We propose to analyze the DNA sequences and protein binding factors which are responsible for elevated transcription of the alpha-1(I) collagen gene in bone cells as compared to fibroblast, and also these sequences and factors responsible for the inhibition of alpha-1(I) synthesis by vitamin D in bone cells but not fibroblasts. We will follow-up on our preliminary evidence that there is a bone-specific enhancer between -3521 and -2291, and a vitamin D responsive element between -2291 and -1665 upstream from the RNA transcription start site. We will analyze DNA sequences using deletions and oligonucleotide directed mutagenesis followed by analysis of transcriptional activity and hormone regulation of the mutants in stably transfected osteosarcoma and fibroblast cell lines, and also in transgenic mice. Subfragments of the promoter will be tested for their effect on the transcription of a weak heterologous promoter. In vitro protein/DNA interactions will be analyzed by gel mobility shifts and footprinting using nuclear extracts from hormone treated and control bone cells and fibroblasts. In vivo chromatin structure will be analyzed using DNase sensitivity studies. Appreciating the different regulatory influences on collagen production in bone and fibroblast is essential for understanding genetic acquired disorders of bone.