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In this study, we report that the Krüppel-like zinc finger transcription factor Gli-similar 3 (Glis3) is induced during the secondary transition of pancreatic development, a stage of cell lineage specification and extensive patterning, and that Glis3(zf/zf) mutant mice develop neonatal diabetes, evidenced by hyperglycemia and hypoinsulinemia. The Glis3(zf/zf) mutant mouse pancreas shows a dramatic loss of beta and delta cells, contrasting a smaller relative loss of alpha, PP, and epsilon cells. In addition, Glis3(zf/zf) mutant mice develop ductal cysts, while no significant changes were observed in acini. Gene expression profiling and immunofluorescent staining demonstrated that the expression of pancreatic hormones and several transcription factors important in endocrine cell development, including Ngn3, MafA, and Pdx1, were significantly decreased in the developing pancreata of Glis3(zf/zf) mutant mice. The population of pancreatic progenitors appears not to be greatly affected in Glis3(zf/zf) mutant mice; however, the number of neurogenin 3 (Ngn3)-positive endocrine cell progenitors is significantly reduced. Our study indicates that Glis3 plays a key role in cell lineage specification, particularly in the development of mature pancreatic beta cells. In addition, we provide evidence that Glis3 regulates insulin gene expression through two Glis-binding sites in its proximal promoter, indicating that Glis3 also regulates beta-cell function.

During liver development, hepatocytes undergo a maturation process that leads to the fully differentiated state. This relies at least in part on the coordinated action of liver-enriched transcription factors (LETFs), but little is known about the dynamics of this coordination. In this context we investigate here the role of the LETF hepatocyte nuclear factor 6 (HNF-6; also called Onecut-1) during hepatocyte differentiation. We show that HNF-6 knockout mouse fetuses have delayed expression of glucose-6-phosphatase (g6pc), which catalyzes the final step of gluconeogenesis and is a late marker of hepatocyte maturation. Using a combination of in vivo and in vitro gain- and loss-of-function approaches, we demonstrate that HNF-6 stimulates endogenous g6pc gene expression directly via a synergistic and interdependent action with HNF-4 and that it involves coordinate recruitment of the coactivator PGC-1alpha. The expression of HNF-6, HNF-4, and PGC-1alpha rises steadily during liver development and precedes that of g6pc. We provide evidence that threshold levels of HNF-6 are required to allow synergism between HNF-6, HNF-4, and PGC-1alpha to induce time-specific expression of g6pc. Our observations on the regulation of g6pc by HNF-6 provide a model whereby synergism, interdependency, and threshold concentrations of LETFs and coactivators determine time-specific expression of genes during liver development.

During liver development, hepatocytes and biliary cells differentiate from common progenitors called hepatoblasts. The factors that control hepatoblast fate decision are unknown. Here we report that a gradient of activin/TGFbeta signaling controls hepatoblast differentiation. High activin/TGFbeta signaling is required near the portal vein for differentiation of biliary cells. The Onecut transcription factors HNF-6 and OC-2 inhibit activin/TGFbeta signaling in the parenchyma, and this allows normal hepatocyte differentiation. In the absence of Onecut factors, the shape of the activin/TGFbeta gradient is perturbed and the hepatoblasts differentiate into hybrid cells that display characteristics of both hepatocytes and biliary cells. Thus, a gradient of activin/TGFbeta signaling modulated by Onecut factors is required to segregate the hepatocytic and the biliary lineages.

During development, the endoderm gives rise to several organs, including the pancreas and liver. This differentiation process requires spatial and temporal regulation of gene expression in the endoderm by a network of tissue-specific transcription factors whose elucidation is far from complete. These factors include the Onecut protein hepatocyte nuclear factor-6 (HNF-6), which controls pancreas and liver development as shown in our previous work on Hnf6 knock-out embryos. In mammals, HNF-6 has two paralogs, Onecut-2 (OC-2) and OC-3, whose patterns of expression in the adult overlap with that of HNF-6. In the present work, we examine the expression profile of the three Onecut factors in the developing mouse endoderm. We show that HNF-6, OC-2, and OC-3 are expressed sequentially, which defines new steps in endoderm differentiation. By analyzing Hnf6 knock-out embryos we find that HNF-6 is required for expression of the Oc3 gene in the endoderm. We show that OC-3 colocalizes with HNF-6 in the endoderm and in embryonic pancreas and liver. Based on transfection, chromatin immunoprecipitation, and whole embryo electroporation experiments, we demonstrate that HNF-6 can bind to and stimulate the expression of the Oc3 gene. This study identifies a regulatory cascade between two paralogous transcription factors, sheds new light on the interpretation of the Hnf6 knock-out phenotype, and broadens the transcription factors network operating during development of the endoderm, liver, and pancreas.

