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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-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.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Ruminy P,Derambure C,Chandrasegaran S,Salier JP

doi

10.1006/jmbi.2001.4788

keywords:

subject

Has Abstract

pub_date

2001-07-13 00:00:00

pages

523-35

issue

3

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(01)94788-6

journal_volume

310

pub_type

杂志文章

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