Identification of a transthyretin enhancer site that selectively binds the hepatocyte nuclear factor-3 beta isoform.
The upstream proximal region of the transthyretin (TTR) promoter and a distal enhancer are sufficient to drive liver-specific expression of the TTR gene, as demonstrated by experiments in transgenic mice. Previous analyses have characterized the binding of a number of liver-enriched transcription factors of the TTR promoter including hepatocyte nuclear factors one (HNF-1), HNF-4, and three distinct HNF-3 proteins (alpha, beta, and gamma), which are members of the winged helix (fork head) family. The TTR enhancer was shown to bind members of the CCAAT/enhancer binding protein (C/EBP) family at two distinct sites (TTR-2 and TTR-3), and an oligonucleotide containing the activation protein one (AP-1) binding sequence competed for recognition to a third enhancer site (TTR-1). In this study, we have carried out a detailed analysis of the transcription factors that recognize the TTR enhancer elements (TTR-1, TTR-2, and TTR-3 oligonucleotide sequences). Analysis of the TTR-1 site demonstrates that the putative AP-1 site in the TTR enhancer binds a ubiquitously expressed factor that is distinct from the AP-1 family of proteins. Next we demonstrate, via gel shift analysis, that the TTR-3 site is recognized by the C/EBP family in liver nuclear extracts. We also show that whereas the TTR-2 enhancer site is capable of binding recombinant C/EBP proteins, it does not bind C/EBP proteins from liver nuclear extracts. The TTR-2 site does, however, contain a variant HNF-3 recognition sequence that exclusively binds the HNF-3 beta isoform. Mutation of this HNF-3 beta-specific recognition sequence caused reductions in TTR enhancer activity. We had previously observed a 95% decrease in HNF-3 alpha expression and a 20% reduction in HNF-3 beta expression in acute phase livers, which correlated with a 60% decrease in TTR gene transcription. We propose that the HNF-3 beta-specific binding site in the TTR enhancer may play a role in maintaining TTR gene expression during the acute phase response in spite of the dramatic reduction in HNF-3 alpha protein levels.
Samadani
U
U
Department of Biochemistry, College of Medicine, University of Illinois at Chicago 60612-7334, USA.
Qian
X
X
Costa
R H
RH
D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D051542:Hepatocyte Nuclear Factor 3-beta; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011228:Prealbumin / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C496502:FOXA2 protein, human; C496503:Foxa2 protein, mouse; D009687:Nuclear Proteins; D011228:Prealbumin; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051542:Hepatocyte Nuclear Factor 3-beta