Identifying the complete set of transcription factors that bind the promoter and other regulatory regions of a gene of interest is an essential step in functional genomics. We have developed an original assay for the systematic detection of hepatocyte nuclear factor-3 (HNF-3) binding sites within cloned promoters. This assay is based on expression of a recombinant enzyme, HNF-3beta/FN, that is comprised of the rat HNF-3beta DNA-binding domain and the non-specific nuclease domain of the FokI restriction enzyme. Southern analysis of target plasmids with proven HNF-3 binding sites showed that HNF-3beta/FN was able to specifically cut both DNA strands in the vicinity of these binding sites, whereas mutagenized binding sites were no longer cleaved. Likewise, as yet undescribed HNF-3 binding sites were detected easily over a distance spanning several thousand bases. The functionality of such binding sites was confirmed by electromobility shift assay. Furthermore, the extent of cleavage by HNF-3beta/FN at a given binding site was tightly correlated with the affinity of a natural HNF-3beta molecule for this site. This novel approach can be extended to other transcription factors for long-range identification of functional transcription factor binding sites in genes.
Copyright 2001 Academic Press.