Characterization of a dominant negative mutant form of the HNF-4 orphan receptor.
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-4alpha1 and HNF-4alpha2, as well as naturally-occurring HNF-4. DN-HNF-4 had no effect on the binding of PPARgamma-RXRalpha 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.
Taylor
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DG
Department of Biotechnology, Parke-Davis Pharmaceutical Research, Ann Arbor, MI 48105, USA.
Haubenwallner
S
S
Leff
T
T
D000595:Amino Acid Sequence; D000818:Animals; D053305:Apolipoprotein C-III; D001056:Apolipoproteins C / Q000235:genetics; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D017930:Genes, Reporter; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011487:Protein Conformation; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D016335:Zinc Fingers
D053305:Apolipoprotein C-III; D001056:Apolipoproteins C; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors