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 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.
Ruminy
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P
INSERM Unit 519 and Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Faculté de Médecine-Pharmacie, 22 Boulevard Gambetta, Rouen cedex, 76183, France.
Derambure
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Chandrasegaran
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Salier
J P
JP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D023281:Genomics; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D014157:Transcription Factors
D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496504:Foxa2 protein, rat; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta; D004247:DNA; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific