Separate binding sites for nuclear factor 1 and a CCAAT DNA binding factor in the mouse alpha 2(I) collagen promoter

Abstract

A factor present in nuclear extracts of NIH-3T3 fibroblasts was identified which binds to a region between -315 and -295 with respect to the start of transcription in the mouse alpha 2(I) collagen promoter. This factor was assayed using three different DNA binding assays. Evidence that this factor is nuclear factor 1 or a related protein is based on competition experiments using fragments of another promoter that contains a nuclear factor 1 binding site as well as a fragment containing a consensus sequence for nuclear factor 1 binding sites. The nucleotide sequence of the binding site in the alpha 2(I) promoter. TCGN5GCCAA, presents similarities to the sequence published previously as a consensus recognition sequence for nuclear factor 1. Methylation interference experiments indicate that the factor interacts at least with two successive G residues in the major groove complementary to the two successive C residues in the GCCAA motif. This factor is different from a factor that binds to a CCAAT element between -84 and -80 in the mouse alpha 2(I) collagen promoter. This was shown by competition experiments using both crude NIH-3T3 fibroblast nuclear extracts and a partially purified CCAAT binding factor. These results suggest that in the alpha 2(I) collagen promoter nuclear factor 1 and a CCAAT binding factor bind to separate sites, despite analogies in their recognition sequences.