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The forkhead box (Fox) proteins are a growing family of transcription factors that have important roles in cellular proliferation and differentiation and in organ morphogenesis. The Fox family members hepatocyte nuclear factor (HNF)-3beta (Foxa2) and HNF-3/forkhead homolog (HFH)-8 (FREAC-1, Foxf1) are expressed in adult pulmonary epithelial and mesenchymal cells, respectively, but these cells display only low expression levels of the proliferation-specific HFH-11B gene (Trident, Foxm1b). The regulation of these Fox transcription factors in response to acute lung injury, however, has yet to be determined. We report here on the use of butylated hydroxytoluene (BHT)-mediated lung injury to demonstrate that HFH-11 protein and RNA levels were markedly increased throughout the period of lung repair. The maximum levels of HFH-11 were observed by day 2 following BHT injury when both bronchiolar and alveolar epithelial cells were undergoing extensive proliferation. Although BHT lung injury did not alter epithelial cell expression of HNF-3beta, a 65% reduction in HFH-8 mRNA levels was observed during the period of mesenchymal cell proliferation. HFH-8-expressing cells were colocalized with platelet endothelial cell adhesion molecule-1-positive alveolar endothelial cells and with alpha-smooth muscle actin-positive peribronchiolar smooth muscle cells.

To assess the role of hepatocyte nuclear factor-3beta (HNF-3beta) in hepatocyte-specific gene transcription, we reported the characterization of the liver phenotype with transgenic mice in which the -3-kb transthyretin (TTR) promoter functioned to increase HNF-3beta expression. During breeding of the TTR-HNF-3beta transgenic mice we noticed that they displayed severe ataxia. In this study, we describe the analysis of our transgenic cerebellar phenotype and demonstrate that ectopic expression of HNF-3beta disrupted cerebellar morphogenesis and caused reduction in cerebellar size. In postnatal cerebellum, the HNF-3beta transgene expression pattern is colocalized to glial fibrillary acidic protein-positive cerebellar astrocytes and Bergmann glial cells. As a result of protracted expression, the transgenic cerebella are impaired in terms of astrocyte dispersal and formation of Bergmann glial cell processes. This caused a disruption in neuronal cell migration to the cortical laminar layers and Purkinje dendritic arbor maturation, thus leading to diminished foliation. Differential hybridization of cDNA arrays was used to identify altered expression of cerebellar genes, which is consistent with the observed defect in transgenic cerebellar morphogenesis and size as well as glial maturation. These include diminished expression of the brain lipid-binding protein, which is required for glial morphological differentiation, and the basic helix-loop-helix NeuroD/Beta2 and homeodomain Engrailed-2 transcription factors, which are required for normal cerebellar morphogenesis and foliation. Undetectable levels of ataxia telangiectasia (ATM), which is required for proper development of the Purkinje dendritic arbor, were found in postnatal transgenic cerebella. Furthermore, the transgenic cerebella displayed levels of insulin-like growth factor binding protein-1 elevated to 22 times greater than those measured for wild-type cerebella, an elevation consistent with the reduction in transgenic cerebellar size.

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 winged helix transcription factor, hepatocyte nuclear factor-3beta (HNF-3beta), mediates the hepatocyte-specific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3beta in regulating these genes remains unknown because homozygous null HNF3beta mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3beta, we created transgenic mice in which the -3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3beta protein. Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3beta on the hepatic expression of the endogenous mouse HNF-3alpha,-3beta, -3gamma, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3beta transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.

During organogenesis, the winged helix hepatocyte nuclear factor 3beta (HNF-3beta) protein participates in regulating gene transcription in the developing esophagus, trachea, liver, lung, pancreas, and intestine. Hepatoma cell transfection studies identified a critical HNF-3beta promoter factor, named UF2-H3beta, and here, we demonstrate that UF2-H3beta is identical to the fetoprotein transcription factor (FTF). In situ hybridization studies of mouse embryos demonstrate that FTF expression initiates in the foregut endoderm during liver and pancreatic morphogenesis (day 9) and that earlier expression of FTF is observed in the yolk sac endoderm, branchial arch and neural crest cells (day 8). Abundant FTF hybridization signals are observed throughout morphogenesis of the liver, pancreas, and intestine and its expression continues in the epithelial cells of these adult organs. In day 17 mouse embryos and adult pancreas, however, expression of FTF becomes restricted to the exocrine acinar and ductal epithelial cells.

