Type I collagen, a heterotrimer consisting of two a1(I) and one a2(I) polypeptides, is the most abundant protein found in skin, bone, and tendons and deregulated synthesis of type I collagen occurs in a number of pathological conditions. The CCAAT-binding factor (CBF) is a critical trans-acting factor that activates the in vitro transcription of both the a1(I) and a2(I) collagen promoters, as well as other promoters. CBF consists of three subunits, CBF-A, CBF-B, and CBF-C, which are all needed for DNA binding. The segment responsible for the formation of the CBF-DNA complex in each subunits is conserved from yeast to human, and in two of them, CBF-A and CBF-C, is homologous to the histone-fold motif. In vivo inhibition of CBF function by a dominant negative CBF mutant expressed in mouse fibroblasts inhibited cell growth and decreased expression of the collagen gene, as well as several other genes that are regulated during cell growth. In the proposed study, a comprehensive analysis will be done using the differential display and cDNA expression array to identify genes whose expression is decreased or increased as a result of inhibition of CBF binding in fibroblasts. The in vivo function of CBF will be studied in different mammalian cell lines using the dominant negative CBF mutant. The transcription activation function of CBF will be analyzed in an in vitro reconstituted system to identify the nuclear factor that associates with CBF and mediates the activation of CBF-dependent transcription. To study the transcriptional activation by recombinant CBF subunits in vivo, altered-specificity CBF mutants, that bind to a mutant CBF binding site will be created to test activity of the a1(I) and a2(I) collagen promoters in mouse fibroblasts.