Requirement of distal and proximal promoter sequences for chromatin organization of the osteocalcin gene in bone-derived cells.

The osteocalcin (OC) gene encodes a 10 Kda bone-specific protein which is expressed with the onset of mineralization during the differentiation of normal diploid osteoblasts. We have previously reported that transcriptional activation of this gene is accompanied by the presence of two DNase I hypersensitive sites, both located in the promoter region spanning key basal (proximal site, -170 to -70) and steroid-dependent enhancer (distal site, -600 to -400) elements. Here, we have examined stably transfected ROS 17/2.8 cell lines carrying OC promoter-reporter transgenes which contain a series of 5'-deletions and determined the effects of these truncations on the chromatin organization. It has been found that: (1) DNase I hypersensitivity at -600 is not a requirement for vitamin D-dependent transcriptional upregulation; (2) basal transcriptional activity and proximal nuclease hypersensitivity depend exclusively on protein-DNA interactions occurring within the proximal promoter region, and (3) within the chromatin context, the proximal 100 bp promoter fragment, containing essential elements such as the OC box (-99 to -76) and TATA box (-44 to -31), is insufficient to support formation of the proximal nuclease hypersensitive site and transcriptional activity.