Characterization of a response element in the 5'-flanking region of the avian (chicken) PTH gene that mediates negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 and binds the vitamin D3 receptor.
Academic Article
Overview
abstract
Analysis of the 5'-flanking region of the avian (chicken) PTH (cPTH) gene has revealed a DNA segment between -74 and -60 that is analogous to the consensus sequence for the vitamin D3 response element (VDRE). The DNA segment consists of two imperfect direct repeats, GGGTCA and GGGTGT, which are separated by a 3-bp spacer. The functionality of the putative VDRE was verified by transfection studies in opossum kidney cells using plasmid constructs that contained various regions of the cPTH gene 5'-flanking sequence and promoter fused to the gene for chloramphenicol acetyl transferase (CAT). Likewise, negative regulation of gene transcription by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] was detected when the cPTH VDRE was inserted immediately upstream from truncated forms of the cPTH or the SV40 early promoter. Using gel mobility shift assays, the cPTH VDRE was compared with the human osteocalcin (hOC) VDRE, which activates gene transcription in the presence of 1,25-(OH)2D3. With a partially purified nuclear extract from dog intestine, the two VDREs produced gel shift patterns that were remarkably similar, with the exception that the binding affinity for the hOC sequence was notably greater. Both VDREs produced two major bound complexes (B1 and B2), which could be completely abolished by the addition of an excess of unlabeled hOC VDRE or a monoclonal antibody specific for the VDR protein. Furthermore, similar protein:DNA complexes were observed when either the cPTH or hOC VDRE were incubated with a mixture of purified preparations of recombinant VDR and retinoid X receptor alpha proteins. Ethylation interference analysis showed that the base contacts made by complexes B1 and B2 with the cPTH VDRE were essentially the same and were restricted primarily to the two half-site sequences.
