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Jaagsiekte sheep retrovirus (JSRV) is the causative agent of ovine pulmonary adenocarcinoma, a contagious lung cancer of sheep that arises from type II pneumocytes and Clara cells of the lung epithelium. Studies of the tropism of this virus have been hindered by the lack of an efficient system for viral replication in tissue culture. To map regulatory regions important for transcriptional activation, an in vivo footprinting method that couples dimethyl sulfate treatment and ligation-mediated PCR was performed in murine type II pneumocyte-derived MLE-15 cells infected with a chimeric Moloney murine leukemia virus driven by the JSRV enhancers (DeltaMo+JS Mo-MuLV). In vivo footprints were found in the JSRV enhancers in two regions previously shown to be important for JSRV long terminal repeat (LTR) activity: a binding site for the lung-specific transcription factor HNF-3beta and an E-box element in the distal enhancer adjacent to an NF-kappaB-like binding site. In addition, in vivo footprints were detected in two downstream motifs likely to bind C/EBP and NF-I. Mutational analysis of a JSRV LTR reporter construct (pJS21luc) revealed that the C/EBP binding site is critical for LTR activity, while the putative NF-I binding element is less important; elimination of these sites resulted in 70% and 40% drops in LTR activity, respectively. Electrophoretic mobility shift assays using nuclear extracts from MLE-15 murine Clara cell-derived mtCC1-2 cells with probes corresponding to the NF-I or C/EBP sites revealed several complexes. Antiserum directed against NF-IA, C/EBPalpha, or C/EBPbeta supershifted the corresponding protein-DNA complexes, indicating that these isoforms, which are also important for the expression of several cellular lung-specific genes, may be important for JSRV expression in lung epithelial cells.

Survival during prolonged food deprivation depends on the activation of hepatic gluconeogenesis. Inappropriate regulation of this process is a hallmark of diabetes and other metabolic diseases. Activation of the genes encoding gluconeogenic enzymes is mediated by hormone-responsive transcription factors such as the cyclic AMP response element binding protein (CREB) and the glucocorticoid receptor (GR). Here we show using cell-type-specific gene ablation that the winged helix transcription factor Foxa2 is required for activation of the hepatic gluconeogenic program during fasting. Specifically, Foxa2 promotes gene activation both by cyclic AMP, the second messenger for glucagon, and glucocorticoids. Foxa2 mediates these effects by enabling recruitment of CREB and GR to their respective target sites in chromatin. We conclude that Foxa2 is required for execution of the hepatic gluconeogenic program by integrating the transcriptional response of the hepatocyte to hormonal stimulation.

A complex network of hepatocyte nuclear transcription factors, including HNF6 and Foxa2, regulates the expression of liver-specific genes. The current model, based on in vitro studies, suggests that HNF6 and Foxa2 interact physically. This interaction is thought to synergistically stimulate Foxa2-dependent transcription through the recruitment of p300/CBP by HNF6 and to inhibit HNF6-mediated transcription due to the interference of Foxa2 with DNA binding by HNF6. To test this model in vivo, we utilized hepatocyte-specific gene ablation to study the binding of HNF6 to its targets in the absence of Foxa2. Chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2(loxP/loxP) Alfp.Cre and control mouse livers, and HNF6 binding to its target, Glut2, was determined by quantitative PCR. In contrast to the current model, we found no significant difference in HNF6 occupancy at the Glut2 promoter between Foxa2-deficient and control livers. In order to evaluate the Foxa2/HNF6 interaction model on a global scale, we performed a location analysis using a microarray with 7,000 mouse promoter fragments. Again, we found no evidence that HNF6 binding to its targets in chromatin is reduced in the presence of Foxa2. We also examined the mRNA levels of HNF6 targets in the liver using a cDNA array and found that their expression was similar in Foxa2-deficient and control mice. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2.

The specification of the vertebrate liver is thought to occur in a two-step process, beginning with the establishment of competence within the foregut endoderm for responding to organ-specific signals, followed by the induction of liver-specific genes. On the basis of expression and in vitro studies, it has been proposed that the Foxa transcription factors establish competence by opening compacted chromatin structures within liver-specific target genes. Here we show that Foxa1 and Foxa2 (forkhead box proteins A1 and A2) are required in concert for hepatic specification in mouse. In embryos deficient for both genes in the foregut endoderm, no liver bud is evident and expression of the hepatoblast marker alpha-fetoprotein (Afp) is lost. Furthermore, Foxa1/Foxa2-deficient endoderm cultured in the presence of exogenous fibroblast growth factor 2 (FGF2) fails to initiate expression of the liver markers albumin and transthyretin. Thus, Foxa1 and Foxa2 are required for the establishment of competence within the foregut endoderm and the onset of hepatogenesis.

HOM/C homeobox (Hox) and forkhead box (Fox) factors are reported to be expressed in the foregut endoderm and are subsequently detected in a spatio-temporal pattern during lung development. Some of these factors were reported to influence the expression of lung marker proteins or to modulate lung development. To clarify the molecular mechanisms for generating functional lung cells from progenitor cell populations, we introduced the forkhead box factors, FoxA1 and FoxA2, and the homeobox factor, HoxB3, into the differentiation process in a multipotent hamster lung epithelial M3E3/C3 cell line. Ectopic expression of FoxA2 promoted differentiation to Clara-like cells with up-regulation of the expression of the lung marker proteins, Clara cell-specific 10-kDa protein and surfactant protein-B. In contrast, FoxA1 repressed the differentiation. HoxB3 transfection induced FoxA2 expression transiently at the pre-differentiation stage. The endogenous HoxB3 expression level decreased at later stages of Clara-like cell differentiation, and the attenuation was enhanced by FoxA2 transfection. HoxB3 is a putative upstream regulator that enhances FoxA2 expression at the pre-differentiation stage. In addition, we found that the expression of HoxA4, HoxA5, and HoxC9 increased differentially during Clara-like cell differentiation. These results suggest that HoxB3 may be a putative positive regulator of FoxA2 expression at the pre-differentiation stage, and those interactions of Fox factors and Hox factors could participate in Clara cell differentiation.

Foxa2 (Hnf3beta) is a winged-helix/forkhead transcription factor that regulates gene expression in the liver, pancreatic islets and adipocytes. It is required for the maintenance of glucose and lipid homeostasis. Hyperinsulinemia-mediated inactivation of Foxa2 by nuclear exclusion has recently been implicated in the development of liver steatosis and insulin resistance in three animal models of diabetes. These abnormalities were cured by adenovirus-mediated expression of a constitutively active form of Foxa2 containing a mutated T156 phosphorylation site, which increases fatty acid oxidation and reduces its biosynthesis. Accordingly, the prevention of phosphorylation of Foxa2 was suggested as a pharmacological target for the treatment of obesity and diabetes.

