The genes for type I collagen are activated in a precise spatial and temporal pattern during embryonic development. This proposal seeks to identify and characterize the lineage specific transcriptional mechanisms which activate these genes in fibroblasts, osteoblasts and odontoblasts. One can postulate that the same genetic programs might also activate these genes in fibrotic diseases, maybe in response to similar cues from the extracellular environment. Previous results have established that defined segments of the 5' flanking sequences of the type I collagen genes confer tissue-specific expression to reporter genes in transgenic mice, that largely mimics expression of the endogenous genes. The following Specific Aims are proposed: (1) Determine by mutagenesis which precise sequences within a short reconstructed alpha2(I) collagen promoter are responsible for this tissue-specificity. This reconstructed promoter contains a segment between -40 and +54 and 5' to this sequence the -315 to -284 segment which is tandemly repeated. (2) Demonstrate in a in vitro transcription system that activation of a reconstructed alpha2(I) collagen promoter is mediated by the sequences which are needed for tissue-specificity in animals. (3) Identify and clone the transcription factor(s) which determine the cell specificity of a minimal alpha2(I) collagen promoter. Approaches to clone such cDNAs include: (a) a Southwestern method to identify and clone cell-specific DNA-binding proteins which bind to a alpha2(I) collagen promoter sequence, which is needed for cell specificity; (b) establishment of a mammalian expression system or a yeast expression system to clone cDNAs which activate a reconstructed alpha2(I) collagen promoter through the sequence that is needed for cell-specificity in transgenic mice (c) test whether the products of two homeobox genes, which show a pattern of expression in embryos that is similar to that of the type I collagen genes, can activate these genes. (4) Identify within the 350 bp promoter the cis-acting elements that are needed for activity in osteoblasts. (5) Identify additional far-upstream, or intragenic or 3' flanking cis-acting enhancer elements that confer high level expression to the alpha2(I) collagen gene. (6) Use transgenic mice which harbor various alpha2(I) collagen promoter constructions to determine at which time during bleomycin induced lung fibrosis, the fibroblasts acquire a more active alpha2(I) collagen promoter and which sequences in the alpha2(I) collagen promoter are needed for this bleomycin-induced activation.
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