage within an established gene regulatory mechanism. This type of regulation could allow the \nsame stromal gene repertoire to respond differently depending on ligand exposure, cellular state, or \nadditional hormonal inputs. \n \nThe integration of ATAC-seq with RNA-seq further supports this interpretation. Most vitamin D-responsive \ntranscripts were associated with nearby accessible chromatin regions (Fig. 3D). Thus, active vitamin D \nappears to act within an epigenomic landscape that is permissive for regulatory engagement. This \nsuggests that the major contribution of 1,25(OH)2D3 is to increase the functional capacity or regulatory \nstrength of accessible regions that are present. Ligand-activated transcription factors often regulate gene \nexpression not only by binding to newly accessible DNA regions but also by redistributing among \naccessible regulatory elements, recruiting cofactors, altering local chromatin activity, or changing \nenhancer-promoter communication  (Liu et al. , 2014; Jin  et al. , 2025) . In such cases, chromatin \naccessibility define s the regulatory territory available to the receptor, whereas ligand exposure \ndetermines how that territory is used. Although this study did not directly measure cofactor recruitment, \nhistone modifications, or chromatin looping, the ATAC-seq patterns observed here are compatible with a \nmodel in which 1,25(OH)2D3 acts on an accessible chromatin landscape to modify transcriptional output. \nFuture integration with VDR chromatin occupancy will be important to distinguish regions that VDR \ndirectly regulates from regions that change accessibility indirectly through downstream transcriptional or \nchromatin-associated mechanisms.  These findings also have relevance for applied pharmacology \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n21 \n \nbecause they illustrate how the action of a chemically defined ligand can be evaluated beyond \nconventional transcriptional endpoints (Pike, 2011; Nguyen et al., 2026). Pharmacologic responses are \noften measured through changes in gene expression, protein abundance, or cellular phenotype; however, \nligand-induced changes in chromatin accessibility represent an earlier or parallel layer of molecular \nregulation (Chen et al., 2024). In this study, 1,25(OH)2D3 produced a prominent increase in ATAC -seq \nsignal intensity without a corresponding large increase in peak number, suggesting that the \npharmacological action of active vitamin D is expressed as a quantitative reinforcement of regulatory \ncompetence rather than broad chromatin remodeling. This distinction is important for understanding how \nbioactive metabolites and nuclear receptor ligands can modulate cellular state through changes in the \nmagnitude of regulatory element accessibility (Etchegaray and Mostoslavsky, 2016). \n \nOpen chromatin provides a permissive state, but accessibility alone is not sufficient to determine whether \na gene will be induced, repressed, or unchanged (Mansisidor and Risca, 2022). Many accessible regions \nremain functionally inactive under a given condition, while others become active only when appropriate \ntranscription factors, cofactors, or signaling pathways are engaged (Chereji et al., 2019; Inge et al., 2024). \nTherefore, the presence of accessible chromatin should be viewed as a necessary but not always \nsufficient feature of transcriptional regulation. In T-HESCs, 1,25(OH)2D3 selectively activates or represses \ntranscriptional programs by acting on a subset of accessible regions that are competent for vitamin D -\nresponsive regulation. This interpretation is especially important for understanding stromal differentiation \nand preparation for decidualization. Decidualization requires the coordinated activation and repression \nof gene networks that support stromal cell transformation, extrace llular matrix remodeling, immune \ncommunication, and endocrine responsiveness (Dunn et al., 2003; Krikun et al., 2004a; Gellersen and \nBrosens, 2014). Although the present study does not directly establish functional effects on pregnancy \noutcomes, implantation, or in vivo decidual success, it provides a chromatin -level framework for \nunderstanding how exposure to an active vitamin D alters the regulatory state of stromal cell s. By \nincreasing accessibility intensity across pre -existing regulatory regions, 1,25(OH)2D3 helps reinforce a \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n22 \n \nchromatin architecture that supports differentiation-associated transcriptional responsiveness. This \nshould be interpreted as a proposed chromatin -priming model rather than direct evidence of improved \nreproductive outcome (Fig. 4D). \n \nIf ligand treatment increases global ATAC -seq signal intensity, one might expect a large number of \nstatistically significant DARs (Wang et al., 2022; Vu et al., 2023). Our results indicate analytical thresholds, \npeak-calling behavior, and the distinction between global signal shifts and region -specific statistical \nchanges. Therefore, the modest number of DARs should not be interpreted as evidence that 1,25(OH)2D3 \nhas little effect on chromatin. Rather, it indicates that the ligand’s effect is more strongly reflected in \nquantitative accessibility enhancement than in extensive gain or loss of discrete accessible regions (Fig. \n4B and C). The genomic distribution of accessible regions and DARs further supports the idea that vitamin \nD signaling acts through both promoter -proximal and distal regulatory elements  (Wang et al., 2023). \nAccessible chromatin regions were distributed across promoters, introns, exons, untranslated regions, \nand intergenic intervals, suggesting that ligand -responsive chromatin regulation is not confined to \npromoters alone (Wiench et al., 2011; Thurman et al., 2012). Distal intronic and intergenic regions include \nenhancers or other regulatory elements that contribute to stromal transcriptional programs (Heinz et al., \n2010; Thurman et al., 2012). Thus, the gene associations reported here should be interpreted as active \nvitamin D acts primarily by reinforcing accessibility within a shared regulatory landscape, while producing \nselective remodeling at a smaller group of responsive loci (Fig. 4D). \n \nSeveral limitations should be acknowledged. First, this study used T -HESCs as a model of human \nendometrial stromal cells. While T -HESCs are useful for mechanistic studies and retain important \nfeatures of stromal biology, they do not fully recapitulate the complexity of primary stromal cells or the in \nvivo endometrial environment. Future studies using primary human endometrial stromal cells, \ndecidualization models, or time-course designs would help determine whether the chromatin accessibility \npatterns observed here are maintained across more physiological contexts. \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n23 \n \nSecond, this study was designed to define chromatin-level responses to a bioactive ligand rather than to \nevaluate reproductive toxicity, drug safety, or clinical pregnancy outcomes. Therefore, the findings should \nbe interpreted as a mechanistic molecular p harmacology study of 1,25(OH)2D3 action in stromal cells. \nFuture studies using primary human endometrial stromal cells, decidualization models, dose -response \ndesigns, or time-course experiments would help determine whether the chromatin accessibility patterns \nobserved here are maintained across more physiology. \nThird, ATAC-seq measures accessible chromatin but does not directly identify the transcription factors \noccupying these regions associated with regulatory activity , and  integration with RNA -seq provides \nimportant correlative evidence but does not prove direct regulatory relationships between specific \naccessible regions and gene expression changes. Functional validation, such as perturbation of \ncandidate regulatory elemen ts, reporter assays, or CRISPR -based enhancer interrogation, would be \nrequired to establish causality. \nLastly, chromatin accessibility is only one layer of genome regulation. Three -dimensional genome \norganization, enhancer-promoter looping, nucleosome