The DNA-binding specificity of the hepatocyte nuclear factor 3/forkhead domain is influenced by amino-acid residues adjacent to the recognition helix.
Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (HNF-3 alpha, -3 beta, and -3 gamma) are known to regulate the transcription of liver-specific genes. The HNF-3 proteins bind to DNA as a monomer through a modified helix-turn-helix, known as the winged helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic forkhead (fkh) protein. We have previously described the isolation, from rodent tissue, of an extensive family of tissue-specific HNF-3/fkh homolog (HFH) genes sharing homology in their winged helix motifs. In this report, we have determined the preferred DNA-binding consensus sequence for the HNF-3 beta protein as well as for two divergent family members, HFH-1 and HFH-2. We show that these HNF-3/fkh proteins bind to distinct DNA sites and that the specificity of protein recognition is dependent on subtle nucleotide alterations in the site. The HNF-3, HFH-1, and HFH-2 consensus binding sequences were also used to search DNA regulatory regions to identify potential target genes. Furthermore, an analysis of the DNA-binding properties of a series of HFH-1/HNF-3 beta protein chimeras has allowed us to identify a 20-amino-acid region, located adjacent to the DNA recognition helix, which contributes to DNA-binding specificity. These sequences are not involved in base-specific contacts and include residues which diverge within the HNF-3/fkh family. Replacement of this 20-amino-acid region in HNF-3 beta with corresponding residues from HFH-1 enabled the HNF-3 beta recognition helix to bind only HFH-1-specific DNA-binding sites. We propose a model in which this 20-amino-acid flanking region influences the DNA-binding properties of the recognition helix.
Overdier
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DG
Department of Biochemistry, College of Medicine, University of Illinois at Chicago 60612-7334.
Porcella
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A
Costa
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000378:metabolism; D051858:Forkhead Transcription Factors; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D051543:Hepatocyte Nuclear Factor 3-gamma; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008168:Lung / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051858:Forkhead Transcription Factors; C496501:Foxa1 protein, rat; C496504:Foxa2 protein, rat; C496507:Foxa3 protein, rat; D051541:Hepatocyte Nuclear Factor 3-alpha; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051543:Hepatocyte Nuclear Factor 3-gamma; D051542:Hepatocyte Nuclear Factor 3-beta; D004247:DNA