THE CCAAT-BINDING PROTEINS CP1 AND NF-I COOPERATE WITH ATF-2 IN THE TRANSCRIPTION OF THE FIBRONECTIN GENE

We have previously proposed a molecular interaction between the liver factors that bind to the cyclic AMP response element (CRE) and CCAAT s ites of the fibronectin (FN) gene based on the following evidence: (i) the close spacing of 20 base pairs between CRE and CCAAT elements is conserved in the FN genes from rats, mice, and humans; (ii) footprinti ng competitions showed that CRE oligonucleotides are able to detach bo th liver factors; (iii) CCAAT binding and transcriptional activity of liver extracts are reduced when the distance between the CRE and CCAAT elements is increased; and (iv) CCAAT-binding is stimulated by the ad dition of a liver extract fraction containing the CRE-binding factor A TF-2. This report provides binding and immunochemical evidence that nu clear factor I (CTF/NF-I) and CP1 (NF-Y or CBF) are the only liver fac tors that bind to the -150 CCAAT element of the FN gene, forming disti nct complexes, We show that these factors bind less efficiently to the CCAAT site of a FN promoter in which the -170 CRE has been disrupted by site directed mutagenesis and that each element contributes positiv ely to the liver transcriptional activity assessed in vitro with a G-l ess cassette construct and in vivo by transfection of hepatoma cells w ith CAT constructs, Furthermore, using a method that combines UV cross linking and immunoprecipitation, we show that antibodies specific to A TF-2 are able to specifically precipitate protein-protein-DNA complexe s containing NF-I and CP1. This simple method preserves weak macromole cular interactions, avoiding the disruptive electrophoresis conditions of gel mobility shifts assays.