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Retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells toward the visceral endoderm lineage is accompanied by increased expression of the Forkhead Box (Fox) transcription factors hepatocyte nuclear factor 3a (HNF-3alpha) and HNF-3beta, suggesting that they play a crucial role in visceral endoderm development. Retinoic acid stimulation results in a cascade of HNF-3 induction in which HNF-3alpha is a primary target for retinoic acid action and its increase is required for subsequent induction of HNF-3beta expression. Increased expression of HNF-3beta precedes activation of its known target genes, including transthyretin (TTR), Sonic hedgehog (Shh), HNF-1alpha, HNF-1beta, and HNF-4alpha. In order to examine whether increased HNF-3 expression is sufficient to induce expression of its downstream target genes without retinoic acid stimulation, we have used adenovirus-based expression vectors to increase HNF-3 protein levels in F9 cells. We demonstrate that adenovirus-mediated increase of HNF-3alpha levels in F9 cells is sufficient to induce activation of endogenous HNF-3beta levels followed by increased TTR and Shh expression. Furthermore, we show that elevated HNF-3beta levels stimulate expression of endogenous TTR and Shh without retinoic acid stimulation. Moreover, ectopic HNF-3 levels in undifferentiated F9 cells are insufficient to induce HNF-3alpha, HNF-1alpha, HNF-1beta, and HNF-4alpha expression, suggesting that their transcriptional activation required other regulatory proteins induced by the retinoic acid differentiation program. Finally, our studies demonstrate the utility of cell infections with adenovirus expressing distinct transcription factors to identify endogenous target genes, which are assembled with the appropriate nucleosome structure.

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.

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due to lower HNF-3 alpha expression levels and that the induction of primarily c-jun homodimers, which are poor transcriptional activators, is insufficient to maintain normal TTR expression levels. We also discuss the role of reduced HNF-3 alpha expression in mediating decreased transcription of HNF-3 target genes which respond negatively to cytokine signalling.

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.

The serum thyroxine-binding protein, transthyretin (TTR), is made by hepatocytes and by choroid plexus epithelium in adults and by yolk sac cells in embryogenesis. Four hepatocyte nuclear factors (HNF-1, -3, and -4 and C/EBP) that are present in liver but not in most other adult tissues bind DNA sites in the TTR gene that are sufficient to direct transgenic expression. Three of these proteins were also found in yolk sac cells, which also express the transgene. A limited transgenic construct is not active in the choroid plexus and a TTR-producing choroid plexus tumor lacks three of the liver-enriched DNA-binding proteins. We conclude that cell-specific expression of TTR is regulated at least in part by the differential cellular distribution of positive-acting transcription factors.

Transthyretin (TTR) is a thyroid hormone transport protein that is secreted by hepatocytes into the serum and by the choroid plexus epithelium into the cerebral spinal fluid. The protein is not made elsewhere in adult animals in significant amounts. We find that the start site for mRNA synthesis is the same in both cell types. The sequences required for mouse TTR expression in cultured hepatocytes include an enhancer at -1.86 to -1.96 kbp and a promoter-proximal region at -70 to -200 bp relative to the mRNA cap site. We demonstrate that in transgenic mice these regulatory regions (approximately 300 bp) are sufficient for quantitatively normal expression of a TTR minigene in hepatocytes, but not for restricted expression in the choroid plexus cells of the brain. Instead, they direct aberrant widespread expression in regions of the brain outside the choroid plexus. With 3 kbp of upstream sequence the TTR minigene is expressed specifically in the choroid plexus as well as in the liver, demonstrating the normal cell type specificity for TTR. These results suggest that 3 kbp of upstream sequence contains positive element(s) required for choroid plexus expression which are distinct from those utilized in the hepatocyte, and may also contain negative element(s) that function to suppress transcription in other brain cell types.

Transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) are expressed at high levels in the liver and also in at least one other cell type. We report here a detailed analysis of the proximal regulatory region of the TTR gene, which has uncovered two new DNA-binding factors that are present mainly (or only) in hepatocytes. One of these new factors, hepatocyte nuclear factor 3 (HNF-3), binds to two sites that are crucial in TTR expression as well as to two additional sites in the alpha 1-AT proximal enhancer region. The second new factor, HNF-4, binds to two sites in TTR that are required for gene activity. We had previously identified binding sites for another hepatocyte-enriched DNA-binding protein (C/EBP or a relative thereof), and additional promoter-proximal sites for that protein in both TTR and alpha 1-AT are also reported here. From these results it seems clear that cell-specific expression is not simply the result of a single cell-specific factor for each gene but the result of a combination of such factors. The variation and distribution of such factors among different cell types could be an important basis for the coordinate expression of the TTR and alpha 1-AT genes in the liver or the discordant transcriptional activation of these genes in a few other cell types. The identification of such cell-enriched factors is a necessary prelude to understanding the basis for cell specificity.

We previously defined two distinct cell-specific DNA elements controlling the transient expression of the transthyretin gene in Hep G2 (human hepatoma) cells: a proximal promoter region (-202 base pairs [bp] to the cap site), and a far-upstream cell-specific enhancer located between 1.6 and 2.15 kilobases (kb) 5' of the cap site (R. H. Costa, E. Lai, and J. E. Darnell, Jr., Mol. Cell. Biol. 6:4697-4708, 1986). In this report, we located the effective transthyretin enhancer element within a 100-bp region between 1.96 and 1.86 kb 5' to the mRNA cap site. In Hep G2 nuclear extracts, three protein-binding sites within this minimal enhancer element were identified by gel mobility and methylation protection experiments. Each binding site was required for full enhancer activity in Hep G2 transient expression assays. Competition experiments in protein-binding assays suggested that two of the three sites were recognized by a similar factor and that the protein interaction with the third site was different. The nuclear protein(s) which bound to the two homologous sites was found mainly or only in cells of hepatic origin, suggesting an involvement of this region in the cell-specific function of this enhancer. The nuclear protein(s) recognizing the third enhancer region was also found in HeLa and spleen cells.

The mouse genomic clone for the prealbumin (transthyretin) gene was cloned, and its upstream regulatory regions were analyzed. The 200 nucleotides 5' to the cap site when placed within a recombinant plasmid were sufficient to direct transient expression in HepG2 (human hepatoma) cells, but this DNA region did not support expression in HeLa cells. The sequence of the 200-nucleotide region is highly conserved between mouse and human DNA and can be considered a cell-specific promoter. Deletions of this promoter region identified a crucial element for cell-specific expression between 151 and 110 nucleotides 5' to the RNA start site. A region situated at about 1.6 to 2.15 kilobases upstream of the RNA start site was found to stimulate expression 10-fold in HepG2 cells but not in HeLa cells. This far upstream element was invertible and increased expression from the beta-globin promoter in HepG2 cells. Unlike the simian virus 40 enhancer, the prealbumin enhancer would not stimulate beta-globin synthesis in HeLa cells, and even the simian virus 40 enhancer did not stimulate the prealbumin promoter in HeLa cells. Thus, we identified in the prealbumin gene two DNA elements that respond in a cell-specific manner: a proximal promoter including a crucial sequence between -108 and -151 nucleotides and a distant enhancer element located between 1.6 and 2.15 kilobases upstream.

Double-stranded oligodeoxynucleotides that represent protein binding sites in the regulatory regions of the mouse genes encoding transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) bound a nuclear protein factor(s) found mainly in hepatocytes. A site in the regulatory region of the gene encoding rat serum albumin and, surprisingly, a region in the simian virus 40 enhancer also bind the same factor. Oligodeoxynucleotide affinity chromatography (with one of the TTR binding sites) allowed a 500-fold purification of the protein. The purified protein protected similar portions of all the regulatory regions, as well as the simian virus 40 core C enhancer element, from digestion with DNase I. A DNA-binding protein previously purified from liver by virtue of its ability to bind to several virus enhancer sequences also binds to TTR, alpha 1-AT, and albumin regulatory sites. Thus, all these binding sites, which contain only minimal sequence similarity, may bind to a single protein, or a similar family of proteins, that activates liver-specific transcription of coordinately expressed genes.

Four different regulatory sites required for transcriptional stimulation by the enhancers of two unrelated liver-specific genes alpha 1-antitrypsin and transthyretin appear to bind the same nuclear protein that is found mainly in the liver. Such proteins may provide a basis for a coordinated, hepatocyte-specific control of gene transcription.