Murine hepatocyte nuclear factor-3alpha (HNF-3alpha) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and participate in embryonic pattern formation. HNF-3alpha also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal, pancreatic and bronchiolar epithelium. We have previously determined that -520 nucleotides upstream of the rat HNF-3alpha gene were sufficient to elicit hepatoma-specific expression in transfection assays and reported on a novel HNF-3alpha expression pattern in the renal pelvis urothelium of the embryonic and adult kidney. We also showed that retinoic acid mediated activation of the HNF-3alpha gene required -4 kb of the HNF-3alpha promoter region in F9 teratocarcinoma transfections. In order to determine regulatory sequences mediating the HNF-3alpha cellular expression pattern in developing mouse embryos, we created transgenic mice bearing the -4 kb HNF-3alpha promoter region driving expression of the beta-galactosidase transgene. Embryonic analysis of two transgenic mouse lines demonstrated that the -4 kb HNF-3alpha promoter sequences were sufficient to elicit transgene expression in the developing liver, intestine, esophagus, nasal epithelial cells and floorplate of the neurotube, but not in the mesodermal notochord or in the lung bud. One of the transgenic lines also exhibited proper expression in the mesonephric ducts and metanephric diverticulum, suggesting that the -4 kb HNF-3alpha promoter region contained a subset of the regulatory sequences necessary for HNF-3alpha expression in the developing kidney.

Murine hepatocyte nuclear factor-3 beta (HNF-3 beta) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and that participate in embryonic pattern formation. HNF-3 beta also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal and bronchiolar epithelial, and pancreatic acinar cells. We have previously identified a liver-enriched transcription factor, HNF-6, which is required for HNF-3 beta promoter activity and also recognizes the regulatory region of numerous hepatocyte-specific genes. In this study we used the yeast one-hybrid system to isolate the HNF-6 cDNA, which encodes a cut-homeodomain-containing transcription factor that binds with the same specificity as the liver HNF-6 protein. Cotransfection assays demonstrate that HNF-6 activates expression of a reporter gene driven by the HNF-6 binding site from either the HNF-3 beta or transthyretin (TTR) promoter regions. We used interspecific backcross analysis to determine that murine Hnf6 gene is located in the middle of mouse chromosome 9. In situ hybridization studies of staged specific embryos demonstrate that HNF-6 and its potential target gene, HNF-3 beta, are coexpressed in the pancreatic and hepatic diverticulum. More detailed analysis of HNF-6 and HNF-3 beta's developmental expression patterns provides evidence of colocalization in hepatocytes, intestinal epithelial, and in the pancreatic ductal epithelial and exocrine acinar cells. The expression patterns of these two transcription factors do not overlap in other endoderm-derived tissues or the neurotube. We also found that HNF-6 is also abundantly expressed in the dorsal root ganglia, the marginal layer, and the midbrain. At day 18 of gestation and in the adult pancreas, HNF-6 and HNF-3 beta transcripts colocalize in the exocrine acinar cells, but their expression patterns diverge in other pancreatic epithelium. HNF-6, but not HNF-3 beta, expression continues in the pancreatic ductal epithelium, whereas only HNF-3 beta becomes restricted to the endocrine cells of the islets of Langerhans. We discuss these expression patterns with respect to specification of hepatocytes and differentiation of the endocrine and exocrine pancreas.

Hepatocyte nuclear factor-3beta (HNF-3beta), a nuclear protein of the winged helix family of transcription factors, is known to play a critical role in the formation of the embryonic node, notochord, and foregut endoderm. HNF-3beta influences the expression of a number of target genes in the respiratory epithelium, activating transcription of thyroid transcription factor-1, surfactant protein-B and clara cell secretory protein. In order to discern the role of HNF-3beta in differentiation and gene expression in the lung, HNF-3beta was expressed in developing respiratory epithelial cells of transgenic mice, under the control of the human surfactant protein C gene promoter. Pulmonary abnormalities were observed in the lungs of fetal mice bearing the HNF-3beta transgene. Differentiation of distal respiratory epithelial cells was arrested in the early pseudoglandular stage. Branching morphogenesis and vasculogenesis were markedly disrupted in association with decreased E-cadherin and vascular endothelial growth factor expression. HNF-3beta limits cellular diversity of developing respiratory epithelium and alters lung morphogenesis in vivo, suggesting that precise temporal-spatial regulation of HNF-3beta expression is critical for respiratory epithelial cell differentiation and lung morphogenesis.

This report demonstrates that Clara cell 10-kDa protein (CC10) mRNA levels are regulated by interferon-gamma (IFN-gamma). An analysis of total lung RNA from mice given IFN-gamma intratracheally showed increased levels of CC10 mRNA compared to control animals but no significant increases in surfactant proteins B and C. These results were confirmed in a Clara cell line, mtCC1-2, generated from the lungs of a transgenic mouse expressing the SV40 large T antigen under the control of a Clara cell-specific promoter. Significant increases in mtCC1-2 CC10 mRNA levels were observed in a time- and a dose-dependent manner. The expression of transacting factors hepatocyte nuclear factors 3 alpha and 3 beta (HNF-3 alpha and HNF-3 beta) were also analyzed, and a transient increase in the expression of HNF-3 beta but not HNF-3 alpha was detected. Deoxyribonuclease I footprint analysis identified a signal transducer and activator of transcription (STAT) binding site (at nucleotides -293 to -284 of CC10) adjacent to two thyroid transcription factor-1 (TTF-1) binding sites, suggesting a potential interaction between STAT1 and TTF-1. This report reinforces the hypothesis that CC10 functions as an anti-inflammatory protein and that increases in CC10 protein may provide additional protection from inflammation and disease in the lung.

The hepatocyte nuclear factor 3alpha (HNF-3alpha) and 3beta proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription factor family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-29 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-29, Caco-2, HepG2, HeLa, A549, and H441). We demonstrate that differentiation of Caco-2 cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3alpha and HNF-3beta mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, 24, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of keratinocyte growth factor in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription factor is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.

The hepatocyte nuclear factor-3 alpha (HNF-3 alpha) and -3 beta proteins share homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and respiratory epithelium. In this study, we used transfection assays to demonstrate that the -520 nucleotides upstream of the rat HNF-3 alpha gene were sufficient for cell-specific expression. We identified binding sites for a liver and kidney-enriched nuclear factor and a kidney-enriched protein that recognizes two distinct promoter elements. We showed that the rat HNF-3 alpha promoter binds the HNF-3 protein isoforms, which may serve an auto- and/or cross-regulatory role. Furthermore, we showed that cotransfection of the thyroid transcription factor-1 expression vector enhanced HNF-3 alpha promoter activity. We discuss these results with respect to the transcriptional induction of the HNF-3 alpha gene in respiratory epithelium during embryogenesis. Because the HNF-3 alpha promoter region bound nuclear factors in kidney extracts, we used in situ hybridization to demonstrate that it was expressed in the urothelium of the renal pelvis in adult and embryonic kidney. We also report on a novel expression pattern of HNF-3 alpha in the epithelium of the urinary bladder, penile urethra, and the prostate gland, and show that its expression in the intestinal epithelium increases from the proximal duodenum to distal ileum. We also demonstrate that HNF-3 alpha is abundantly expressed in the colonic epithelium. Furthermore, we use the HNF-3 DNA binding consensus sequence to identify putative target genes in the renal pelvis and gut epithelium.

