Type I collagen, a heterotrimer consisting of two(alpha(l) and one alpha2(l) polypeptides, is the most abundant protein found in skin, bone, and tendons. Changes in the synthesis of type 1 collagen occur in a number of pathological conditions. To understand the molecular mechanism of the regulation of type I collagen gene expression, several cis-acting elements and their cognate DNA-binding proteins have been identified in the alphal(l) and alpha2(l) collagen gene promoters. One critical transacting factor is a CCAAT binding factor (CBF) that activates in vitro transcript ion of both the alpha(l) and alpha(l) collagen promoters as well as other promoters. CBF is a multimeric protein and consists of three different polypeptides, CBF-A, CBF-B, and CBF-C, all which are needed for DNA binding. The cDNA clones for CBF-A, CBF-B, and CBF-C polypeptides have been isolated. These three recombinant CBF subunits, expressed from their cDNAs, together form a CBF-DNA complex, and all three CBF subunits are present in the DNA- protein complex. Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor. In the proposed study, recombinant CBF subunits will be used to analyze different steps of association of the three subunits for formation of the CBF protein. The reconstituted CBF will be used to identify consensus DNA sequences for CBF binding by the PCR-mediated random site selection method. The contact of the CBF molecule in DNA will be revealed by various DNA-protein interaction assays to determine whether CBF interacts with the minor or major groove of DNA. A photo-crosslinking method will be used to crosslink the CBF subunits to DNA and to identify the specific DNA contact in the subunits of CBF. A CBF-dependent promoter will be constructed to determine whether transcriptional activation of the promoter by CBF is dependant upon distances and orientations of the CBF-binding site with respect to the TATA box. Dominant negative mutants of the CBF subunits that inhibit DNA binding of the wild-type CBF will be expressed in fibroblast cells under a tight control by a tetracycline-responsive promoter to inactivate the endogenous CBF protein. in this study, the role of CBF in transcription activation of both the type I collagen genes and in fibroblast cell growth will be determined.