An elevated plasma fibrinogen level is an independent risk factor for cardiovascular disease. Therefore, an understanding of the regulation of fibrinogen expression is important. Inflammation and genetic variation of the fibrinogen beta gene regulate plasma fibrinogen levels, and there are indications that inflammation and genetic variation interact. The aim of our study was to gain more understanding of the regulation of the inflammatory response of the fibrinogen beta gene and to determine the effects of genetic variation. Luciferase reporter gene assays in hepatoma cells, mutation analysis, and electrophoretic mobility shift assays were used to investigate the transcriptional regulation of the fibrinogen beta promoter. We identified a hepatocyte nuclear factor-3 (HNF-3) site located just upstream of previously identified interleukin-6 (IL6)-responsive sequences. This HNF-3 site is essential for a full response of the promoter to IL6, which is a new function for HNF-3. The activity of the CCAAT box/enhancer-binding protein site (located 18 nucleotides downstream of the HNF-3 site and important to the IL6 response) depends on the integrity of the HNF-3 site and vice versa, explaining the necessity of HNF-3 in the IL6 response of the fibrinogen beta promoter. Furthermore, small interfering RNA to HNF-3 reduces the fibrinogen beta mRNA levels. The rare T allele of the -148C/T polymorphism, which is present between the binding sites of HNF-3 and CCAAT box/enhancer-binding protein, interferes with this mechanism, and this polymorphism is in our assay system the only genetic determinant of IL6-induced promoter activity among six polymorphisms in the fibrinogen beta promoter.

Foxa1 and Foxa2 are closely related family members of the Foxa group of transcription factors that are coexpressed in subsets of respiratory epithelial cells throughout lung morphogenesis. Shared patterns of expression, conservation of DNA binding, and transcriptional activation domains indicate that they may serve complementary functions in the regulation of gene expression during lung morphogenesis. Whereas branching morphogenesis of the fetal lung occurs normally in the Foxa2Delta/Delta and Foxa1-/- mice, deletion of both Foxa1 and Foxa2 (in Foxa2Delta/Delta, Foxa1-/- mice) inhibited cell proliferation, epithelial cell differentiation, and branching. Dilation of terminal lung tubules and decreased branching were observed as early as embryonic day 12.5. Foxa1 and Foxa2 regulated Shh (sonic hedgehog) and Shh-dependent genes in the respiratory epithelial cells that influenced the expression of genes in the pulmonary mesenchyme that are required for branching morphogenesis. Epithelial cell differentiation, as indicated by lack of expression of surfactant protein B, surfactant protein C, the Clara cell secretory protein, and Foxj1, was inhibited. Foxa family members regulate signaling and transcriptional programs required for morphogenesis and cell differentiation during formation of the lung.

Aim: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells., Methods: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentiation of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay., Results: Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3beta (HNF-3beta, albumin (ALB), cytokeratin 18 (CK18), transthyretin (TTR), glucose-6-phosphate (G-6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3? ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen., Conclusion: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

N-Oct-3 is a neuronal transcription factor widely expressed in the developing mammalian central nervous system, and necessary to maintain neural cell differentiation. The key role of N-Oct-3 in the transcriptional regulation of a multiplicity of genes is primarily due to the structural plasticity of its so-called 'POU' (acronym of Pit, Oct, Unc) DNA-binding domain. We have recently reported about the unusual dual neuro-specific transcriptional regulation displayed by N-Oct-3 [Blaud,M., Vossen,C., Joseph,G., Alazard,R., Erard,M. and Nieto,L. (2004) J. Mol. Biol., 339, 1049-1058]. To elucidate the underlying molecular mechanisms, we have now made use of molecular modeling, DNA footprinting and electrophoretic mobility shift assay techniques. This combined approach has allowed us to uncover a novel mode of homodimerization adopted by the N-Oct-3 POU domain bound to the neuronal aromatic amino acids de-carboxylase and corticotropin-releasing hormone gene promoters and to demonstrate that this pattern is induced by a structural motif that we have termed 'NORE' (N-Oct-3 responsive element), comprising the 14 bp sequence element TNNRTAAATAATRN. In addition, we have been able to explain how the same structural motif can also induce the formation of a heterodimer in association with hepatocyte nuclear factor 3beta(/Forkhead box a2). Finally, we discuss the possible role of the NORE motif in relation to neuroendocrine lung tumor formation, and in particular the development of small cell lung cancer.

Background: The Foxa family (a1, a2, and a3) of proteins are transcription factors that are central to endodermal development. Recently, Foxa1 has been shown to regulate the transcription of several murine and human prostate specific genes involved in differentiated function by interacting with DNA promoter sequences and androgen receptors. Currently, the developmental expression pattern of Foxa proteins in the murine prostate is unknown., Methods: Male CD-1 mice (embryonic, prepubertal, pubertal, and adult) were used for immunohistochemical analysis of Foxa1, a2, and a3. Immunofluorescence was also performed for androgen receptor and cytokeratin 14 expression. Prostate tissue from pre-pubertal, pubertal, and adult mice were analyzed by Western blot and RT-PCR analysis for Foxa1, a2, and a3 expression., Results: Strong Foxa1 immunoreactivity was observed in epithelial cells throughout prostate development, growth, and adult differentiation. Prominent Foxa2 protein expression was only observed in the early stages of prostate development and was exclusively localized to epithelial cells of the forming buds. RT-PCR analysis identified low Foxa2 mRNA expression levels in the ventral and dorsolateral lobes of the adult prostate, with Foxa2 epithelial cell expression being localized to periurethral regions of the murine adult prostatic complex. Foxa3 expression was not observed in the murine prostate., Conclusions: Foxa proteins represent epithelial cell markers in the murine prostate gland. The early expression of Foxa1 and a2 proteins in prostate formation suggests that these proteins play an important role in normal prostate development, in addition to differentiated secretory function.

The differentiation of insulin-producing beta-cells has been investigated in great detail; however, little is known about the factors that delineate the second-most abundant endocrine lineage, the glucagon-producing alpha-cell. Here we utilize a novel YAC-based Foxa3Cre transgene to delete the winged helix transcription factor Foxa2 (formerly HNF-3beta) in the pancreatic primordium during midgestation. The resulting Foxa2(loxP/loxP); Foxa3Cre mice are severely hypoglycemic and die within the first week of life. Mutant mice are hypoglucagonemic secondary to a 90% reduction of glucagon expression. While the number of mature glucagon-positive alpha-cells is dramatically reduced, specification of alpha-cell progenitors is not affected by Foxa2 deficiency. By marker gene analysis, we show that the expression of the alpha-cell transcription factors Arx, Pax6, and Brn4 does not require Foxa2 in the transcriptional hierarchy governing alpha-cell differentiation.