We used immunohistochemical analysis to localize thyroid transcription factor-1 (TTF-1), hepatocyte nuclear factor-3beta (HNF-3beta), prosurfactant proteins B and C (pro-SP-B, pro-SP-C), surfactant protein B (SP-B), and Clara cell secretory protein (CCSP) in developing mouse lung. TTF-1 and HNF-3beta were expressed at the onset of lung morphogenesis (gestational Day 10) and throughout fetal lung development, being detected in the nuclei of airway epithelial cells. TTF-1 was most prominent in distal airway epithelial cells in embryonic lung and HNF-3beta in proximal bronchial and bronchiolar epithelial cells. Pro-SP-B and pro-SP-C were first detected on gestational Day 11, being localized to the cytoplasm of airway epithelial cells. Expression of both pro-proteins was confined to distal airway epithelial cells from gestational Day 12 to Day 16. From gestational Day 17 and thereafter, pro- SP-B was detectable in Type II cells and bronchiolar epithelial cells, whereas pro-SP-C was restricted to Type II cells. SP-B peptide was first detected on gestational Day 17 in the cytoplasm of Type II cells and within the lumen of distal airways. SP-B peptide was detectable only in the cytoplasm of Type II cells in adult lung. CCSP was first detected on gestational Day 17, being localized to the cytoplasm of columnar epithelial cells lining the conducting airways. Pro-SP-B, SF-B, pro-SP-C, and CCSP staining increased before birth. The early expression of TTF-1 and HNF-3beta, preceding and overlapping that of pro-SP-B, mature SP-B, pro-SP-C, and CCSP, supports a regulatory role for TTF-1 and HNF-3beta in lung-specific gene expression.

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) regulate the transcription of numerous liver-enriched genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of proteins known to be critical for determination events in embryogenesis. We have demonstrated previously that two binding sites in the -184 HNF-3 beta promoter are recognized by widely distributed factors and that there is also a critical autoregulatory site, we identified a binding site for a cell-specific factor, LF-H3 beta, that may function in restricting HNF-3 beta gene expression to hepatocytes. Our present study demonstrates that members of the C/EBP and proline and acidic amino acid-rich subfamilies of basic region leucine zipper transcription factors bind the LF-H3 beta site, and cotransfection of HepG2 cells shows that these factors are able to activate an HNF-3 beta promoter reporter construct. The LF-H3 beta-C/EBP binding sequence also confers HNF-3 beta promoter stimulation in response to interleukin (IL)-1 and IL-6. Upstream of this HNF-3 beta proximal promoter region, an IFN-stimulated response element core sequence (-231 to -210) was found that mediates transcriptional induction by IFN-gamma but not IFN-alpha. Gel mobility supershift assay demonstrates that an IFN-gamma-induced protein-DNA complex is disrupted by an antibody specific for interferon regulatory factor-1/interferon-stimulated gene factor-2. Consistent with this finding, we observed that IFN-gamma induction requires ongoing protein synthesis. Surprisingly, the effect of the three cytokines (IL-1, IL-6, and IFN-gamma) in combination as assayed by the same model is not synergistic. HNF-3beta joins the C/EBP family on the list of liver-enriched transcription factors, the expression of which is modulated by cytokines.

Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta and gamma) regulate transcription of the transthyretin (TTR) and numerous other liver-specific genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of developmental regulatory proteins including the Drosophila homeotic fork head (fkh) protein. The mammalian HNF-3/fkh family consists of at least thirty distinct members and is expressed in a variety of different cellular lineages. In addition to the winged helix motif, several HNF-3/fkh family members also share homology within transcriptional activation region II and III sequences. In the present study we further define the sequence boundaries of the HNF-3 beta N-terminal transcriptional activation domain to extend from amino acids 14 to 93 and include conserved region IV and V sequences. We also demonstrate that activity of the HNF-3 N-terminal domain was diminished by mutations which altered a putative alpha-helical structure located between amino acid residues 14 and 19. However, transcriptional activity was not affected by mutations which eliminated two conserved casein kinase I sites or increased the number of acidic amino acid residues in the N-terminal domain. Furthermore, we determined that the nuclear localization signal overlaps with the winged helix DNA-binding motif. These results suggest that conserved sequences within the winged helix motif of the HNF-3/fkh family may be involved not only in DNA recognition, but also in nuclear targeting.