positioning, transcription factor cooperativity, and \ninteractions with progesterone or estrogen signaling pathways are all likely to influence how vitamin D \nsignaling is interpreted in stromal cells. These layers will be important to examine in future studies, \nparticularly because endometrial stromal biology is shaped by multiple converging endocrine signals. \n \nDespite these limitations, this study provides important insight into the epigenomic action of active vitamin \nD in human endometrial stromal cells. The accessible chromatin landscape of T -HESCs was largely \nshared between vehicle - and 1,25(OH)2D3-treated cells, indicating that ligand exposure acts primarily \nwithin an established permissive regulatory environment rather than by broadly creating a new one. \n1,25(OH)2D3 enhanced ATAC-seq signal intensity across accessible regions and induced a subset of \nsignificant accessibility changes at selected loci. Integration with public cistromic data further showed \nthat many 1,25(OH)2D3-associated chromatin occupancy changes occurred within open chromatin \nregions, while integration with RNA-seq data showed that most ligand-responsive transcripts were also \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n24 \n \nassociated with nearby accessible chromatin regions. Collectively, these observations support a model \nin which active vitamin D modulates transcription mainly through an accessible chromatin landscape, \nwith both cistromic and transcriptomic responses occur ring largely within open chromatin and with \nselective chromatin remodeling contributing at a smaller subset of loci. More broadly, these findings \nhighlight chromatin accessibility as an important and relatively underexplored layer of regulation in human \nendometrial stromal cells. \n \nIn summary, this study identifies 1,25(OH)2D3 as a chemically defined modulator of chromatin \naccessibility in human endometrial stromal cells and highlights an understudied epigenomic dimension \nof vitamin D pharmacology in uterine biology. 1,25(OH)2D3 enhances accessibility intensity within an \nestablished regulatory landscape. This supports a model in which active vitamin D operates through a \npermissive stromal chromatin architecture that supports transcriptional responsiveness during stromal \ndifferentiation and preparation for decidualization. These findings also emphasize that chromatin \naccessibility is not merely a static backdrop, but an important regulatory framework in human endometrial \nstromal cells, a cell type in which this layer of regulation has r eceived comparatively limited attention.  \nThese findings demonstrate the utility of chromatin accessibility profiling for defining molecular responses \nto bioactive ligands and provide a mechanism for future studies examining how vitamin D signaling \ninteracts with VDR occupancy, three -dimensional genome organization, and ovarian steroid hormone \npathways to regulate endometrial stromal cell function. \n \nFunding \nThis study was supported by the Intramural Research Program of the National Institute of Environmental \nHealth Sciences 1ZIAES103311 (FJD). \n \nCRediT authorship contribution statement \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n25 \n \nMyeongJin Yi: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, \nValidation, Visualization, Writing – original draft, Writing – review & editing.  Bostan Hamed : Data \ncuration, Formal analysis, Software, Validation. Francesco J . DeMayo: Conceptualization, Funding \nacquisition, Resources, Supervision, Writing – original draft, Writing – review & editing. \n \nDeclaration of Competing Interest \nThe authors declare that they have no known competing financial interests or personal relationships that \ncould have appeared to influence the work reported in this paper. \n \nAcknowledgments \nThis research was supported in part by the Intramural Research Program of the National Institutes of \nHealth (NIH). The contributions of the NIH author(s) were made as part of their official duties as NIH \nfederal employees, are in compliance with