Growth hormone (GH) regulates the expression of many genes in the liver, and for some genes this regulation may be mediated through liver-enriched transcription factors (LETFs). As part of the long-term goal to investigate the role of LETFs in GH regulation of gene expression in the liver, in this study we determined the effect of GH administration on the expression of 10 LETFs, including hepatocyte nuclear factor (HNF)-1alpha, HNF-1beta, HNF-3alpha, HNF-3beta, HNF-3gamma, HNF-4alpha, HNF-6, CCAAT/enhancer-binding protein (C/EBP) alpha, C/EBPbeta, and albumin D-element binding protein (DBP) in the bovine liver. Eighteen non-lactating and non-pregnant Angus cows were assigned randomly to three groups (n=6 per group) and each cow received a single intramuscular injection of 500 mg slow-release recombinant bovine GH. Liver biopsy samples were taken from group 1 cows 6 h after GH administration, from group 2 cows 24 h after GH administration, and from group 3 cows 1 week after GH administration. Liver biopsies were also collected from group 3 cows 1 day before GH administration, serving as pre-GH controls. The LETF mRNAs in these liver samples were quantified using ribonuclease protection assays with probes generated from bovine LETF cDNAs cloned by standard reverse transcription-polymerase chain reaction. The levels of HNF-3gamma and HNF-6 mRNAs were higher (P< 0.05) in the cows 24 h and 1 week after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-4alpha mRNA were higher (P< 0.05) in the cows 1 week after GH administration than in the other three groups of cows. The levels of C/EBPalpha mRNA were higher (P< 0.05) in the cows 24 h after GH administration than in the untreated cows or the cows 6 h after GH administration. The levels of HNF-3alpha mRNA were higher (P< 0.05) in the cows 6 h after GH administration but were lower (P< 0.05) in the cows 24 h or 1 week after GH administration compared with those in the untreated cows. The levels of DBP mRNA were higher (P< 0.05) in the cows 6 h after GH administration but were lower (P< 0.05) in the cows 24 h after GH administration compared with those in the untreated cows. The levels of HNF-1alpha, HNF-3alpha, and C/EBPbeta mRNAs were not different (P>0.05) between groups. The expression of HNF-1beta mRNA was not detectable. Thus, the expression of six LETFs including HNF-3gamma , HNF-3beta, HNF-4alpha, HNF-6, C/EBPalpha, and DBP mRNAs in the bovine liver is regulated by GH, and these six LETFs may play a role in mediating GH regulation of gene expression in the liver. Among the 10 LETFs, the response of HNF-3gamma to GH is most significant. Cloning and sequencing the promoter region of this gene revealed multiple putative binding elements for signal transducers and activators of transcription 5 (STAT5), suggesting that GH regulation of HNF-3gamma expression in the liver may be mediated through direct binding of STAT5 to the HNF-3gamma promoter.

The regulation of fat and glucose metabolism in the liver is controlled primarily by insulin and glucagon. Changes in the circulating concentrations of these hormones signal fed or starvation states and elicit counter-regulatory responses that maintain normoglycaemia. Here we show that in normal mice, plasma insulin inhibits the forkhead transcription factor Foxa2 by nuclear exclusion and that in the fasted (low insulin) state Foxa2 activates transcriptional programmes of lipid metabolism and ketogenesis. In insulin-resistant or hyperinsulinaemic mice, Foxa2 is inactive and permanently located in the cytoplasm of hepatocytes. In these mice, adenoviral expression of Foxa2T156A, a nuclear, constitutively active Foxa2 that cannot be inhibited by insulin, decreases hepatic triglyceride content, increases hepatic insulin sensitivity, reduces glucose production, normalizes plasma glucose and significantly lowers plasma insulin. These changes are associated with increased expression of genes encoding enzymes of fatty acid oxidation, ketogenesis and glycolysis. Chronic hyperinsulinaemia in insulin-resistant syndromes results in the cytoplasmic localization and inactivation of Foxa2, thereby promoting lipid accumulation and insulin resistance in the liver. Pharmacological intervention to inhibit phosphorylation of Foxa2 may be an effective treatment for type 2 diabetes.

Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor and acts as a coregulator of various nuclear receptors. Herein, we examined a novel cross talk between SHP and a forkhead transcription factor HNF3 (hepatocyte nuclear factor 3/Foxa. Transient transfection assay demonstrated that SHP inhibited the transcriptional activity of all three isoforms of HNF3, HNF3alpha, beta, and gamma. In vivo and in vitro protein interaction studies showed that SHP physically interacted with HNF3. Adenovirus-mediated overexpression of SHP significantly decreased the mRNA levels of glucose-6-phosphase (G6Pase), cholesterol 7-alpha-hydroxylase (CYP7A1), and phosphoenolpyruvate carboxykinase (PEPCK) in HepG2 cells and rat primary hepatocytes. Moreover, the mRNA level of G6Pase was notably increased by down-regulation of SHP with small interfering RNA. Interestingly, HNF3 transactivity was still repressed by SHPDelta128-139 that fails to repress nuclear receptors. Mapping of interaction domain revealed that SHP interacted with forkhead DNA binding domain of HNF3alpha. Gel mobility shift and chromatin immunoprecipitation assays demonstrated that SHP inhibits DNA binding of HNF3. These results suggest that SHP is involved in the regulation of G6Pase, CYP7A1, and PEPCK gene expression via novel mechanism of inhibition of HNF3 activity and expand the role of SHP as a coregulator of other family of transcription factors in addition to nuclear receptors.

An 18-bp enhancer controls cell-specific expression of the calcitonin/calcitonin gene-related peptide gene. The enhancer is bound by a heterodimer of the bHLH-Zip protein USF-1 and -2 and a cell-specific factor from thyroid C cell lines. In this report we have identified the cell-specific factor as the forkhead protein Foxa2 (previously HNF-3beta). Binding of Foxa2 to the 18-bp enhancer was demonstrated using electrophoretic mobility shift assays. The cell-specific DNA-protein complex was selectively competed by a series of Foxa2 DNA binding sites, and the addition of Foxa2 antiserum supershifted the complex. Likewise, a complex similar to that seen with extracts from thyroid C cell lines was generated using an extract from heterologous cells expressing recombinant Foxa2. Interestingly, overexpression of Foxa2 activated the 18-bp enhancer in heterologous cells but only in the presence of the adjacent helix-loop-helix motif. Likewise, coexpression of USF proteins with Foxa2 yielded greater activation than by Foxa2 alone. Unexpectedly, Foxa2 overexpression repressed activity in the CA77 thyroid C cell line, suggesting that Foxa2 may interact with additional cofactors. The stimulatory role of Foxa2 at the calcitonin/calcitonin gene-related peptide gene enhancer was confirmed by short interfering RNA-mediated knockdown of Foxa2. As seen with Foxa2 overexpression, the effect of Foxa2 knockdown also required the adjacent helix-loop-helix motif. These results provide the first evidence for combinatorial control of gene expression by bHLH-Zip and forkhead proteins.

