DNA-SEQUENCES DOWNSTREAM FROM THE VITAMIN-D RESPONSE ELEMENT OF THE RAT OSTEOCALCIN GENE ARE REQUIRED FOR LIGAND-DEPENDENT TRANSACTIVATION

The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (VDRE) have been shown to augment transcriptional ac tivation by 1,25-dihydroxyvitamin D-3 [1,25-(OH)(2)D-3]. These DNA seq uences, however, are unable to bind the VDR or mediate 1,25-(OH)(2)D-3 responsiveness independently of the VDRE. To further characterize thi s region, the functional properties of a series of mutant oligonucleot ides were examined in transiently transfected ROS 17/2.8 cells. When t hese mutant oligonucleotides were expressed upstream of the heterologo us herpes simplex virus thymidine kinase promoter, the bases between - 420 and -414 of the rat osteocalcin gene were identified as critical f or maximal transactivation by 1,25-(OH)(2)D-3. Furthermore, mutation o f these sequences in the context of the native osteocalcin promoter an d enhancer totally abolished the ability of the VDRE to mediate 1,25-( OH)(2)D-3 responsiveness. These bases, which are essential for the 1,2 5-(OH)(2)D-3 responsiveness of the rat osteocalcin gene, are also pres ent in a similar position, relative to the VDRE, in the human osteocal cin gene. To explore whether these sequences could enhance transactiva tion by other inducible transcription factors, they were examined for their ability to synergize with the chick vitellogenin estrogen respon se element and the rat somatostatin cAMP response element. When placed upstream to the herpes simplex virus thymidine kinase promoter and tr ansfected into ROS 17/2.8 cells, these sequences were able to enhance transcriptional responsiveness to 17 beta-estradiol and forskolin, res pectively, demonstrating that they also contribute to transactivation by other inducible transcription factors.