IDENTIFICATION OF FUNCTIONAL ELEMENTS AND RECONSTITUTION OF THE ALPHA-1(VI) COLLAGEN PROMOTER

To gain insight into the regulatory mechanisms of collagen VI synthesi s we have characterized the cis-acting elements of the chicken alpha1( 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 prote ins that interact with the three sites were identified by gel retardat ion assays in combination with the use of various oligonucleotide comp etitors as well as specific antibodies raised against well characteriz ed transcription factors. Site A was found to be a target for transcri ptional activator AP1, whereas sites B and C were shown to be recogniz ed each by two distinct nuclear proteins which belong to the Spl multi gene 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 re porter gene. After transfection into chicken fibroblasts, this constru ct exhibited a high relative promoter activity when compared to a larg e genomic fragment containing the basic alpha1(VI) collagen promoter. Thus, the three sites are sufficient to induce transcription of this g ene.