Birth defects, which occur in one out of 20 live births, often affect multiple organs that have common developmental origins. Human and mouse studies indicate that haploinsufficiency of the transcription factor TBX1 disrupts pharyngeal arch development, resulting in the cardiac and craniofacial features associated with microdeletion of 22q11 (del22q11), the most frequent human deletion syndrome. Here, we have generated an allelic series of Tbx1 deficiency that reveals a lower critical threshold for Tbx1 activity in the cardiac outflow tract compared with other pharyngeal arch derivatives, including the palatal bones. Mice hypomorphic for Tbx1 failed to activate expression of the forkhead transcription factor Foxa2 in the pharyngeal mesoderm, which contains cardiac outflow precursors derived from the anterior heart field. We identified a Fox-binding site upstream of Tbx1 that interacted with Foxa2 and was necessary for pharyngeal mesoderm expression of Tbx1, revealing an autoregulatory loop that may explain the increased cardiac sensitivity to Tbx1 dose. Downstream of Tbx1, we found a fibroblast growth factor 8 (Fgf8) enhancer that was dependent on Tbx1 in vivo for regulating expression in the cardiac outflow tract, but not in pharyngeal arches. Consistent with its role in regulating cardiac outflow tract cells Tbx1 gain of function resulted in expansion of the cardiac outflow tract segment derived from the anterior heart field as marked by Fgf10. These findings reveal a Tbx1-dependent transcriptional and signaling network in the cardiac outflow tract that renders mouse cardiovascular development more susceptible than craniofacial development to a reduction in Tbx1 dose, similar to humans with del22q11.

Toward the end of gestation in mammals, the fetal lung undergoes a process of differentiation that is required for transition to air breathing at birth. Respiratory epithelial cells synthesize the surfactant proteins and lipids that together form the pulmonary surfactant complex necessary for lung function. Failure of this process causes respiratory distress syndrome, a leading cause of perinatal death and morbidity in newborn infants. Here we demonstrate that expression of the forkhead gene Foxa2 in respiratory epithelial cells of the peripheral lung controls pulmonary maturation at birth. Newborn mice lacking Foxa2 expression in the lung develop severe pulmonary disease on the first day of life, with all of the morphological, molecular, and biochemical features of respiratory distress syndrome in preterm infants, including atelectasis, hyaline membranes, and the lack of pulmonary surfactant lipids and proteins. RNA microarray analysis at embryonic day 18.5 demonstrated that Foxa2-regulated expression of a group of genes mediating surfactant protein and lipid synthesis, host defense, and antioxidant production. Foxa2 regulates a complex pulmonary program of epithelial cell maturation required for transition to air breathing at birth.

The regulation of insulin secretion by pancreatic beta cells is perturbed in several diseases, including adult-onset (type 2) diabetes and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). The first mouse model for PHHI has a conditional deletion of the gene encoding the winged-helix transcription factor Foxa2 (Forkhead box a2, formerly Hepatocyte nuclear factor 3beta) in pancreatic beta cells. Using isolated islets, we found that Foxa2 deficiency resulted in excessive insulin release in response to amino acids and complete loss of glucose-stimulated insulin secretion. Most PHHI cases are associated with mutations in SUR1 (Sulfonylurea receptor 1) or KIR6.2 (Inward rectifier K(+) channel member 6.2), which encode the subunits of the ATP-sensitive K(+) channel, and RNA in situ hybridization of mutant mouse islets revealed that expression of both genes is Foxa2 dependent. We utilized expression profiling to identify additional targets of Foxa2. Strikingly, one of these genes, Hadhsc, encodes short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase, deficiency of which has been shown to cause PHHI in humans. Hadhsc is a direct target of Foxa2, as demonstrated by cotransfection as well as in vivo chromatin immunoprecipitation experiments using isolated islets. Thus, we have established Foxa2 as an essential activator of genes that function in multiple pathways governing insulin secretion.

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Recent studies have led to a model of the molecular pathway that specifies the endoderm during vertebrate gastrulation. The HMG box transcription factor Sox17 is a key component of this pathway and is essential for endoderm formation; however, the molecular events controlled by Sox17 are largely unknown. We have identified several direct transcriptional targets of Sox17, including Foxa1 and Foxa2. We show that beta-catenin, a component of Wnt signaling pathway, physically interacts with Sox17 and potentiates its transcriptional activation of target genes. We identify a motif in the C terminus of Sox17, which is conserved in all the SoxF subfamily of Sox proteins, and this motif is required for the ability of Sox17 to both transactivate target genes and bind beta-catenin. Nuclear beta-catenin is present in endoderm cells of the gastrula, and depletion of beta-catenin from embryos results in a repression of Sox17 target genes. These data suggest that in a mechanism analogous to Tcf/Lef interacting with beta-catenin, Sox17 and beta-catenin interact to transcribe endodermal target genes.

Sclerosing hemangioma (PSH) is a rare pulmonary tumor, in which two types of tumor cells could be histologically discerned--surface and stromal tumor cells. Nine tumor-tissue specimens from six female Japanese patients were studied, focusing on the distribution of several transcription factors related to lung epithelial development and surfactant proteins and comparing the ultrastructural features of the tumor cells. The immunohistochemical analysis revealed that the surfactant proteins of surfactant apoprotein A, surfactant protein B, and prosurfactant protein C were distributed in many of the surface-lining cells and in a small number of stromal-tumor cells. In addition, the nuclei of the tumor cells stained positive for thyroid transcription factor 1 (TTF)-1, hepatocyte nuclear factor (HNF)-3 alpha, and HNF-3 beta. In situ hybridization staining for TTF-1 showed similar positive signals. Ultrastructurally, two types of tumor cells showed similar features, but stromal tumor cells lost the definitive apico-lateral differentiation compared with the surface tumor cells and showed restricted surface differentiation between the adjacent tumor cells, forming small lumina accompanied by microvilli and occasional multi-vesicle or multi-lamellar bodies. Conclusively, the real tumoral population being undifferentiated stromal cells, the lining cells are fully differentiated type-II pneumonocytes. PSH is a proliferation of rather fetal type-II pneumonocytes (pneumocytoma or pneumoblastoma?).

N Oct-3, a neurospecific POU protein, homodimerizes in a non-cooperative fashion on the neuronal aromatic l-amino acid decarboxylase gene promoter and generates heterodimers with HNF-3beta. Several other neuronal gene promoters, the corticotropin releasing hormone and the aldolase C gene promoters also contain overlapping binding sites for N Oct-3 and HNF-3beta. We have demonstrated that N Oct-3 presents a non-cooperative homodimerization on these two additional targets and can also give rise to heterodimers with HNF-3beta. Surprisingly, despite the high degree of conservation of the respective POU subunits, the ubiquitous POU protein Oct-1 can only form monomers even in the presence of either N Oct-3 or HNF-3beta on these DNA targets. Our data indicate that this difference is correlated with the specific ability of a portion of the N Oct-3 linker to fold as an alpha-helix, a property shared by class III POU proteins. These results suggest that this novel binding pattern permits the heterodimerization of N Oct-3 and HNF-3beta on the neuronal promoters, which could be a key issue in the development of the nervous system and possibly tumors of neural origin.

We showed previously that CCAAT/enhancer binding protein alpha (C/EBP alpha), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP alpha target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3beta (HNF3 beta), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP alpha. We found down-regulation of HNF3 beta expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 beta, as well as hypermethylation of the HNF3 beta promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 beta expression. Conditional expression of HNF3 beta led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 beta is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.

