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

Philippe Ruminy, Céline Derambure, Srinivasan Chandrasegaran, Jean Philippe Salier

Research output: Contribution to journalArticle

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 languageEnglish (US)
Pages (from-to)523-535
Number of pages13
JournalJournal of Molecular Biology
Volume310
Issue number3
DOIs
StatePublished - Jul 13 2001

Fingerprint

Hepatocyte Nuclear Factors
Binding Sites
Transcription Factors
Nucleic Acid Regulatory Sequences
DNA
Enzymes
Genomics
Genes
Plasmids

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 journalArticle

@article{2a1566b34f704d93919be05de41082b1,
title = "Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease",
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.",
keywords = "Chimeric nuclease, Cleavage, Detection, Genomics, Transcription factor",
author = "Philippe Ruminy and C{\'e}line Derambure and Srinivasan Chandrasegaran and Salier, {Jean Philippe}",
year = "2001",
month = "7",
day = "13",
doi = "10.1006/jmbi.2001.4788",
language = "English (US)",
volume = "310",
pages = "523--535",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",
number = "3",

}

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

UR - http://www.scopus.com/inward/record.url?scp=0035854460&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035854460&partnerID=8YFLogxK

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 -