Identification of a transthyretin enhancer site that selectively binds the hepatocyte nuclear factor-3 beta isoform

Gene Expr. 1996;6(1):23-33.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins / metabolism*
  • Enhancer Elements, Genetic*
  • HeLa Cells
  • Hepatocyte Nuclear Factor 3-beta
  • Humans
  • Mice
  • Nuclear Proteins / metabolism*
  • Prealbumin / genetics*
  • Recombinant Fusion Proteins / metabolism
  • Transcription Factors / metabolism*
  • Tumor Cells, Cultured


  • CCAAT-Enhancer-Binding Proteins
  • DNA-Binding Proteins
  • FOXA2 protein, human
  • Foxa2 protein, mouse
  • Nuclear Proteins
  • Prealbumin
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Hepatocyte Nuclear Factor 3-beta