Inherited defects in signaling pathways downstream of the insulin receptor have long been suggested to contribute to human type 2 diabetes mellitus. Here we describe a mutation in the gene encoding the protein kinase AKT2/PKBbeta in a family that shows autosomal dominant inheritance of severe insulin resistance and diabetes mellitus. Expression of the mutant kinase in cultured cells disrupted insulin signaling to metabolic end points and inhibited the function of coexpressed, wild-type AKT. These findings demonstrate the central importance of AKT signaling to insulin sensitivity in humans.

The specification of distinct neuronal cell-types is controlled by inducing signals whose interpretation in distinct areas along the central nervous system provides neuronal progenitors with a precise and typical expression code of transcription factors. To gain insights into this process, we investigated the role of Otx2 in the specification of identity and fate of neuronal progenitors in the ventral midbrain. To achieve this, Otx2 was inactivated by Cre recombinase under the transcriptional control of En1. Lack of Otx2 in the ventrolateral and posterior midbrain results in a dorsal expansion of Shh expression and in a dorsal and anterior rotation of the midbrain-hindbrain boundary and Fgf8 expression. Indeed, in this mutant correct positioning of the ventral site of midbrain-hindbrain boundary and Fgf8 expression are efficiently controlled by Otx1 function, thus allowing the study of the identity and fate of neuronal progenitors of the ventral midbrain in the absence of Otx2. Our results suggest that Otx2 acts in two ways: by repressing Nkx2.2 in the ventral midbrain and maintaining the Nkx6.1-expressing domain through dorsal antagonism on Shh. Failure of this control affects the identity code and fate of midbrain progenitors, which exhibit features in common with neuronal precursors of the rostral hindbrain even though the midbrain retains its regional identity and these neuronal precursors are rostral to Fgf8 expression. Dopaminergic neurons are greatly reduced in number, red nucleus precursors disappear from the ventral midbrain where a relevant number of serotonergic neurons are generated. These results indicate that Otx2 is an essential regulator of the identity, extent and fate of neuronal progenitor domains in the ventral midbrain and provide novel insights into the mechanisms by which neuronal diversity is generated in the central nervous system.

Cytochrome P450 3A4 (CYP3A4) is the major cytochrome P450 present in adult human liver and is involved in the metabolism of over 50% of therapeutic compounds currently in use. Since expression levels of CYP3A4 are regulated by many of these compounds, this raises the potential for drug-drug interactions and subsequent altered efficacy or toxicity of the individual compounds at the dose prescribed. Hence, understanding the molecular mechanisms of CYP3A4 regulation is of key importance in predicting and understanding such interactions. To examine this we have used DNase I footprinting and bioinformatic analysis to identify putative transcription factor binding sites within the 250 base pairs of promoter proximal to the transcription start site. We identified several protected fragments within this region that corresponded to putative binding sites for Sp1, AP2, CCAAT/enhancer binding protein (C/EBPalpha), and hepatic nuclear factor-3 (HNF3), as well as confirming previously identified C/EBPalpha, pregnane X receptor (PXR), and HNF3 binding sites. Sequential site-directed mutagenesis of C/EBPalpha, Sp1, HNF3, and PXR binding sites was next used to examine the role of these sites in basal CYP3A4 expression. Disruption of the C/EBPalpha, HNF3, and PXR binding sites all affected basal expression. Finally, the role of these sites was examined in activation of CYP3A4 expression by rifampicin, metyrapone, clotrimazole, and phenobarbital. Disruption of any of these sites either led to an altered pattern of activation by the xenobiotic, as altered maximal activation, or altered the EC(50) value of activation. Such effects were xenobiotic-specific, with each disrupted site playing a role in the activation of some of the xenobiotics.

Early in vertebrate development, the processes of gastrulation lead to the formation of the three germ layers: ectoderm, mesoderm, and endoderm. The mechanisms leading to the segregation of the endoderm and mesoderm are not well understood. In mid-blastula stage zebrafish embryos, single marginal cells can give rise to both endoderm and mesoderm (reviewed by Warga and Stainier [2002] The guts of endoderm formation. In: Solnica-Krezel L, editor. Pattern formation in zebrafish. Berlin: Springer-Verlag. p 28-47). By the late blastula stage, however, single marginal cells generally give rise to either endoderm or mesoderm. To investigate this segregation of the blastoderm into cells with either endodermal or mesodermal fates, we analyzed the role of Notch signaling in this process. We show that deltaC, deltaD, and notch1 are expressed in the marginal domain of blastula stage embryos and that this expression is dependent on Nodal signaling. Activation of Notch signaling from an early stage leads to a reduction of endodermal cells, as assessed by sox17 and foxA2 expression. We further find that this reduction in endoderm formation by the activation of Notch signaling is preceded by a reduction in the expression of bonnie and clyde (bon) and faust/gata5, two genes necessary for endoderm formation (Reiter et al. [1999] Genes Dev 13:2983-2995; Reiter et al. [2001] Development 128:125-135; Kikuchi et al. [2001] Genes Dev 14:1279-1289). However, activation of Notch signaling in bon mutant embryos leads to a further reduction in endodermal cells, also arguing for a bon-independent role for Notch signaling in endoderm formation. Altogether, these results suggest that Notch signaling plays a role in the formation of the endoderm, possibly in its segregation from the mesoderm., (Copyright 2004 Wiley-Liss, Inc.)

Hepatocyte nuclear factors (HNF) play a critical role in development of the liver. Their roles during liver tumorigenesis and progression of hepatocellular carcinomas (HCC) are, however, poorly understood. To address the role of HNFs in tumor progression, we generated a new experimental model in which a highly differentiated slow-growing transplantable mouse HCC (sgHCC) rapidly gives rise in vivo to a highly invasive fast-growing dedifferentiated variant (fgHCC). This in vivo model has allowed us to investigate the fundamental mechanisms underlying HCC progression. A complete loss of cell polarity, a decrease in cell-cell and cell-extracellular matrix (ECM) adhesion, elevation of telomerase activity, and extinction of liver-specific gene expression accompanies tumor progression. Moreover, cells isolated from fgHCCs acquired the ability to proliferate rapidly in culture. These alterations were coupled with a reduced expression of several liver transcription factors including HNF4, a factor essential for hepatocyte differentiation. Forced re-expression of HNF4alpha1 in cultured fgHCC cells reversed the progressive phenotype and induced fgHCC cells to re-establish an epithelium and reform cell-ECM contacts. Moreover, fgHCC cells that expressed HNF4alpha1 also re-established expression of the profile of liver transcription factors and hepatic genes that are associated with a differentiated hepatocyte phenotype. Importantly, re-expression of HNF4alpha1 in fgHCC reduced the proliferation rate in vitro and diminished tumor formation in congenic recipient mice. In conclusion, loss of HNF4 expression is an important determinant of HCC progression. Forced expression of this factor can promote reversion of tumors toward a less invasive highly differentiated slow-growing phenotype.

