Identification of functional elements and reconstitution of the alpha 1(VI) collagen promoter

To gain insight into the regulatory mechanisms of collagen VI synthesis we have characterized the cis-acting elements of the chicken alpha 1(VI) collagen promoter. Footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated A, B, and C, that were protected from DNase I digestion. The nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitors as well as specific antibodies raised against well characterized transcription factors. Site A was found to be a target for transcriptional activator AP1, whereas sites B and C were shown to be recognized each by two distinct nuclear proteins which belong to the Sp1 multigene family. To address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites A, B, and C were placed in front of a reporter gene. After transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic alpha 1(VI) collagen promoter. Thus, the three sites are sufficient to induce transcription of this gene.