A network of liver-enriched transcription factors controls differentiation and morphogenesis of the liver. These factors interact via direct, feedback, and autoregulatory loops. Previous work has suggested that hepatocyte nuclear factor (HNF)-6/OC-1 and HNF-3alpha/FoxA1 participate coordinately in this hepatic network. We investigated how HNF-6 controls the expression of Foxa1. We observed that Foxa1 expression was upregulated in the liver of Hnf6(-/-) mouse embryos and in bipotential mouse embryonic liver (BMEL) cell lines derived from embryonic Hnf6(-/-) liver, suggesting that HNF-6 inhibits the expression of Foxa1. Because no evidence for a direct repression of Foxa1 by HNF-6 was found, we postulated the existence of an indirect mechanism. We found that the expression of a mediator and targets of the transforming growth factor beta (TGF-beta) signaling was increased both in Hnf6(-/-) liver and in Hnf6(-/-) BMEL cell lines. Using these cell lines, we demonstrated that TGF-beta signaling was increased in the absence of HNF-6, and that this resulted from upregulation of TGF-beta receptor II expression. We also found that TGF-beta can stimulate the expression of Foxa1 in Hnf6(+/+) cells and that inhibition of TGF-beta signaling in Hnf6(-/-) cells down-regulates the expression of Foxa1. In conclusion, we propose that Foxa1 upregulation in the absence of HNF-6 results from increased TGF-beta signaling via increased expression of the TGF-beta receptor II. We further conclude that HNF-6 inhibits Foxa1 by inhibiting the activity of the TGF-beta signaling pathway. This identifies a new mechanism of interaction between liver-enriched transcription factors whereby one factor indirectly controls another by modulating the activity of a signaling pathway.

The hepatocyte nuclear factor (HNF) 4alpha gene possesses two promoters, proximal P1 and distal P2, whose use results in HNF4alpha1 and HNF4alpha7 transcripts, respectively. Both isoforms are expressed in the embryonic liver, whereas HNF4alpha1 is almost exclusively in the adult liver. A 516-bp fragment, encompassing a DNase I-hypersensitive site associated with P2 activity that is still retained in adult liver, contains functional HNF1 and HNF6 binding sites and confers full promoter activity in transient transfections. We demonstrate a critical role of the Onecut factors in P2 regulation using site-directed mutagenesis and embryos doubly deficient for HNF6 and OC-2 that show reduced hepatic HNF4alpha7 transcript levels. Transient transgenesis showed that a 4-kb promoter region is sufficient to drive expression of a reporter gene in the stomach, intestine, and pancreas, but not the liver, for which additional activating sequences may be required. Quantitative PCR analysis revealed that throughout liver development HNF4alpha7 transcripts are lower than those of HNF4alpha1. HNF4alpha1 represses P2 activity in transfection assays and as deduced from an increase in P2-derived transcript levels in recombinant mice in which HNF4alpha1 has been deleted and replaced by HNF4alpha7. We conclude that although HNF6/OC-2 and perhaps HNF1 activate the P2 promoter in the embryo, increasing HNF4alpha1 expression throughout development causes a switch to essentially exclusive P1 promoter activity in the adult liver.

