Identification of a hormone-responsive promoter immediately upstream of exon 1c in the human vitamin D receptor gene

To gain insight into the molecular regulation of the human vitamin D-3 receptor (hVDR), we have cloned and sequenced the 5' flanking region of exon Ic and examined promoter activity of this region in breast cancer cells. Sequence analysis of the first 1300 bp upstream of exon Ic reveals several characteristics of a class II promoter, including GC-rich regions and the presence of a TATA box at -29 bp. Putative transcription factor binding sites identified in this potential hVDR promoter include AP-2, Sp-l, and glucocorticoid response elements. No consensus vitamin D-3 (VDRE) or estrogen (ERE) responsive elements were identified in the promoter sequence. Primer extension analysis performed with a primer specific for exon Ic confirms that transcription initiated in the 5' flanking region of exon Ic occurs in MCF-7 cells. Transient transfection of MCF-7 cells with this putative promoter region cloned into the pRLnull luciferase reporter vector generates significant reporter gene activity that is enhanced by treatment with forskolin, retinoic acid, and 17 beta-estradiol. The enhancement of exon Ic promoter activity by 17 beta-estradiol is blocked by the selective estrogen response modifier (SERM) tamoxifen and is not observed in estrogen receptor-negative breast cancer cells. In summary, we have cloned and characterized a TATA containing promoter upstream of exon Ic of the hVDR and provide evidence that this region represents a hormonally regulated hVDR promoter.