Background/aims: The organic anion transporting polypeptides (OATPs) mediate the uptake of numerous amphipathic compounds into hepatocytes. Our aim was to study the expression and regulation of OATP8 (OATP1B3, SLC21A8/SLCO1B3) and OATP-C (OATP1B1, SLC21A6/SLCO1B1) in hepatocellular carcinomas (HCC)., Methods: RNA and protein levels in 13 paired HCC and adjacent non-tumor liver samples were quantified by real-time polymerase chain reaction or Western blot, respectively. The OATP8 and OATP-C gene promoters were characterized by luciferase reporter assays and electrophoretic mobility shift assays (EMSA)., Results: The expression of OATP8 was decreased in 60% of HCC compared to surrounding non-tumor liver tissue, on both the mRNA and protein levels. Expression of the liver-enriched transcription factor hepatocyte nuclear factor 3beta (HNF3beta) was increased in 70% of HCC and correlated inversely with OATP8 mRNA (r=-0.75, P<0.05) and protein. In contrast to OATP8, expression of OATP-C was not significantly decreased in HCC. In transfected Huh7 cells, OATP8 promoter activity was inhibited by 70% when HNF3beta was cotransfected. An HNF3beta binding site was located at nt -39/-23 by EMSA. The OATP-C promoter was not inhibited by HNF3beta., Conclusions: HNF3beta represses transcription of the OATP8 but not the OATP-C gene, providing a mechanism for reduced expression of OATP8 in HCC.

The airways are lined by several distinct epithelial cells that play unique roles in pulmonary homeostasis; however, the mechanisms controlling their differentiation in health and disease are poorly understood. The winged helix transcription factor, FOXA2, is expressed in the foregut endoderm and in subsets of respiratory epithelial cells in the fetal and adult lung. Because targeted mutagenesis of the Foxa2 gene in mice is lethal before formation of the lung, its potential role in lung morphogenesis and homeostasis has not been determined. We selectively deleted Foxa2 in respiratory epithelial cells in the developing mouse lung. Airspace enlargement, goblet cell hyperplasia, increased mucin and neutrophilic infiltration were observed in lungs of the Foxa2-deleted mice. Experimental goblet cell hyperplasia caused by ovalbumin sensitization, interleukin 4 (IL4), IL13 and targeted deletion of the gene encoding surfactant protein C (SP-C), was associated with either absent or decreased expression of Foxa2 in airway epithelial cells. Analysis of lung tissue from patients with a variety of pulmonary diseases revealed a strong inverse correlation between FOXA2 and goblet cell hyperplasia. FOXA2 is required for alveolarization and regulates airway epithelial cell differentiation in the postnatal lung.

Prediction of transcription-factor target sites in promoters remains difficult due to the short length and degeneracy of the target sequences. Although the use of orthologous sequences and phylogenetic footprinting approaches may help in the recognition of conserved and potentially functional sequences, correct alignment of the short transcription-factor binding sites can be problematic for established algorithms, especially when aligning more divergent species. Here, we report a novel phylogenetic footprinting approach, CONREAL, that uses biologically relevant information, that is, potential transcription-factor binding sites as represented by positional weight matrices, to establish anchors between orthologous sequences and to guide promoter sequence alignment. Comparison of the performance of CONREAL with the global alignment programs LAGAN and AVID using a reference data set, shows that CONREAL performs equally well for closely related species like rodents and human, and has a clear added value for aligning promoter elements of more divergent species like human and fish, as it identifies conserved transcription-factor binding sites that are not found by other methods. CONREAL is accessible via a Web interface at http://conreal.niob.knaw.nl/.

We have previously shown that 17-beta-estradiol (E2) and genistein increase the expression of apolipoprotein A-I (apoA-I), the major protein component of HDL, in Hep G2 cells. To elucidate the mechanism mediating the increase in apoA-I gene expression by these compounds, plasmid constructs containing serial deletions of the apoA-I promoter region were generated. The smallest region maintaining response to E2 and genistein spanned the -220 to -148 sequence, and the estrogen antagonist ICI182,780 completely inhibited the E2 and genistein effect. Nuclear extracts from cells treated with E2 and genistein showed increased binding to site B oligonucleotide (-169 to -146), and nuclear extracts from genistein-treated cells showed increased binding to an early growth response factor 1 (Egr-1) oligonucleotide compared to control cells. An increase in the concentrations of Egr-1 and hepatocyte nuclear factor-3beta was observed in nuclear extracts of cells treated with both compounds compared to control cells. Treatment with a specific inhibitor of mitogen-activated protein (MAP) kinase, but not with other inhibitors, abolished the stimulation of apoA-I gene expression by E2 and genistein. These results indicate that the MAP kinase pathway is involved in the regulation of apoA-I gene expression by genistein and E2, possibly through downstream regulation of transcription factors binding to the promoter region.

The winged-helix transcription factor HNF3beta/FoxA2 is expressed in embryonic organizing centers of the gastrulating mouse, frog, fish, and chick. In the mouse, HNF3beta is required for the formation of the mammalian node and notochord, and can induce ectopic floor plate formation when misexpressed in the developing neural tube; HNF3beta expression in the extraembryonic endoderm is also necessary for the proper morphogenesis of the mammalian primitive streak. In the frog Xenopus laevis, several lines of evidence suggest that the related winged-helix factor Pintallavis functions as the ortholog of mammalian HNF3beta in both axial mesoderm and neurectoderm; the role of Xenopus HNF3beta itself, however, has not been clearly defined, and is the subject of this study. HNF3beta is widely expressed in the vegetal pole but, as previously suggested, is excluded from the gastrula-stage mesoderm. We find that expression of an HNF3beta-Engrailed repressor fusion protein induces ectopic axes and inhibits head formation in Xenopus embryos, while ectopic HNF3beta inhibits mesoderm and anterior endoderm formation in explant assays and in vivo. Our studies suggest that HNF3beta target genes function to limit the extent of mesoderm formation in the Xenopus gastrula, and point to related roles for Xenopus HNF3beta and the extraembryonic component of mammalian HNF3beta during vertebrate gastrulation.

Hepatocyte nuclear factors 3 alpha, beta, and gamma (Foxa-1, -2, and -3) are transcriptional activators of important metabolic genes in the liver that are suppressed by the actions of insulin. Here, we show that the activation of phosphatidylinositol 3-kinase-Akt by insulin induces Foxa-2 phosphorylation, nuclear exclusion, and inhibition of Foxa-2-dependent transcriptional activity. Foxa-2 physically interacts with Akt, a key mediator of the phosphatidylinositol 3-kinase pathway and is phosphorylated at a single conserved site (T156) that is absent in Foxa-1 and Foxa-3 proteins. This Akt phosphorylation site in Foxa-2 is highly conserved from mammals to insects. Mutant Foxa-2T156A is resistant to Akt-mediated phosphorylation, nuclear exclusion, and transcriptional inactivation of Foxa-2-regulated gene expression. These results implicate an evolutionarily conserved mechanism in the regulation of Foxa-2-dependent transcriptional control by extracellular signals such as insulin.

