Characterization of an enhancer required for 1,25-dihydroxyvitamin D3-dependent transactivation of the rat osteocalcin gene.

The sequences in the rat osteocalcin gene that lie 3' to the vitamin D response element (VDRE) contain a GGTTTGG motif (-420 to -414) that is essential for transcriptional activation of osteocalcin-CAT (OC-CAT) fusion genes by 1,25(OH)2D3. A second copy of this motif, present on the antisense strand is unable to compete for nuclear protein binding to the VDRE-associated motif, suggesting that the core element extends beyond the GGTTTGG motif. In order to examine the base requirements for both function and nuclear protein interactions with the VDRE-associated GGTTTGG enhancer motif, deletion and substitution of flanking sequences was performed in the context of both the native osteocalcin promoter and a heterologous viral promoter. These data demonstrate that the base requirements for protein-DNA interactions and transactivation are located between -430 and -414. The position of the element with respect to the VDRE is flexible and insertion of additional copies either 5' or 3' to the VDRE further enhances transactivation, both in the context of the native osteocalcin promoter and a heterologous viral promoter. These data demonstrate that VDR-dependent transactivation of the rat osteocalcin gene requires not only the VDRE (-456 to -442) but also sequences between -430 and -414. The protein(s) that interacts with these sequences is capable of enhancing transcription in both a position and orientation-independent fashion.