TY - JOUR
T1 - Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease
AU - Ruminy, Philippe
AU - Derambure, Céline
AU - Chandrasegaran, Srinivasan
AU - Salier, Jean Philippe
N1 - Funding Information:
Our thanks to A. Le Cam (Inserm, Montpellier) for a critical reading of the manuscript. We are indebted to S. Cereghini (Institut Pasteur, Paris), T. Grange (Institut Jacques Monod, Paris) and M. Zakin (Institut Pasteur, Paris) for kindly providing various plasmids with HNF-3 binding sites and related information and to J.E. Darnell Jr (New York) for a gift of anti-HNF-3 antisera. P. R. is the recipient of a doctoral fellowship from Section de l’Eure de la Ligue contre le Cancer. This work was supported, in part, by Association pour la Recherche sur le Cancer (attribution 5378 to J.P.S.) and the University of Rouen, France. S.C. is supported by a grant from the National Institutes of Health (GM53923).
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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-3β/FN, that is comprised of the rat HNF-3β 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-3β/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-3β/FN at a given binding site was tightly correlated with the affinity of a natural HNF-3β 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.
AB - 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-3β/FN, that is comprised of the rat HNF-3β 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-3β/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-3β/FN at a given binding site was tightly correlated with the affinity of a natural HNF-3β 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.
KW - Chimeric nuclease
KW - Cleavage
KW - Detection
KW - Genomics
KW - Transcription factor
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U2 - 10.1006/jmbi.2001.4788
DO - 10.1006/jmbi.2001.4788
M3 - Article
C2 - 11439020
AN - SCOPUS:0035854460
VL - 310
SP - 523
EP - 535
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 3
ER -