Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 523-535 |
| Number of pages | 13 |
| Journal | Journal of Molecular Biology |
| Volume | 310 |
| Issue number | 3 |
| DOIs | |
| State | Published - Jul 13 2001 |
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Keywords
- Chimeric nuclease
- Cleavage
- Detection
- Genomics
- Transcription factor
ASJC Scopus subject areas
- Virology
Cite this
Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease. / Ruminy, Philippe; Derambure, Céline; Chandrasegaran, Srinivasan; Salier, Jean Philippe.
In: Journal of Molecular Biology, Vol. 310, No. 3, 13.07.2001, p. 523-535.Research output: Contribution to journal › Article
}
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
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
VL - 310
SP - 523
EP - 535
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 3
ER -