The pancreas develops from the endoderm to give rise to ducts, acini and islets of Langerhans. This process involves extracellular signals of the Transforming Growth Factor beta (TGFbeta) family. The aim of this work was to study the effects of activin A, a member of this family, whose potential role in pancreas differentiation is controversial. To this end, we used pancreatic explants from E12.5 mouse embryos. In culture these explants exhibited spontaneous growth, epithelial morphogenesis and endocrine and exocrine differentiation. Exposure to activin A did not affect exocrine or endocrine differentiation. Surprisingly, activin A induced in the explants the appearance of a large contractile structure surrounded by a cylindrical epithelium, a thick basal lamina and a smooth muscle layer. This structure, the formation of which was prevented by follistatin, was typical of an intestinal wall. Consistent with this interpretation, activin A rapidly induced in the explants the mRNAs for fatty acid binding proteins (FABPs), which are markers of the intestine, but not of the pancreas. We also found that induction of the FABPs was preceded by induction of Sonic hedgehog (Shh), a known inducer of intestinal differentiation in the endoderm. Activin B induced neither Shh nor intestinal differentiation. The activin A-mediated intestinal differentiation was blocked by cyclopamine, an inhibitor of Hedgehog signaling, and it was mimicked by Shh. We conclude that activin A does not appear to affect the exocrine or endocrine components of the pancreas, but that it can promote differentiation of pancreatic tissue into intestine via a Shh-dependent mechanism. These findings illustrate the plasticity of differentiation programs in response to extracellular signals in the pancreas and they shed new light on the regulation of pancreas and intestinal development.

During pancreatic organogenesis endocrine cells arise from non self-renewing progenitors that express Ngn3. The precursors that give rise to Ngn3+ cells are presumably located within duct-like structures. However, the nature of such precursors is poorly understood. We show that, at E13-E18, the embryonic stage during which the major burst of beta-cell neogenesis takes place, pancreatic duct cells express Hnf1beta, the product of the maturity-onset diabetes of the young type 5 (MODY5) gene. Ngn3+ cells at this stage invariably cluster with mitotically competent Hnf1beta+ cells, and are often intercalated with these cells in the epithelium that lines the lumen of primitive ducts. We present several observations that collectively indicate that Hnf1beta+ cells are the immediate precursors of Ngn3+ cells. We furthermore show that Hnf1beta expression is markedly reduced in early pancreatic epithelial cells of Hnf6-deficient mice, in which formation of Ngn3+ cells is defective. These findings define a precursor cellular stage of the embryonic pancreas and place Hnf1beta in a genetic hierarchy that regulates the generation of pancreatic endocrine cells.

Mouse genetic models have helped to identify transcription factors that are expressed by hemopoietic cells and control their differentiation into lymphoid cells. However, little is known on transcription factors that are involved in this process, but are expressed in nonhemopoietic cells of the microenvironment. We show in this study that inactivation of the gene coding for hepatocyte nuclear factor-6 (HNF-6) in mice led to B lymphopenia in the bone marrow and spleen. This phenotype disappeared shortly after birth when fetal B lymphopoiesis is no longer active, pointing to a defect in fetal liver. Indeed, the number of B cells was decreased in this organ as well. An analysis of B cell developmental markers in fetal liver cells showed that B lymphopoiesis was impaired just beyond the pre-pro B cell stage. Hemopoietic cells from hnf6(-/-) fetal liver could reconstitute the lymphoid system when injected into scid mice. Because parenchymal cells, but not hemopoietic cells, expressed hnf6 in normal liver, we concluded that HNF-6 controls B lymphopoiesis in fetal liver and that HNF-6 exerts this control indirectly by acting in parenchymal cells. The involvement, in the B cell defect of hnf6(-/-) fetuses, of genes known to exert such an indirect control was ruled out by expression analysis, including microarrays, and by in vivo rescue experiments. This work identifies HNF-6 as the first noncell-intrinsic transcription factor known to control B lymphopoiesis specifically in fetal liver.

