Binding of 1,25-dihydroxyvitamin D3 receptors to the 5′-flanking region of the bovine parathyroid hormone gene

To further define the binding site for receptors for 1,25(OH)2D3 (VDR) in the bovine PTH gene and to study the interactions of transcription factors with VDR, Southwestern and gel shift assays were used. Data from the former indicated binding of VDR to DNA fragments spanning the regions −451 to −348 bp and −668 to −452 bp. Studies using gel shift assays confirmed binding to the −451 to −348 bp fragment and specificity was shown by using excess concentrations of unlabelled −451 to −348 bp fragment to compete for binding, whereas excess unlabelled −347 to +50 bp did not compete. Binding was also observed with the −668 to −452 bp fragment but excess concentrations of unlabelled −668 to −452 or −451 to −348 bp fragments did not compete for binding to radiolabelled fragments. These data indicate the presence of two binding domains within this region; the upstream element having a lower affinity for VDR than the downstream element. In addition, there was no interaction between VDR and consensus sequences for AP1, AP2, AP3 and SP1. The putative vitamin D3 response element (VDRE) contains two similar hexameric steroid response element-like half-sites placed as AGGTCA-related direct repeats. The upstream repeat is at −461 to −456 bp and the downstream element is at −449 to −444 bp. The presence of these half-sites is consistent with our experimental data in which cleavage with SspI at −452 bp resulted in two DNA fragments which bound VDR. Journal of Endocrinology (1994) 142, 53–60