Genome-wide identification of vitamin D receptor–regulated enhancer RNAs reveals epigenetic regulation of CYP24A1 transcription

Vitamin D (VD) exerts diverse physiological and pathological effects primarily through the vitamin D receptor (VDR), a ligand-dependent transcription factor. However, the limited number of known VDR target genes does not fully explain the broad spectrum of VD actions. In this study, we performed a comprehensive genome-wide analysis to identify enhancer RNAs (eRNAs) involved in VD-dependent gene regulation. Using integrated ChIP-seq, RNA-seq, and NET-CAGE analyses in human HCT116 and HaCaT cells, we mapped VDR binding sites and identified VD-responsive transcripts. VDR binding was strongly dependent on vitaminD and predominantly localized to enhancer regions marked by H3K27ac, indicating association with active chromatin. Through stepwise filtering, we identified a limited number of high-confidence eRNA candidates associated with VDR-bound regulatory regions. Among these, eRNAs located at the CYP24A1 locus were robustly induced by VD and correlated with strong transcriptional activation of the gene. Functional analyses using BET inhibition and LNA-mediated knockdown demonstrated that these eRNAs contribute to VD-dependent transcription. Furthermore, CRISPR/dCas9-mediated induction of specific eRNAs enhanced CYP24A1 expression, supporting a direct regulatory role. In contrast, newly identified target genes such as DLX2 and SYT8 exhibited modest, cell type–dependent responses to VD. Our findings reveal that VDR-mediated gene regulation involves a limited but functionally significant set of eRNAs, particularly at super-enhancer regions, and highlight a unique epigenetic mechanism underlying VD action.