Identification of the key regions within the mouse pro-α2(I) collagen gene far-upstream enhancer

Studies using transgenic mice have shown that the mouse pro-alpha2(I) collagen gene contains a far-upstream enhancer, which directs expression in the majority of collagen I-producing cells during development and in response to tissue injury. In this study, we have investigated the minimal functional region required for the enhancer effect and studied the role of the three hypersensitive sites (HS3-HS5) that overlap this region. The results of deletion experiments indicate that the minimal functional unit of this enhancer is a 1.5-kb region between -17.0 and -15.45 kb from the transcription start site. This region includes the core sequences of HS3 and HS4 but not HS5. The HS4 sequences are essential for the functional integrity of the enhancer, whereas HS3 represents tissue-specific elements that direct expression in mesenchymal cells of internal tissues and body wall muscles. The HS3 region appears to bind a complex of transcription factors illustrated by large regions of protected sequences. A 400-bp sequence located between -17.0 and -16.6 is also essential for the enhancer because its deletion results in increased susceptibility to the chromatin environment.