Site-directed mutagenesis of hepatocyte nuclear factor (HNF) binding sites in the mouse transthyretin (TTR) promoter reveal synergistic interactions with its enhancer region.
The transthyretin (TTR) gene is regulated by two DNA regions which elicit hepatocyte-specific expression: a proximal promoter and distal enhancer. The TTR promoter and enhancer are composed of at least eight DNA binding sites for three different hepatocyte nuclear factors (HNF), CCAAT/enhancer binding protein (C/EBP), and AP-1/cJun. Site directed mutations within each of the HNF binding sites in the TTR promoter were introduced to evaluate their contribution to transcriptional activity in hepatoma cells. The data indicate that the strong affinity HNF-3-S binding site (-106 to -94) is absolutely required for TTR promoter activity since several mutations in this site eliminate TTR expression in the context of its enhancer. Conversion of a second weak affinity HNF3-W site (-140 to -131) in the TTR promoter to a high affinity site resulted in higher levels of expression. TTR mutations that disrupted several weak affinity sites (HNF1, HNF3-W, and HNF4) only slightly diminished expression levels in the presence of the TTR enhancer. In contrast, when we deleted the TTR enhancer from these HNF mutant constructs, TTR expression decreased to undetectable levels. This result suggests cooperation between the factors binding to the TTR promoter and enhancer regions. These results also demonstrate that the HNF3-S site alone is not sufficient to activate TTR transcription, but rather requires the participation of three cell-specific factors to elicit minimal promoter activity. The complexity of this promoter design and the requirement for a minimal number of cell-specific factors to achieve transcription allows us to propose a model which may explain the maintenance of tissue-specific expression of TTR.
Costa
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RH
Department of Biochemistry (M/C 536), University of Illinois, College of Medicine, Chicago 60612.
Grayson
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DR
D000818:Animals; D001483:Base Sequence; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D005786:Gene Expression Regulation / Q000502:physiology; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D051541:Hepatocyte Nuclear Factor 3-alpha; D051542:Hepatocyte Nuclear Factor 3-beta; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins; D011228:Prealbumin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C496499:FOXA1 protein, human; C496502:FOXA2 protein, human; C496500:Foxa1 protein, mouse; C496503:Foxa2 protein, mouse; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; D051541:Hepatocyte Nuclear Factor 3-alpha; D051557:Hepatocyte Nuclear Factor 4; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; C403128:MLX protein, human; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011228:Prealbumin; C501970:Tcfl4 protein, mouse; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051542:Hepatocyte Nuclear Factor 3-beta; D051539:Hepatocyte Nuclear Factor 1-beta