The pancreas derives from cells in the ventral and dorsal foregut endoderm that express the transcription factor Pdx-1. These specified cells give rise to the precursors of the endocrine, ductal, and exocrine pancreatic cells. The identification of transcription factors that regulate the onset of Pdx-1 expression is therefore essential to understand pancreas development. No such factor that acts both in the ventral and in the dorsal endoderm is known. We showed previously that the Onecut transcription factor HNF-6 promotes differentiation of the endocrine cell precursors in which it stimulates expression of the proendocrine gene Ngn-3. By analyzing the phenotype of HNF-6 null mice, we now demonstrate that HNF-6 also controls an earlier step in pancreas development. Indeed, the pancreas of Hnf6(-/-) mice was hypoplastic. This did not result from decreased proliferation or from increased apoptosis, but from retarded pancreatic specification of endodermal cells. The onset of Pdx-1 expression was delayed both in the ventral and in the dorsal endoderm, leading to a reduction in the number of endodermal cells expressing Pdx-1 at the time of pancreatic budding. In normal embryos, HNF-6 was detected in the endoderm prior to the expression of Pdx-1. Moreover, HNF-6 could directly stimulate the Pdx1 promoter. Our data therefore identify HNF-6 as the first factor known to control Pdx-1 expression both in the ventral and in the dorsal endoderm. We conclude that HNF-6 controls the timing of pancreas specification and that HNF-6 acts upstream of Pdx-1 in this developmental process. Together with the known role of HNF-6 in pancreatic endocrine cell differentiation, our data point to HNF-6 as a key regulator of pancreas development.

Aims/hypothesis: Embryonic stem cells, when grown as embryoid bodies, spontaneously generate insulin-producing cells which could be used in therapy of diabetes mellitus, provided that their selection and differentiation are optimized. To achieve such optimization, one needs to know whether the differentiation of cells in embryoid bodies mimicks that of pancreatic beta cells in embryos. To address this question we verified if the differentiation of the insulin-producing cells in embryoid bodies requires Hepatocyte Nuclear Factor-6 (HNF-6), a transcription factor known to control pancreatic endocrine differentiation in embryos., Methods: We generated mouse Hnf6-/- embryonic stem cells and grew them as embryoid bodies. The expression of HNF-6, insulin, and transcription factors that are regulated by HNF-6 in developing pancreas was compared in wild-type and Hnf6-/- embryoid bodies., Results: No difference was observed in the expression of insulin between wild-type and Hnf6-/-embryoid bodies. In both cases insulin was expressed in the outer layer of cells, which is similar to the visceral endoderm. In wild-type embryoid bodies HNF-6 was transiently expressed in the outer layer of cells, but was not co-expressed with insulin. The expression of genes that are targets of HNF-6 in developing pancreas was unaffected in Hnf6-/-embryoid bodies., Conclusion/interpretation: In contrast to the development of pancreatic beta cells, the differentiation of insulin-producing cells in embryoid bodies did not require HNF-6. Thus, the differentiation mechanism of insulin-producing cells in embryoid bodies differs from that of the beta cells and it is likely to resemble that of insulin-producing cells in the visceral endoderm.

The rat alpha-fetoprotein ( afp ) gene is controlled by three enhancers whose function depends on their interaction with liver-enriched transcription factors. The afp enhancer III, located at -6 kb, is composed of three regions that act in synergy. Two of these regions, called s1 and s2, contain a putative binding site for hepatocyte nuclear factor-6 (HNF-6). This factor is the prototype of the ONECUT family of cut-homoeodomain proteins and is a known regulator of liver gene expression in adults and during development. We show here that the two splicing isoforms of HNF-6 bind to a site in the s1 region and in the s2 region. The core sequence of the s1 site corresponds to none of the known HNF-6 binding sites. Nevertheless, the binding properties of the s1 site are identical with those of the s2 site and of previously characterized HNF-6 binding sequences. The HNF-6 consensus should therefore be rewritten as DRRTCVATND. Binding of HNF-6 to the s1 and s2 sites requires both the cut and the homoeo domains, is co-operative and induces DNA bending. HNF-6 strongly stimulates the activity of the afp enhancer III in transient transfection experiments. This effect requires the stereo-specific alignment of the two HNF-6 sites. Moreover, HNF-6 stimulates the enhancer in synergy with the retinoic-acid-receptor-related orphan receptor alpha (RORalpha), which binds to a neighbouring site in the s1 region. Thus expression of the afp gene requires functional interactions between HNF-6 molecules and between HNF-6 and RORalpha.