agency policy requirements, and are considered Works of the \nUnited States Government. However, the conclusions presented in this paper are those of the author(s) \nand do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. \nThe authors appreciate the support from Epigenomics and the DNA Sequencing Core (1ZICES102545), \nas well as the Integrative Bioinformatics Supportive Group (1ZICES103371). \n \nAppendix A. Supplementary material \nSupplementary Table 1 \nSupplementary Table 2  \nSupplementary Table 3  \n \nSupplementary data statement \nSupplementary data associated with this article can be found in the online version. \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n26 \n \nData availability \nThe raw and processed data files generated in this study are available in NCBI GEO with the accession \nnumbers: ATAC-seq (GSE326063). This study utilized publicly available RNA-seq data files (GSE254251) \nfrom the following samples: GSM8036787, GSM8036788, GSM8036789, GSM8036790, GSM8036791, \nand GSM8036792, and CUT&RUN data files (GSE306127).  \n \nThe following datasets were generated: \nContributor(s) Year Dataset title \nDatabase and \nIdentifier \nYi, MyeongJin; Bostan, \nHamed; DeMayo, Francesco J \n2026 \n Epigenomic changes in chromatin accessibility \nfollowing 1,25-dihydroxyvitamin D3 treatment in \nhuman endometrial stromal cells \nGSE326063 \n \n  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n27 \n \nReferences \nBarakat, T.S., Halbritter, F., Zhang, M., Rendeiro, A.F., Perenthaler, E., Bock, C., Chambers, I., 2018. \nFunctional Dissection of the Enhancer Repertoire in Human Embryonic Stem Cells. Cell Stem \nCell 23, 276-288 e278. \nBuenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., Greenleaf, W.J., 2013. Transposition of native \nchromatin for fast and sensitive epigenomic profiling of open chromatin, DNA -binding proteins \nand nucleosome position. Nat Methods 10, 1213-1218. \nBuenrostro, J.D., Wu, B., Chang, H.Y., Greenleaf, W.J., 2015. ATAC -seq: A Method for Assaying \nChromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109, 21 29 21-21 29 29. \nChen, Y., Liang, R., Li, Y., Jiang, L., Ma, D., Luo, Q., Song, G., 2024. 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Impact of vitamin D deficiency on defective endometrial decidualization and the \nrepressive role of vitamin D receptor (VDR) in the epigenom ic network. Pharmacol Res 227, \n108162. \nZhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers, R.M., \nBrown, M., Li, W., Liu, X.S., 2008. Model -based analysis of ChIP-Seq (MACS). Genome Biol 9, \nR137. \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n34 \n \nFIGURES \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n35 \n \nFig. 1. 1,25(OH)2D3 enhances chromatin accessibility signal intensity in T-HESCs. The analysis \nshows that 1,25(OH)2D3 treatment is associated with increased accessibility signal without widespread \nreorganization of the global accessible chromatin landscape. (A) Overall profiles and heatmaps of ATAC-\nseq signal centered on called accessible regions (center ± 2 kb) and (B) surrounding transcription start \nsites (TSS ± 2 kb) in vehicle- and 1,25(OH)2D3-treated T-HESCs. (C) Overlap of open chromatin peaks \nidentified by ATAC-seq in vehicle- and 1,25(OH)2D3-treated cells. (D) Representative genome browser \nloci of ATAC -seq signal at VDR, CYP24A1, PRL, and EFL1 regions from vehicle - and 1,25(OH)2D3-\ntreated cells. RefSeq gene models are shown below. \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n36 \n \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n37 \n \nFig. 2. Differentially accessible regions (DARs) identified in response to 1,25(OH)2D3 in T-HESCs. \n(A) Clustered heatmaps and average signal profiles comparing ATAC -seq intensity patterns between \nvehicle- and 1,25(OH)2D3-treated cells across DARs. (B) Volcano plot showing differential chromatin \naccessibility between 1,25(OH)2D3-treated and vehicle -treated T -HESCs. Peaks were classified as \nincreased or decreased accessible regions based on adjusted p-value and fold-change thresholds. (C) \nComparison of genomic features of regions between loss and gain of accessibility.  (D) Metaplot and \nheatmap of ATAC -seq signal across regions with increased accessibility in response to 1,25(OH)2D3, \nshowing elevated signal intensity in ligand-treated cells compared with vehicle-treated cells. (E) Metaplot \nand heatmap of ATAC-seq signal across regions with decreased accessibility in response to 1,25(OH)2D3. \nThe result shows that 1,25(OH)2D3 induces a distinct set of accessibility changes, with pronounced signal \nenhancement at selected ligand-responsive regions. \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n38 \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n39 \n \nFig. 3. Integration of accessible chromatin -associated genes with vitamin D -responsive \ntranscriptomic changes in T -HESCs. (A) The number of overlapped peaks between ATAC -seq and \npublicly available CUT&RUN. (B) Enriched binding motifs from cistromic modification peaks within the \nopen chromatin areas in T-HESCs by HOMER de novo analysis. (C) Venn diagram showing overlap of \ngenes annotated to ATAC -seq peaks in vehicle - and 1,25(OH) 2D3-treated T -HESCs. Peaks were \nannotated to the nearest gene within 100 kb of the transcription start site. A total of 19,789 genes were \nassociated with open chromatin regions in vehicle-treated cells, whereas 19,114 genes were associated \nwith open chromatin regions in 1,25(OH)2D3-treated cells. Most annotated genes were shared between \nconditions, with 18,322 genes common to both datasets.  (D) Overlap between 1,25(OH)2D3-responsive \nDEGs and genes associated with open chromatin regions in vehicle- or 1,25(OH)2D3-treated cells. Among \n626 DEGs, 540 overlapped with vehicle-associated open chromatin genes, whereas 530 overlapped with \n1,25(OH)2D3-associated open chromatin genes. The high proportion of overlap indicates that most ligand-\nresponsive transcripts are associated with nearby accessible  chromatin regions present in either \ncondition. (E) Ingenuity Pathway Analysis (IPA) of the 530 differentially expressed genes associated with \nopen chromatin regions in 1,25(OH)2D3-treated T-HESCs. Causal network analysis identified VDR as the \nmost significant predicted activated master regulator. (F) IPA canonical pathway analysis of the same \ngene set revealed enrichment of pathways related to transcriptional regulation, immune -associated \nsignaling, and vitamin D receptor activity.\nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n40 \n \n \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\n41 \n \nFig. 4. Schematic summary of selective chromatin remodeling and transcriptomic response to \n1,25(OH)2D3 in T-HESCs. (A) Summarized transcript information on the overlap between DAR s. (B) \nUpregulated DARs overlap with 1,25(OH)2D3 DEGs, and downregulated DARs overlap with 1,25(OH)2D3 \nDEGs. (C) Representative ligand-responsive accessible regions selected for locus -specific quantitative \nvalidation. Corresponding validation of the enhancer peak region of GPAT3 and MAMDC2. (D) Proposed \nworking model of 1,25(OH)2D3-dependent chromatin-transcription coupling in T-HESCs. 1,25(OH)2D3 \nreinforces a permissive chromatin landscape in human endometrial stromal cells.  \nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\nA                                                 B                                                  C\nD                                                                        \nFigure. 1\nVehicle             1,25(OH)2D3                  Vehicle           1,25(OH)2D3\nVehicle\n1,25(OH)2D3\nVehicle\n1,25(OH)2D3 \nVehicle\n1,25(OH)2D3\nVehicle\n1,25(OH)2D3\nVDR\nCYP24A1\nPRL\nEFL1\n80.85% of vehicle\n90.15% of 1,25(OH)2D3\nCombined peaks \n140659\nVehicle 129246 \n1,25(OH)2D3 115916\n24743      104503    11413\nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\nA                                                    B                                   C\n  \n                                                                     \n                                                                                        \n                                                                    \n                                                        D                                     E        \n-2.0     center    2.0  -2.0    center    2.0        -2.0      center   2.0  -2.0    center   2.0\ngene distance (Kb)\n1,25(OH)2D3      Vehicle              1,25(OH)2D3       Vehicle\nUpregulated Accessible Regions  Downregulated Accessible Regions\nDown                    Up\n369                        110\nFigure. 2\n13.28\n30.00\n4.55\n2.17\n1.82\n10.57\n17.27\n73.98\n46.36\n0%\n10%\n20%\n30%\n40%\n50%\n60%\n70%\n80%\n90%\n100%\nDown_DAR Up_DAR\npromoter 5'UTR 3'UTR\nexon intron intergenic\n1,25(OH)2D3          Vehicle\nCluster\n1\n2\n3\n4\nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\nA                                        B\n                                                                \n                                                            \n                                                                            \nC                                       E                                              \nD                                                                  F\nFigure. 3\n19114 Genes in 1,25(OH)2D3 \nOpen Chromatin\n19782 Genes in Vehicle\nOpen Chromatin\n792          18322       1460\n95.86% of 1,25(OH)2D3\n92.62% of vehicle \nContrast DEGs\nGEO: GSE254251\n1,25(OH)2D3 vs. \nvehicle\n626 (Up: 386, \nDown: 240)\n19242    540  86\n86.26% of DEGs84.66% of DEGs \n18584     530  96\n9        521    19\n112286       3630   2462\n59.59% of 1,25(OH)2D3 \nCUT&RUN Peaks\nBest Match P-value % of targets\nFra1(bZIP)/BT549-Fra1 1e-163 17.30%\nVDR/MA0693.2 1e-38 24.63%\nTEAD2/MA1121.1 1e-26 14.10%\nTEAD4/MA0809.2 1e-20 0.34%\nNR1I3/MA1534.1 1e-17 4.13%\nPB0121.1_Foxj3_2 1e-17 1.21%\nTEAD3(TEA)/HepG2-TEAD3 1e-17 0.40%\nPU.1(ETS)/ThioMac-PU.1 1e-15 3.57%\nPB0110.1_Bcl6b_2 1e-14 18.14%\nNFkB-p65(RHD)/GM12787-p65 1e-14 3.11%\nSp5(Zf)/mES-Sp5.Flag 1e-14 6.93%\nRUNX1(Runt)/Jurkat-RUNX1 1e-12 1.27%\nVDR(NR),DR3/GM10855-VDR+vitD 1e-12 0.75%\nZNF692(Zf)/HEK293-ZNF692.GFP 1e-12 0.40%\nCausal Network\nCanonical Pathways\nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint \n\nUpregulated Accessible Regions – \nLigand DEG overlap\nDownregulated Accessible Regions – \nLigand DEG overlap\nFigure. 4\nA                                                                 B      \n                \n \n \nC\nD\nAnnotated_gene Peak loci DEG FC\nBNC1 2-10kb -1.51291\nCHSY3 <2kb_promoter -1.42111\nENOX1 10-25kb 5.4048\nGPAT3 25-100kb 1.90342\nKIAA1324L 25-100kb 3.13663\nAnnotated_gene Peak loci DEG FC\nMAMDC2 2-10kb 1.57345\nDAR_class Peaks Annotated\n_genes\nDEG \noverlap \nannotated \ngenes\nUp_DAR 110 108 5\nDown_DAR 369 162 1\nSummary of overlap between DARs \n(chromatin) and DEGs (transcriptome) \nVehicle\n1,25(OH)2D3\n0\n1000\n2000\n3000\n4000\nGPAT3 Enhancer\nRelative mRNA expression\n(fold change)\n✱\nVehicle\n1,25(OH)2D3\n0.0\n0.5\n1.0\n1.5\n2.0\nMAMDC2 Enhancer\nRelative mRNA expression\n(fold change)\n✱\n- + - +1,25(OH)2D3\nEndometrial Stromal Cell Chromatin \nArchitecture Outcome\nVehicle (basal status)\nPre-accessible chromatin landscape\nATAC\nsignal\nGenome track intensity\nVDR\n1,25(OH)2D3\n• Predominant increase in intensity across \npre-accessible regions\n• Strengthened accessibility\nATAC\nsignal\nHigher intensity \nat most pre-\nexisting regions\nGenome track intensity\nVDR\n1,25(OH)2D3 reinforces a permissive chromatin architecture\nCistromic and transcriptomic \nmodifications in endometrial \nstromal cells within \nchromatin accessible \nregulatory elements \nCoregulators\nNucleosome\nAccessible elements\n1,25(OH)2D3\nChromatin loops connecting \nregulatory to target genes\nand is also made available for use under a CC0 license. \nwas not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 \nThe copyright holder for this preprint (whichthis version posted May 9, 2026. ; https://doi.org/10.64898/2026.05.06.723064doi: bioRxiv preprint","source_license":"Public-Domain","license_restricted":false}