Temporal stratification of promoter-proximal versus enhancer VDR binding directs vitamin D-responsive transcription.

BACKGROUND

The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activates the vitamin D receptor (VDR) in immune cells, altering the epigenome to drive diverse responses. However, how the temporal dynamics, genomic positioning, and inducibility of VDR binding are integrated to determine transcriptional outcomes remains incompletely understood.

RESULTS

Here, we present a time-resolved analysis of VDR-mediated gene regulation in human THP-1 monocytes, integrating VDR ChIP-seq profiles at 40 min, 4, 8, and 24 h with matched transcriptome data following stimulation with 1,25(OH)2D3. Time-resolved VDR ChIP-seq revealed > 22,000 binding sites, with > 5,100 showing ligand-inducible occupancy that expanded over time. This expansion parallels a marked increase in vitamin D-responsive genes, evolving from a limited early response to a broad late transcriptional program. Using distance- and topologically associated domain-informed assignment strategies, we classify vitamin D target genes according to their spatial and temporal association with VDR binding, distinguishing promoter-proximal and enhancer-dominated regulatory architectures. Early vitamin D target genes are characterized by frequent VDR occupancy at transcription start site regions, often supported by coordinated enhancer binding, whereas late-responsive genes are predominantly associated with distal enhancers or lack assignable VDR-driven regulatory models. Importantly, promoter‑proximal VDR binding is consistently stronger and more inducible than enhancer‑only binding and is associated with higher basal expression and greater transcriptional responsiveness.

CONCLUSIONS

Together, these findings define a hierarchical, multi-layered model of vitamin D signaling in which early, promoter-proximal VDR binding is strongly associated with establishment of a transcriptionally competent core program, while later enhancer-mediated and indirect mechanisms progressively expand and diversify the response.