Cell-specific in vivo DNA-protein interactions at the proximal promoters of the proα1(I) and the proα2(I) collagen genes

We performed in vivo dimethylsulfate footprinting of the 220 bp mouse proximal proα1(I) collagen promoter and the 350 bp mouse proximal proα2(I) collagen promoter in BALB/3T3 fibroblasts, primary mouse skin fibroblasts, S-194 B cells, NMuLi liver epithelial cells and RAG renal adenocarcinoma cells and in vitro DNase I footprinting of these promoters using nuclear extracts of these different cell types. Whereas proα1(I) and proα2(I) collagen RNAs were present in BALB/3T3 fibroblasts and primary fibroblasts, these RNAs could not be detected in the three other cell lines. Comparison of in vitro DNase I footprints for each of the two proximal collagen promoters indicated that the patterns of protection were very similar with the different nuclear extracts, suggesting that the DNA binding proteins binding to these promoters were present in all cell types tested. In contrast, in vivo footprints over these proximal promoters were cell-specific, occurring only in fibroblast cells and not in the other three cell types. The in vivo footprints were generally located within the in vitro footprinted regions. Our results suggest that although all cell types tested contained nuclear proteins that can bind to the proximal proα1(I) and proα2(I) collagen promoters in vitro, it is only in fibroblasts that these proteins bind to their cognate sites in vivo. We discuss possible regulatory mechanisms in type I collagen genes that can contribute to the cell-specific in vivo protein-DNA interactions at the proximal promoters.