Characterization of a novel hexameric repeat DNA sequence in the promoter of the immediate early gene, IEX-1, that mediates 1α, 25-dihydroxyvitamin D₃-associated IEX-1 gene repression

1α, 25-Dihydroxyvitamin D₃(1α, 25(OH)₂D₃), the active metabolite of vitamin D₃, mediates anti-proliferative effects in cells by regulating the expression of 1α, 25(OH)₂D₃-responsive genes. The expression of the proliferation-promoting Immediate Early gene X-1 (IEX-1) is reduced by 1α, 25(OH)₂D₃ through unknown mechanisms. Here we report the presence of a novel inhibitory hexameric repeat DNA response element in the promoter of the human IEX-1 gene that mediates 1α, 25(OH)₂D₃-associated IEX-1 gene repression. To localize a vitamin D sensitive DNA response element we transfected the keratinocyte-like cell line, HaCaT, (referred as HaCaT) with a series of plasmids containing full-length and truncated IEX-1 promoter elements fused to the luciferase reporter gene in the absence or presence of 1α, 25(OH)₂D₃, and we performed electrophoretic gel mobility assays in the presence of receptors for 1α, 25(OH)₂D₃ (vitamin D receptor, VDR) and 9-cis-retinoic acid (RXRα). We mapped a negative response element between nt -405 and -391(15 bp) of the IEX-1 promoter (5′-TGAACC AGG GAGTCA-3′) that mediates transcriptional inhibition in response to 1α, 25(OH)₂D₃ and which requires expression of both nuclear receptors for 1α, 25(OH)₂D₃ and 9-cis-retinoic acid. Our data indicate that the physiological repression of IEX-1 gene expression by 1α, 25(OH)₂D₃ is directly mediated by nuclear VDR/RXRα heterodimers through a specific transcriptional element.