DNASE-I HYPERSENSITIVE SITES IN PROMOTER ELEMENTS ASSOCIATED WITH BASAL AND VITAMIN-D-DEPENDENT TRANSCRIPTION OF THE BONE-SPECIFIC OSTEOCALCIN GENE

Nuclease hypersensitive sites were mapped in the proximal promoter of the osteocalcin gene, which is expressed only in bone cells exhibiting the mature osteoblast phenotype. Nuclei from proliferating and conflu ent rat osteosarcoma (ROS) 17/2.8 cells were subjected to DNase I dige stion, and hypersensitivity was assayed by the indirect end-labeling m ethod, using osteocalcin gene probes. Hypersensitive sites were detect ed in two promoter domains: -590 to -390, which spans the vitamin D re sponsive element, and -170 to -70, which spans the TATA box and the CC AAT-containing OC box domain. Together, these elements regulate basal and vitamin D enhanced osteocalcin gene transcription. We observed a p arallel relationship between the intensity of bands representing the h ypersensitive sites and the extent to which the osteocalcin gene is tr anscribed. Both in confluent cultures and in response to vitamin D, wh en osteocalcin transcription was upregulated, the hypersensitive bands were significantly intensified. Additionally, the bands were decrease d under conditions that downregulate osteocalcin gene transcription. A functional relationship between the presence of hypersensitive sites and osteocalcin gene transcription is further supported by the absence of hypersensitivity in nonosseous cells that do not express osteocalc in, although these proliferating cells exhibited hypersensitivity in a cell cycle regulated histone gene promoter. Our results suggest the i nvolvement of chromatin structure in transcriptional responsiveness of the osteocalcin gene to physiologic modulation.