(A) Quantitative RT-PCR analysis of CST5 RNA expression in SW480-ADH cells treated with 1α,25(OH)₂D₃ or vehicle that were pretreated for 30 minutes with actinomycin D (2 μg/ml) or cycloheximide (8 μg/ml) as indicated. (B) Scheme of the human CST5 gene promoter showing putative VDR binding sites grouped in regions A–D. (C) Activation of CST5 promoter constructs by 1α,25(OH)₂D₃ in SW480-ADH cells and in HEK293T cells cotransfected with an expression vector for wild-type VDR. Twenty-four hours after transfection, cells were treated with vehicle or 1α,25(OH)₂D₃ (10^–7 M) for an additional 48 hours. The empty pGL3 vector was used as control. (D) The activation of the CST5 promoter by 1α,25(OH)₂D₃ requires a transcriptionally competent VDR. SW480-R cells were cotransfected with the promoter construct pGL3-1867 or pGL3-251 and either the wild-type VDR or the mutant ΔAF2-VDR (VDR^ΔAF2), or an empty vector. The cells were then treated with 1α,25(OH)₂D₃ (10^–7 M) or vehicle for 48 hours. As control, the consensus 4xVDRE-DR3-Tk-Luc (VDRE) reporter construct was cotransfected with the wild-type or mutant VDR. Values correspond to promoter induction by 1α,25(OH)₂D₃ in 3 independent experiments done in triplicate. (E) 1α,25(OH)₂D₃ induces VDR binding to and an active chromatin conformation of the CST5 promoter in vivo. ChIP assay showing the induction by 1α,25(OH)₂D₃ of VDR binding, SMRT corepressor release, and increased histone H4 acetylation (AcH4) of the CST5 gene promoter in SW480-ADH cells. The CYP24 gene was used as control. The promoter regions studied are indicated.