DNA binding specificity of the CCAAT-binding factor CBF/NF-Y

CBF is a heterotrimeric protein that binds to DNA containing CCAAT motifs, Here we have analyzed interactions of recombinant CBF with DNA using hydroxyl radical footprinting and methylation interference assays, In the CBF-DNA complex, three separate DNA regions are protected from hydroxyl radical cleavage, one located over and immediately adjacent to the CCAAT motif itself and the other two located on both sides of the CCAAT motif, The methylation interference assay showed, however, that only in the CCAAT motif region methylation of bases was able to interfere with the for mation of a CBF-DNA complex, suggesting that CBF makes sequence-specific contacts only in the CCAAT motif region, To further determine the specific DNA sequences necessary for CBF binding, we employed a polymerase chain reaction-mediated random binding site selection method, This analysis showed that CBF binding to DNA requires the CCAAT sequence and other specific sequences immediately flanking both ends of the CCAAT motif, We also showed that the nature of the flanking nucleotide sequences affects the affinity of CBF for DNA, Interestingly, most of the CCAAT motifs present in various higher eukaryotic promoters correspond to the CBF binding sites that were selected, consistent with the hypothesis that these motifs are binding sites for CBF and, hence, that CBF could regulate transcription of numerous eukaryotic genes.