The hypothesis behind this application is that type I collagen genes are activated during embryonic development in a discrete number of cell types by some of the same transcriptional mechanisms which also control the differentiated phenotypes of these cells. The objective of the proposal is to elucidate the mechanisms that control expression of these genes in fibroblasts and osteoblasts and, consequently, broader genetic programs in these cell types. Previous work has indicated that separate cis-acting elements in the type I collagen genes control expression of reporter genes in different type I collagen producing cells. One of these elements in the mouse proa1(I) gene controls osteoblast expression; another one is a potent far-upstream enhancer in the proa2(I) gene which controls expression mainly in fibroblasts. This application is divided into two parts. One proposes to delineate the minimal elements in the proa2(I) far-upstream enhancer which control expression of a reporter gene in fibroblastic cells of transgenic mice. It is expected that this minimal segment can then be used to identify fibroblast-specific or fibroblast-enriched DNA binding transcription factors which can activate the fibroblast elements in the proa2(I) gene. Subsequently, cDNAs for these DNA binding proteins will be cloned and characterized. The second part of the project will further define the DNA sequence in the proa1(I) gene critical for osteoblast expression in transgenic mice. It is expected that this sequence can be used to identify osteoblast-specific or osteoblast-enriched DNA binding proteins which control expression of the proa1(I) gene in osteoblasts. Also in this case, cDNAs for these polypeptides will then be cloned and characterized. Together, these two studies will enhance our understanding of the molecular determinants underlying the differentiation of mesenchymal cell precursors.
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