Aims/hypothesis: Glucokinase plays a key role in glucose homeostasis and the expression of its gene is differentially regulated in pancreatic beta cells and in the liver through distinct promoters. The factors that determine the tissue-specific expression of the glucokinase gene are not known. Putative binding sites for hepatocyte nuclear factor (HNF)-6, the prototype of the ONECUT family of transcription factors, are present in the hepatic promoter of the glucokinase gene and hnf6 knockout mice are diabetic [corrected]. We hypothesized that HNF-6 controls the activity of the hepatic glucokinase promoter., Methods: We tested the binding of recombinant HNF-6 to DNA sequences from the mouse hepatic glucokinase promoter in vitro and the effect of HNF-6 on promoter activity in transfected cells. We investigated in vivo the role of HNF-6 in mice by examining the effect of inactivating the hnf6 gene on glucokinase gene-specific deoxyribonuclease I hypersensitive sites in liver chromatin and on liver glucokinase mRNA concentration., Results: HNF-6 bound to the hepatic promoter of the glucokinase gene and stimulated its activity. Inactivation of the hnf6 gene did not modify the pattern of deoxyribonuclease I hypersensitive sites but was associated with a decrease of liver glucokinase mRNA to half the control value., Conclusions/interpretation: Although HNF-6 is not required to open chromatin of the hepatic promoter of the glucokinase gene, it stimulates transcription of the glucokinase gene in the liver. This could partly explain the diabetes observed in hnf6 knockout mice.

During liver development, hepatoblasts differentiate into hepatocytes or biliary epithelial cells (BEC). The BEC delineate the intrahepatic and extrahepatic bile ducts, and the gallbladder. The transcription factors that control the development of the biliary tract are unknown. Previous work has shown that the onecut transcription factor HNF6 is expressed in hepatoblasts and in the gallbladder primordium. We now show that HNF6 is also expressed in the BEC of the developing intrahepatic bile ducts, and investigate its involvement in biliary tract development by analyzing the phenotype of Hnf6(-/-) mice. In these mice, the gallbladder was absent, the extrahepatic bile ducts were abnormal and the development of the intrahepatic bile ducts was perturbed in the prenatal period. The morphology of the intrahepatic bile ducts was identical to that seen in mice whose Hnf1beta gene has been conditionally inactivated in the liver. HNF1beta expression was downregulated in the intrahepatic bile ducts of Hnf6(-/-) mice during development. Furthermore, we found that HNF6 can stimulate the Hnf1beta promoter. We conclude that HNF6 is essential for differentiation and morphogenesis of the biliary tract and that intrahepatic bile duct development is controlled by a HNF6-->HNF1beta cascade.

Transcription factors of the ONECUT class, whose prototype is HNF-6, contain a single cut domain and a divergent homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. The cut domain is required for DNA binding. The homeodomain is required either for DNA binding or for transcriptional stimulation, depending on the target gene. Transcriptional stimulation by the homeodomain involves the F48M50 dyad. We investigate here how HNF-6 stimulates transcription. We identify transcriptionally active domains of HNF-6 that are conserved among members of the ONECUT class and show that the cut domain of HNF-6 participates to DNA binding and, via a LXXLL motif, to transcriptional stimulation. We also demonstrate that, on a target gene to which HNF-6 binds without requirement for the homeodomain, transcriptional stimulation involves an interaction of HNF-6 with the coactivator CREB-binding protein (CBP). This interaction depends both on the LXXLL motif of the cut domain and on the F48M50 dyad of the homeodomain. On a target gene for which the homeodomain is required for DNA binding, but not for transcriptional stimulation, HNF-6 interacts with the coactivator p300/CBP-associated factor but not with CBP. These data show that a transcription factor can act via different, sequence-specific, mechanisms that combine distinct modes of DNA binding with the use of different coactivators.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class of cut homeodomain transcription factors. During mouse development, HNF-6 is expressed in the epithelial cells that are precursors of the exocrine and endocrine pancreatic cells. We have investigated the role of HNF-6 in pancreas differentiation by inactivating its gene in the mouse. In hnf6(-/-) embryos, the exocrine pancreas appeared to be normal but endocrine cell differentiation was impaired. The expression of neurogenin 3 (Ngn-3), a transcription factor that is essential for determination of endocrine cell precursors, was almost abolished. Consistent with this, we demonstrated that HNF-6 binds to and stimulates the ngn3 gene promoter. At birth, only a few endocrine cells were found and the islets of Langerhans were missing. Later, the number of endocrine cells increased and islets appeared. However, the architecture of the islets was perturbed, and their beta cells were deficient in glucose transporter 2 expression. Adult hnf6(-/-) mice were diabetic. Taken together, our data demonstrate that HNF-6 controls pancreatic endocrine differentiation at the precursor stage and identify HNF-6 as the first positive regulator of the proendocrine gene ngn3 in the pancreas. They also suggest that HNF-6 is a candidate gene for diabetes mellitus in humans.

