Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease

Identifying the complete set of transcription factors that bind the promote r and other regulatory regions of a gene of interest is an essential step i n functional genomics. We have developed an original assay for the systemat ic detection of hepatocyte nuclear factor-3 (HNF-3) binding sites within cl oned promoters. This assay is based on expression of a recombinant enzyme, HNF-3 beta /F-N, that is comprised of the rat HNF-3 beta DNA-binding domain and the non-specific nuclease domain of the FokI restriction enzyme. South ern analysis of target plasmids with proven HNF-3 binding sites showed that HNF-S beta /F-N was able to specifically cut both DNA strands in the vicin ity of these binding sites, whereas mutagenized binding sites were no longe r cleaved. Likewise, as yet undescribed HNF-3 binding sites were detected e asily over a distance spanning several thousand bases. The functionality of such binding sites was confirmed by electromobility shift assay. Furthermo re, the extent of cleavage by HNF-3 beta /F-N at a given binding site was t ightly correlated with the affinity of a natural HNF-3 beta molecule for th is site. This novel approach can be extended to other transcription factors for long-range identification of functional transcription factor binding s ites in genes. (C) 2001 Academic Press.