A Potent Far-Upstream Enhancer in the Mouse Proα2(I) Collagen Gene Regulates Expression of Reporter Genes in Transgenic Mice

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse proα2(I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse proα2(I) collagen gene, cloned upstream of either the Escherichia coli β-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5′ flanking sequences were cloned upstream of the 350-bp proximal proα2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse proα1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse proα2(I) collagen gene.