Journal Article

Characterization of a Dominant Negative Mutant Form of the HNF-4 Orphan Receptor

David G. Taylor, Sabine Haubenwallner and Todd Leff

in Nucleic Acids Research

Volume 24, issue 15, pages 2930-2935
Published in print August 1996 | ISSN: 0305-1048
Published online August 1996 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/24.15.2930
Characterization of a Dominant Negative Mutant Form of the HNF-4 Orphan Receptor

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The HNF-4 orphan receptor is a member of the nuclear receptor family of transcription factors and a major regulator of genes involved in carbohydrate and lipid metabolism. As an initial step in characterizing the role of HNF-4 in the regulation of metabolism, we have generated a dominant negative form of HNF-4 (DN-HNF-4) that contains a defective DNA-binding domain. In gel mobility shift assays, DN-HNF-4 did not bind an oligonucleotide probe representing an essential HNF-4 binding site, C3P contained in the human apo CIII promoter, but did prevent the binding of two recombinant isoforms, HNF-4α1 and HNF-4α2, as well as naturally-occurring HNF-4. DN-HNF-4 had no effect on the binding of PPARγ-RXRα heterodimers to a PPAR response element. In transfected HepG2 cells, DN-HNF-4 dramatically reduced constitutive transcriptional activity of the human apo CIII promoter and abolished the positive transcriptional activity caused by plasmids expressing either isoform of HNF-4. These results indicate that DN-HNF-4 is a selective dominant negative mutant which forms defective heterodimers with wild-type HNF-4, thereby preventing DNA binding and subsequent transcriptional activation by HNF-4.

Journal Article.  3237 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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