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.

A 100-amino-acid DNA-binding motif, known as the winged helix, was first identified in the mammalian hepatocyte nuclear factor-3 (HNF-3) and Drosophila fork head family of transcription factors. Subsequently, more than 40 different genes that contain the winged helix motif have been identified. In the studies described here, we have determined the murine chromosomal location of eight members of this gene family, HFH-1, HFH-3, HFH-4, HFH-5, HFH-6, HFH-8, BF-1, and BF-2, by interspecific backcross analysis. These genes, designated HNF-3 fork head homolog 1 (Hfh1), Hfh3, Hfh4, Hfh5, Hfh6, Hfh8, Hfh9, and Hfh10, respectively, mapped to 6 different mouse autosomes and are thus well dispersed throughout the mouse genome. Based on this mapping information, we predict the chromosomal location of these genes in humans and discuss the potential of these genes as candidates for uncloned mouse mutations.

The hepatocyte nuclear factor-3 (HNF-3)/forkhead (fkh) proteins consist of an extensive family of tissue-specific and developmental gene regulators which share homology within the winged helix DNA binding motif. We report on the isolation of a new family member, HNF-3/forkhead homolog 8 (HFH-8), from lung cDNA libraries and the derivation of the complete amino acid sequences for the HFH-8 protein as well as previously identified HFH-1 and HFH-4 proteins. The HFH proteins contain several sequence motifs found in activation domains of other transcription factors and HNF-3/fkh family members. In situ hybridization with the HNF-3, HFH-4, and HFH-8 probes in adult lung demonstrate that the HNF-3/fkh cellular expression patterns are regionally specified. Whereas HNF-3 alpha and HNF-3 beta are normally coexpressed in the hepatocyte, their expression patterns in the lung are different. The HNF-3 alpha and HFH-4 genes are coexpressed in the bronchiolar epithelium (clara cells), whereas the HNF-3 beta probe exhibits prominent hybridization with the smooth muscle surrounding arterioles and bronchioles. In contrast, HFH-8 probes labeled the type II pneumocyte cells lining the respiratory surfaces of terminal bronchioles and alveolar sac. We have identified an HNF-3 consensus DNA binding sequence in the proximal surfactant protein B (SPB) promoter region (SPB-f2, -78 to -88). SPB gene transcription is restricted to bronchiolar and alveolar epithelium which colocalizes with the expression pattern of the HNF-3 alpha and HFH-8 genes, respectively. We show that the SPB-f2 sequence is recognized by both HNF-3 alpha and HFH-8 proteins and that these cDNA expression vectors activate the SPB promoter in cotransfection assays through the HNF-3 consensus sequence. Our results suggest that SPB promoter activity is regulated by HNF-3 alpha and HFH-8 proteins in a cell type-specific manner.

IGF binding protein-1 is an important short-term modulator of IGF bioavailability. Hepatic transcription of IGFBP-1 is increased by glucocorticoids and suppressed by insulin. We previously identified adjacent glucocorticoid and insulin response sequences approximately 90 bp 5' to the RNA cap site in the IGFBP-1 promoter. This insulin response sequence contains a sequence highly related (10/12 bases) to a consensus HNF-3 binding sequence. Gel shift and supershift studies confirm that this sequence binds HNF-3 alpha, beta and gamma. Co-expression of HNF-3 beta enhances IGFBP-1 promoter activity in NIH-3T3 cells. Mutation of this HNF-3 binding sequence disrupts this effect as well as the ability of glucocorticoids to stimulate and of insulin to inhibit IGFBP-1 promoter activity in H4IIE and HepG2 hepatoma cells. HNF-3 binding at this site may play an important role in the multihormonal regulation of hepatic IGFBP-1 gene expression.