Mouse embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of developing blastocysts and have multi-lineage differentiation ability. We previously reported that ES cells can be made to differentiate into hepatocytes possessing high metabolic activities by transfection of hepatocyte nuclear factor-3beta (HNF-3beta). In the present study, we investigated the expression of hepatobiliary organic anion transporters and bilirubin uridine diphosphate glucuronosyltransferase (ugt1a1) in undifferentiated and differentiating HNF-3beta-transfected ES (HNF-3beta-ES) cells. The expression of organic anion transporting polypeptide 1 (oatp1), multidrug resistance-associated protein 1 (mrp1), mrp2, mrp3, and ugt1a1 was not seen in the undifferentiated HNF-3beta-ES cells by RT-PCR, whereas all were expressed in differentiating HNF-3beta-ES cells. Protein expression for oatp1, mrp1, mrp2, mrp3, and ugt1a1 was also observed in the differentiating HNF-3beta-ES cells by Western blotting. An immunofluorescence examination revealed that oatp1 was co-located with desmoplakin, a marker for the basolateral (sinusoidal) membrane, and mrp2 was co-localized with CD26, a marker for the apical (canalicular) membrane, though they were both expressed throughout most of the cell membranes.

Background: Prothrombin is a key component in blood coagulation. Overexpression of prothrombin leads to an increased risk of venous thrombosis. Therefore, the study of the transcriptional regulation of the prothrombin gene may help to identify mechanisms of overexpression., Objectives: The aim of our study was to localize the regions within the prothrombin enhancer responsible for its activity, to identify the proteins binding to these regions, and to establish their functional importance., Methods: We constructed a set of prothrombin promoter 5' deletion constructs containing the firefly luciferase reporter gene, which were transiently transfected in HepG2, HuH7 and HeLa cells. Putative transcription factor (TF) binding sites were evaluated by electrophoretic mobility shift assays. The functional importance of each TF binding site was evaluated by site directed mutagenesis and transient transfection of the mutant constructs., Results: We confirmed the major contribution of the enhancer region to the transcriptional activity of the prothrombin promoter. Analysis of this region revealed putative binding sites for hepatocyte nuclear factor HNF4, HNF3-beta and specificity protein(Sp)1. We identified six different TFs binding to three evolutionary conserved sites in the enhancer: HNF4-alpha (site 1), HNF1-alpha, HNF3-beta and an as yet unidentified TF (site 2) and the ubiquitously expressed TFs Sp1 and Sp3 (site 3). Mutagenesis studies showed that loss of binding of HNF3-beta resulted in a considerable decrease of enhancer activity, whereas loss of HNF4-alpha or Sp1/Sp3 resulted in milder reductions., Conclusions: The prothrombin enhancer plays a major role in regulation of prothrombin expression. Six different TFs are able to bind to this region. At least three of these TFs, HNF4-alpha, HNF3-beta and Sp1/Sp3, are important in regulation of prothrombin expression.

Hepatocyte nuclear factors-3 (Foxa-1-3) are winged forkhead transcription factors that regulate gene expression in the liver and pancreatic islets and are required for normal metabolism. Here we show that Foxa-2 is expressed in preadipocytes and induced de novo in adipocytes of genetic and diet-induced rodent models of obesity. In preadipocytes Foxa-2 inhibits adipocyte differentiation by activating transcription of the Pref-1 gene. Foxa-2 and Pref-1 expression can be enhanced in primary preadipocytes by growth hormone, suggesting that the antiadipogenic activity of growth hormone is mediated by Foxa-2. In differentiated adipocytes Foxa-2 expression leads to induction of gene expression involved in glucose and fat metabolism, including glucose transporter-4, hexokinase-2, muscle-pyruvate kinase, hormone-sensitive lipase, and uncoupling proteins-2 and -3. Diet-induced obese mice with haploinsufficiency in Foxa-2 (Foxa-2+/-) develop increased adiposity compared with wild-type littermates as a result of decreased energy expenditure. Furthermore, adipocytes of these Foxa-2+/- mice exhibit defects in glucose uptake and metabolism. These data suggest that Foxa-2 plays an important role as a physiological regulator of adipocyte differentiation and metabolism.

Side population (SP) cells are a rare subset of cells found in various tissues that are highly enriched for stem cell activity. SP cells can be isolated by dual-wavelength flow cytometry because of their capacity to efflux Hoechst dye, a process mediated by the ATP-binding cassette transporter breast cancer resistance protein (Bcrp) 1. By performing flow cytometry of enzymedigested mouse lung stained with Hoechst dye, we found that SP cells comprise 0.03-0.07% of total lung cells and are evenly distributed in proximal and distal lung regions. By RT-PCR, we found that lung SP cells express hepatocyte nuclear factor-3beta, but not thyroid transcription factor-1. Surface marker analysis revealed lung SP cells to be stem cell antigen 1 positive, Bcrp1 positive, lineage marker negative, and heterogeneous at the CD45 locus. As expected, we did not detect lung SP cells in Bcrp1-deficient animals. We, therefore, employed nonisotopic in situ hybridization and immunostaining for Bcrp1 as a strategy to localize these cells in vivo. Expression was observed in distinct lung cell types: bronchial and vascular smooth muscle cells and round cells within the distal air space. We confirmed the expression of Bcrp1 in primary bronchial smooth muscle cell cultures (BSMC) and in lavaged distal airway cells, but neither possessed the capacity to efflux Hoechst dye. In BSMC, Bcrp1 was localized to an intracellular compartment, suggesting that the molecular site of Bcrp1 expression regulates SP phenotype.

The Forkhead box (Fox) transcription factor Foxa2 (HNF-3beta) and related family members Foxa1 (HNF-3alpha) and Foxa3 (HNF-3gamma) act in concert with other hepatocyte nuclear factors (HNF) to coordinately regulate liver-specific gene expression. To circumvent the hepatic functional redundancy of the Foxa proteins, we used the T-77 transgenic (TG) mouse line in which the -3-kb transthyretin (TTR) promoter functioned to increase hepatocyte expression of the Foxa2 cDNA. Adult TG mice exhibited reduced hepatic glycogen and progressive liver injury, but maintained normal serum levels of glucose, insulin, and glucagon. In this study, we further characterized the postnatal liver defect in TTR-FoxA2 TG mice. The postnatal TG mice displayed significant reduction in serum glucose levels and in hepatocyte glycogen storage without increased serum levels of ketone bodies and free fatty acid suggesting that they are not undergoing a starvation response. We show that TG liver developed a substantial transient steatosis, which reached a maximum at postnatal day 5 and is associated with increased expression of hepatic genes involved in fatty acid and triglyceride synthesis, lipid beta-oxidation, and amino acid biosynthesis. Furthermore, transmission electron microscopy analysis of postnatal TG liver revealed extensive mitochondrial membrane damage, which is likely due to reactive oxygen species generated from lipid beta-oxidation. In conclusion, our model proposes that in response to reduction in hepatocyte glycogen storage, the TTR-Foxa2 TG mice survive by maintaining sufficient serum levels of glucose through gluconeogenesis using deaminated amino acids with dicarboxylate products of peroxisomal lipid beta-oxidation shuttled through the tricarboxylic acid cycle.

