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

Vitamin D (VD) exerts diverse physiological effects primarily through the vitamin D receptor (VDR), a ligand-dependent transcription factor. Given the broad spectrum of VD actions, presence of uncharacterized target genes regulated by VD is likely. 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 target transcripts. VDR binding was strongly dependent on 1,25(OH) 2 D 3 and predominantly localized to enhancer regions marked by H3K27ac, indicating its association with active chromatin. Using 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 were correlated with strong transcriptional activation of the gene. Functional analyses using inhibition of bromodomain and extraterminal proteins and locked nucleic acid-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. Our findings reveal that VDR-mediated gene regulation involves a limited but functionally significant set of eRNAs, particularly in super-enhancer regions, and highlight a unique epigenetic mechanism underlying VD action.