We present evidence demonstrating that the liver-enriched transcription factor HNF-3 alpha is activated upon retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells. We have detected increases in the DNA binding activity and mRNA level of HNF-3 alpha. Both are reflections of the actual activation mechanism at the level of transcriptional initiation, which we showed with the help of HNF-3 alpha promoter constructs. Time course studies clearly show that HNF-3 alpha activation is a transient event. Employing Northern blots, HNF-3 alpha mRNA can be detected between 16 and 24 hours post-differentiation, reaches its zenith at approximately 1 day, and then declines to virtually undetectable levels. F9 cells can give rise to three distinct differentiated cell types; visceral endoderm, parietal endoderm, and primitive endoderm. We have clearly shown that HNF-3 alpha stimulation occurs upon primitive endoderm formation. In addition, the transcription factor is also activated during the induction of cell lineages that give rise to parietal and visceral endoderm. HNF-3 alpha stimulation upon visceral endoderm differentiation is accompanied by the activation of HNF-3 target genes such as transthyretin, suggesting that HNF-3 alpha is involved in the developmental activation of this gene. In contrast, HNF-3 alpha target genes in parietal and primitive endoderm have yet to be identified. However, the stimulation of HNF-3 alpha during primitive endoderm formation, which is an extremely early event during murine embryogenesis, points towards a role for the factor in crucial determination processes that occur early during development.

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.

Hepatocyte nuclear factor (HNF)-3 alpha, -3 beta, and -3 gamma are liver transcription factors that mediate the coordinate expression of a number of hepatocyte-specific genes. The HNF-3 proteins share DNA-binding-domain homology among themselves and with the Drosophila homeotic protein forkhead (fkh). The HNF-3/fkh DNA-binding domain constitutes an uncharacterized protein motif that recognizes its cognate DNA binding site as a monomer. Additional HNF-3/fkh-related proteins are known to be required for determination events during embryogenesis in Drosophila and Xenopus. In this report, we describe the isolation of nine additional HNF-3/fkh homologue (HFH) clones from rodent tissue cDNAs by using both low-stringency hybridization and a polymerase chain reaction protocol. Many of the HFH genes exhibit a tissue-restricted expression pattern and are transcribed in tissues other than liver, including brain, kidney, lung, and intestine. The HNF-3/fkh motif therefore comprises a large gene family of transcription factors that play a role in tissue-specific gene regulation and development.

The hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (alpha, beta, and gamma) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions II and III) and a fourth segment of homology at the amino terminus (region IV). Furthermore, the HNF-3 proteins demonstrate homology with the Drosophila homeotic gene fork head in regions I, II, and III, suggesting that HNF-3 may be its mammalian homolog. In order to define HNF-3 beta protein domains involved in transcriptional activation, we have used a reporter gene, whose transcription is dependent on HNF-3 binding, for hepatoma cell cotransfection assays with expression vectors that produced different truncated HNF-3 beta proteins. A position-independent activation domain which contained conserved regions II and III was identified at the carboxyl terminus of the HNF-3 beta protein (amino acids 361 to 458). Moreover, site-directed mutations that altered the sequences within regions II and III demonstrated their importance to transactivation. The region II-III domain does not possess amino acid sequences in common with other transcription factors and may define a novel activation motif. HNF-3 beta amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-III domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1.

The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type-specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor.

Hepatocyte-specific gene expression requires the interaction of many proteins with multiple binding sites in the regulatory regions. HNF-3 is a site found to be important in the maximal hepatocyte-specific expression of several genes. We find that liver nuclear extracts contain three major binding activities for this site, which we call HNF-3A, HNF-3B, and HNF-3C. Purification from rat liver nuclear extracts of HNF-3A and HNF-3C reveals that each activity corresponds to a distinct polypeptide, as determined by SDS-PAGE. Peptide sequence derived from the most abundant species, HNF-3A, was used for synthesizing probes with which to isolate a cDNA clone of this protein. The encoded protein contains 466 amino acids (48.7 kD) and has binding properties identical to those of the purified protein. A 160-amino-acid region that does not resemble the binding domain of any known transcription factor is essential for DNA binding. The mRNA for HNF-3A is present in the rat liver but not in brain, kidney, intestine, or spleen, and the basis for this difference is cell-specific regulation of HNF-3A gene transcription.

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.

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.

We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).