Turning a negative into a positive: Vitamin D receptor interactions with the avian parathyroid hormone response element

1,25-Dihydroxyvitamin D-3 [1,25-(OH)(2)D-3] negatively regulates expression of the avian PTH (aPTH) gene transcript, and a vitamin D response element (VDRE) near the promoter of the aPTH gene had previously been identified. T he present report assessed whether the negative activity imparted by the aP TH VDRE could be converted to a positive transcriptional response through s elective mutations introduced into the element. The tested sequences were d erived from individual and combined mutations to 2 bp in the 3'-half of the direct repeat element, GGGTCAggaGGGTGT. Cold competition experiments using mutant and wild-type oligonucleotides in the mobility shift assay revealed minor differences in the ability of any of these sequences to compete for binding to a heterodimer complex comprised of recombinant proteins. Ethylat ion interference footprint analysis for each of the mutants produced unique patterns over the 3'-half-sites that were distinct from the weak, wild-typ e footprint. Transcriptional outcomes evaluated from a chloramphenicol acet yltransferase reporter construct utilizing the aPTH promoter found that the individual T-->A mutant produced an attenuated negative transcriptional re sponse while the G-->C mutant resulted in a reproducibly weak positive tran scriptional outcome. The double mutant, however, yielded a 4-fold increase in transcription, similar to the 7-fold increase observed from an analogous construct using the human osteocalcin VDRE. UV light crosslinking to gappe d oligonucleotides assessed the polarity of heterodimer binding to the wild -type and double mutant sequences and was consistent with the vitamin D rec eptor preferentially binding to the 5'-half of both elements. Finally, DNA affinity chromatography was used to immobilize heterodimer complexes bound to the wild-type and double mutant sequences as bait to identify proteins t hat may preferentially interact with these DNA-bound heterodimers. This ana lysis revealed the presence of a p160 protein that specifically interacted with the heterodimer bound to the wild-type VDRE, but was absent from compl exes bound to response elements associated with positive transcriptional ac tivity. Thus, the sequence of the individual VDRE appears to play an active role in dictating transcriptional responses that may be mediated by alteri ng the ability of a vitamin D receptor heterodimer to interact with accesso ry factor proteins.