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 differentiat ion of normal diploid osteoblasts. We have previously reported that tr anscriptional activation of this gene is accompanied by the presence o f two DNase I hypersensitive sites, both located in the promoter regio n spanning key basal (proximal site, -170 to -70) and steroid-dependen t enhancer (distal site, -600 to -400) elements. Here, we have examine d stably transfected ROS 17/2.8 cell lines carrying OC promoter-report er transgenes which contain a series of 5'-deletions and determined th e effects of these truncations on the chromatin organization. It has b een found that: (1) DNase I hypersensitivity at -600 is not a requirem ent for vitamin D-dependent transcriptional upregulation; (2) basal tr anscriptional activity and proximal nuclease hypersensitivity depend e xclusively on protein-DNA interactions occurring within the proximal p romoter region, and (3) within the chromatin context, the proximal 100 bp promoter fragment, containing essential elements such as the OC bo x (-99 to -76) and TATA box (-44 to -31), is insufficient to support f ormation of the proximal nuclease hypersensitive site and transcriptio nal activity. (C) 1996 Wiley-Liss, Inc.