3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) is a potent hepatocarcinogen in rats and a weak carcinogen in mice, whereas o-aminoazotoluene (OAT) is a potent hepatocarcinogen in mice but weak hepatocarcinogen in rats. They significantly suppress glucocorticoid induction of tyrosine aminotransferase (TAT) in the liver of sensitive animals and have minor effect on the induction of this enzyme in the liver of resistant animals (3'-MeDAB-treated mice and OAT-treated rats). The inhibitory effect of these carcinogens is realized at the level of gene transcription (decreased accumulation of TAT mRNA). This effect is mediated via reduction of DNA-binding activity of transcription factor HNF3 (without decrease of its content) without any involvement of the glucocorticoid receptor. It was shown that carcinogens influence DNA-binding activity of HNF3 via an unknown nuclear factor.

The MAP1B (Mtap1b) promoter presents two evolutionary conserved overlapping homeoproteins and Hepatocyte nuclear factor 3beta (HNF3beta/Foxa2) cognate binding sites (defining putative homeoprotein/Fox sites, HF1 and HF2). Accordingly, the promoter domain containing HF1 and HF2 is recognized by cerebellum nuclear extracts containing Engrailed and Foxa2 and has regulatory functions in primary cultures of embryonic mesmetencephalic nerve cells. Transfection experiments further demonstrate that Engrailed and Foxa2 interact physiologically in a dose-dependent manner: Foxa2 antagonizes the Engrailed-driven regulation of the MAP1B promoter, and vice versa. This led us to investigate if Engrailed and Foxa2 interact directly. Direct interaction was confirmed by pull-down experiments, and the regions participating in this interaction were identified. In Foxa2 the interacting domain is the Forkhead box DNA-binding domain. In Engrailed, two independent interacting domains exist: the homeodomain and a region that includes the Pbx-binding domain. Finally, Foxa2 not only binds Engrailed but also Lim1, Gsc and Hoxa5 homeoproteins and in the four cases Foxa2 binds at least the homeodomain. Based on the involvement of conserved domains in both classes of proteins, it is proposed that the interaction between Forkhead box transcription factors and homeoproteins is a general phenomenon.

Haploinsufficiency of Tbx1 is likely a major determinant of cardiac and craniofacial birth defects associated with DiGeorge syndrome. Although mice deficient in Tbx1 exhibit pharyngeal and aortic arch defects, the developmental program and mechanisms through which Tbx1 functions are relatively unknown. We identified a single cis-element upstream of Tbx1 that recognized winged helix/forkhead box (Fox)-containing transcription factors and was essential for regulation of Tbx1 transcription in the pharyngeal endoderm and head mesenchyme. The Tbx1 regulatory region was responsive to signaling by Sonic hedgehog (Shh) in vivo. We show that Shh is necessary for aortic arch development, similar to Tbx1, and is also required for expression of Foxa2 and Foxc2 in the pharyngeal endoderm and head mesenchyme, respectively. Foxa2, Foxc1, or Foxc2 could bind and activate transcription through the critical cis-element upstream of Tbx1, and Foxc proteins were required, within their expression domains, for Tbx1 transcription in vivo. We propose that Tbx1 is a direct transcriptional target of Fox proteins and that Fox proteins may serve an intermediary role in Shh regulation of Tbx1.

Interferon gamma (IFN-gamma), a potent cytokine inducing a wide range of immunologic activities, is increased in the airway secondary to viral infection or during an inflammatory response. This increase in IFN-gamma concentration may alter the expression of specific airway epithelial cell genes that regulate adaptation of airway inflammatory responses. One protein induced by IFN-gamma is Clara cell secretory protein (CCSP), which may contribute to the attenuation of airway inflammation. This study was done to investigate the molecular mechanism by which IFN-gamma stimulates the expression of the CCSP gene in mouse transformed Clara cells and transgenic mice. Deletion mapping and linker-scanning mutations demonstrated that IFN-gamma-induced expression of CCSP was regulated, in part, at the level of transcription. In vitro and in vivo studies verified that the minimal IFN-gamma-responsive segment was localized to the proximal 166 bp of the 5'-flanking region. Additionally, IFN-gamma-induced expression of CCSP was mediated indirectly through an interferon regulatory factor-1-mediated increase in hepatocyte nuclear factor-3beta.

In previous studies we used transgenic mice or recombinant adenovirus infection to increase hepatic expression of forkhead box A2 (FoxA2, previously called hepatocyte nuclear factor 3beta [HNF-3beta]), which caused diminished hepatocyte glycogen levels and reduced expression of glucose homeostasis genes. Because this diminished expression of FoxA2 target genes was associated with reduced levels of the Cut-Homeodomain HNF-6 transcription factor, we conducted the present study to determine whether there is a functional interaction between HNF-6 and FoxA2. Human hepatoma (HepG2) cotransfection assays demonstrated that HNF-6 synergistically stimulated FoxA2 but not FoxA1 or FoxA3 transcriptional activity, and protein-binding assays showed that this protein interaction required the HNF-6 Cut-Homeodomain and FoxA2 winged-helix DNA binding domains. Furthermore, we show that the HNF-6 Cut-Homeodomain sequences were sufficient to synergistically stimulate FoxA2 transcriptional activation by recruiting the p300/CBP coactivator proteins. This was supported by the fact that FoxA2 transcriptional synergy with HNF-6 was dependent on retention of the HNF-6 Cut domain LXXLL sequence, which mediated recruitment of the p300/CBP proteins. Moreover, cotransfection and DNA binding assays demonstrated that increased FoxA2 levels caused a decrease in HNF-6 transcriptional activation of the glucose transporter 2 (Glut-2) promoter by interfering with the binding of HNF-6 to its target DNA sequence. These data suggest that at a FoxA-specific site, HNF-6 serves as a coactivator protein to enhance FoxA2 transcription, whereas at an HNF-6-specific site, FoxA2 represses HNF-6 transcription by inhibiting HNF-6 DNA binding activity. This is the first reported example of a liver-enriched transcription factor (HNF-6) functioning as a coactivator protein to potentiate the transcriptional activity of another liver factor, FoxA2.

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3beta, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2-given its importance for midline development also in the mouse-may be one such component.

Hepatitis B virus (HBV) transgenic mice expressing rat hepatocyte nuclear factor 3beta (HNF3beta) were generated by breeding HBV transgenic mice with transgenic mice that constitutively overexpress the rat HNF3beta polypeptide in the liver. HBV 3.5-, 2.4- and 2.1-kb transcripts were reduced 2- to 4-fold in these mice relative to the HBV transgenic mouse controls. In contrast, the abundance of viral replication intermediates was profoundly reduced in HBV transgenic mice by overexpression of HNF3beta. This results, in part, from the preferential reduction in the level of the pregenomic 3.5-kb RNA relative to the precore 3.5-kb RNA. Therefore, it is apparent that increased expression of HNF3beta modestly reduces viral transcription and dramatically inhibits replication in vivo in the HBV transgenic mouse. This suggests that altering the activity of this transcription factor in vivo in chronic HBV carriers might be therapeutically beneficial.

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.