Chromatin and transcriptional dynamics underlying the immune-modulatory effects of vitamin D3 in vivo

Vitamin D₃ is an essential micronutrient that supports innate immunity and modulates adaptive responses, with in vitro studies implicating epigenetic mechanisms. Yet, in vivo evidence for such regulation remains scarce. Here, we assessed genome-wide epigenomic and transcriptomic responses to vitamin D₃ in a high responder from the VitDHiD repeated-measures, non-randomized interventional N-of-1 study, in which the participant received monthly oral boluses of 80,000 IU vitamin D₃ over three months. Peripheral blood mononuclear cells (PBMCs) were collected at baseline (day 0) and at 24 and 48 h post-supplementation (days 1 and 2) for ATAC-seq and RNA-seq profiling. ATAC-seq identified > 3,500 regions with increased chromatin accessibility, predominantly at promoters, indicating a strong epigenomic activation. Motif enrichment revealed immune-regulatory transcription factors, but not vitamin D receptor (VDR) motifs, suggesting indirect or cooperative modes of regulation. RNA-seq detected 380 vitamin D-responsive genes with time-dependent expression changes, including DUSP6 and FOS, enriched in pathways related to innate immunity and interferon signaling. Integrative analysis linked 306 differentially expressed genes to vitamin D-sensitive chromatin regions. Many of these overlap with VDR binding sites or promoters of neighboring non-target genes, suggesting potential long-range regulation. Shared enhancers and promoters among neighboring genes indicated redundancy in regulatory architecture. Analysis of PBMCs from 13 participants in the VitDPAS non-randomized interventional cohort study, each receiving a single vitamin D₃ bolus, confirmed the main findings from the N-of-1 study but also revealed pronounced inter-individual variability in epigenomic and transcriptomic responses. Together, these results provide in vivo evidence that vitamin D signaling directly and indirectly modulates chromatin accessibility and gene expression, emphasizing its role as an epigenetic regulator of immune function and supporting its potential in personalized nutrition and immune health strategies.