HNF-6 is a tissue-restricted transcription factor that participates in the regulation of several genes in liver. We reported earlier that in adult rats, HNF-6 mRNA concentration in liver drops to almost undetectable levels after hypophysectomy and returns to normal after 1 week of GH treatment. We now show that this results from a rapid effect of GH, and we characterize its molecular mechanism. In hypophysectomized rats, HNF-6 mRNAs increased within 1 h after a single injection of GH. The same GH-dependent induction was reproduced on isolated hepatocytes. To determine whether GH regulates hnf6 expression at the gene level, we studied its promoter. DNA binding experiments showed that 1) the transcription factors STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) bind to sites located around -110 and -650, respectively; and 2) STAT5 binding is induced and HNF-4 binding affinity is increased in liver within 1 h after GH injection to hypophysectomized rats. Using transfection experiments and site-directed mutagenesis, we found that STAT5 and HNF-4 stimulated transcription of an hnf6 gene promoter-reporter construct. Furthermore, GH stimulated transcription of this construct in cells that express GH receptors. Consistent with our earlier finding that HNF-6 stimulates the hnf4 and hnf3beta gene promoters, GH treatment of hypophysectomized rats increased the liver concentration of HNF-4 and HNF-3beta mRNAs. Together, these data demonstrate that GH stimulates transcription of the hnf6 gene by a mechanism involving STAT5 and HNF-4. They show that HNF-6 participates not only as an effector, but also as a target, to the regulatory network of liver transcription factors, and that several members of this network are GH regulated.

Glucocorticoids exert their effects on gene transcription through ubiquitous receptors that bind to regulatory sequences present in many genes. These glucocorticoid receptors are present in all cell types, yet glucocorticoid action is controlled in a tissue-specific way. One mechanism for this control relies on tissue-specific transcriptional activators that bind in the vicinity of the glucocorticoid receptor and are required for receptor action. We now describe a gene-specific and tissue-specific inhibitory mechanism through which glucocorticoid action is repressed by a tissue-restricted transcription factor, hepatocyte nuclear factor-6 (HNF-6). HNF-6 inhibits the glucocorticoid-induced stimulation of two genes coding for enzymes of liver glucose metabolism, namely 6-phosphofructo-2-kinase and phosphoenolpyruvate carboxykinase. Binding of HNF-6 to DNA is required for inhibition of glucocorticoid receptor activity. In vitro and in vivo experiments suggest that this inhibition is mediated by a direct HNF-6/glucocorticoid receptor interaction involving the amino-terminal domain of HNF-6 and the DNA-binding domain of the receptor. Thus, in addition to its known property of stimulating transcription of liver-expressed genes, HNF-6 can antagonize glucocorticoid-stimulated gene transcription.

Transcription factors of the ONECUT class, whose prototype is hepatocyte nuclear factor (HNF)-6, are characterized by the presence of a single cut domain and by a peculiar homeodomain (Lannoy, V. J., Bürglin, T. R., Rousseau, G. G., and Lemaigre, F. P. (1998) J. Biol. Chem. 273, 13552-13562). We report here the identification and characterization of human OC-2, the second mammalian member of this class. The OC-2 gene is located on human chromosome 18. The distribution of OC-2 mRNA in humans is tissue-restricted, the strongest expression being detected in the liver and skin. The amino acid sequence of OC-2 contains several regions of high similarity to HNF-6. The recognition properties of OC-2 for binding sites present in regulatory regions of liver-expressed genes differ from, but overlap with, those of HNF-6. Like HNF-6, OC-2 stimulates transcription of the hnf-3beta gene in transient transfection experiments, suggesting that OC-2 participates in the network of transcription factors required for liver differentiation and metabolism.

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6alpha (465 residues) and beta (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24-q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situ hybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6beta-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5' rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.

Hepatocyte nuclear factor-6 (HNF-6) contains a single cut domain and a homeodomain characterized by a phenylalanine at position 48 and a methionine at position 50. We describe here two isoforms of HNF-6 which differ by the linker that separates these domains. Both isoforms stimulated transcription. The affinity of HNF-6alpha and HNF-6beta for DNA differed, depending on the target sequence. Binding of HNF-6 to DNA involved the cut domain and the homeodomain, but the latter was not required for binding to a subset of sites. Mutations of the F48M50 dyad that did not affect DNA binding reduced the transcriptional stimulation of constructs that do not require the homeodomain for DNA binding, but did not affect the stimulation of constructs that do require the homeodomain. Comparative trees of mammalian, Drosophila, and Caenorhabditis elegans proteins showed that HNF-6 defines a new class, which we call ONECUT, of homeodomain proteins. C. elegans proteins of this class bound to HNF-6 DNA targets. Thus, depending on their sequence, these targets determine for HNF-6 at least two modes of DNA binding, which hinge on the homeodomain and on the linker that separates it from the cut domain, and two modes of transcriptional stimulation, which hinge on the homeodomain.

Protein C is a vitamin K-dependent zymogen of a serine protease that inhibits blood coagulation by proteolytic inactivation of factors Va and VIIIa. Individuals affected by protein C deficiency are at risk for venous thrombosis. One such affected individual was shown earlier to carry a -14 T --> C mutation in the promoter region of the protein C gene. It is shown here that the region around this mutation corresponds to a binding site for the transcription factor hepatocyte nuclear factor (HNF)-6 and that this site completely overlaps an HNF-1 binding site. HNF-6 and HNF-1 bound in a mutually exclusive manner. The -14 T --> C mutation reduced HNF-6 binding. In transient transfection experiments, HNF-6 transactivated the wild-type protein C promoter and introduction of the mutation abolished transactivation by HNF-6. Similar experiments showed that wild-type protein C promoter activity was reduced by cotransfection of an HNF-1 expression vector. This inhibiting effect of HNF-1 was reversed to a stimulatory effect when promoter sequences either upstream or downstream of the HNF-6/HNF-1 site were deleted. It is concluded that HNF-6 is a major determinant of protein C gene activity. Moreover, this is the first report describing the putative involvement of HNF-6 and of an HNF-6 binding site in human pathology.

Hepatocyte nuclear factor-6 (HNF-6) is a liver-enriched transcription factor that contains a single cut domain and a novel type of homeodomain. Here we have studied the developmental expression pattern of HNF-6 in the mouse. In situ hybridization experiments showed that HNF-6 mRNA is detected in the liver at embryonic day (E) 9, at the onset of liver differentiation. HNF-6 mRNA disappeared transiently from the liver between E12.5 and E15. In transfection experiments HNF-6 stimulated the expression of HNF-4 and of HNF-3 beta, two transcription factors known to be involved in liver development and differentiation. HNF-6 was detected in the pancreas from E10.5 onward, where it was restricted to the exocrine cells. HNF-6 was also detected in the developing nervous system. Both the brain and the spinal cord started to express HNF-6 at E9-9.5 in postmitotic neuroblasts. Later on, HNF-6 was restricted to brain nuclei, to the retina, to the ventral horn of the spinal cord, and to dorsal root ganglia. Our observations that HNF-6 contributes to the control of the expression of transcription factors and is expressed at early stages of liver, pancreas, and neuronal differentiation suggest that HNF-6 regulates several developmental programs.

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

Transcription of the human growth hormone gene can start in vitro and in vivo 197 base pairs upstream from the cap site of growth hormone mRNA (Courtois, S. J., Lafontaine, D., and Rousseau, G. G. (1992) J. Biol. Chem. 267, 19736-19743). We have now characterized the mRNA that originates from this optional promoter and have found that it occurs in human hypophysis and placenta but not in 10 other tissues. This mRNA contains an open reading frame for a protein of 107 residues that shares sequence similarity with three domains of hepatic nuclear factor-1alpha. With antibodies directed against a peptide corresponding to the C terminus of this protein, immunoreactive material was detected in a subset of cells of the adenohypophysis. When fused to the DNA-binding domain of the yeast transcription factor GAL4, the protein stimulated transcription from a GAL4-sensitive reporter gene in transiently transfected pituitary and placental cells.