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Heterozygous mutations in the human POU-homeobox TCF2 (vHNF1, HNF1beta) gene are associated with maturity-onset diabetes of the young, type 5, and abnormal urogenital tract development. Recently, pancreas atrophies have been reported in several maturity-onset diabetes of the young type 5 patients, suggesting that TCF2 is required not only for adult pancreas function but also for its normal development. Tcf2-deficient mice die before gastrulation because of defective visceral endoderm formation. To investigate the role of this factor in pancreas development, we rescued this early lethality by tetraploid aggregation. We show that TCF2 has an essential function in the first steps of pancreas development, correlated with its expression domain that demarcates the entire pancreatic buds from the earliest stages. Lack of TCF2 results in pancreas agenesis by embryonic day 13.5. At earlier stages, only a dorsal bud rudiment forms transiently and expresses the transcription factors Ipf1 and Hlxb9 but lacks the key transcription factor involved in the acquisition of a pancreatic fate, Ptf1a, as well as all endocrine precursor cells. Regional specification of the gut also is perturbed in Tcf2-/- embryos as manifested by ectopic expression of Shh and lack of Ihh and Ipf1 in the posterior stomach and duodenum. Our results highlight the requirement of Tcf2 for ensuring both accurate expression of key regulator molecules in the stomach-duodenal epithelium and proper acquisition of the pancreatic fate. This study provides further insights into early molecular events controlling pancreas development and may contribute to the development of cell-replacement strategies for diabetes.

The prevalence of type 2 diabetes mellitus in the Oji-Cree of northwestern Ontario is the third highest in the world. A private mutation, G319S, in HNF1A, which encodes hepatic nuclear factor-1alpha (HNF-1alpha), was associated with Oji-Cree type 2 diabetes and was found in approximately 40% of affected subjects. The G319S mutation reduced the in vitro ability of HNF-1alpha to activate transcription by approximately 50%, with no effect on DNA binding or protein stability. There was no evidence of a dominant negative effect of the mutant protein. The impact of the G319S mutation at the population level was assessed by classifying subjects with type 2 diabetes according to HNF1A genotype and plotting the cumulative age of onset of diabetes. Disease onset was modeled satisfactorily by two-parameter sigmoidal functions for all diabetic subjects and all three HNF1A genotypes. Pairwise statistical comparisons showed significant between-genotype differences in t50 (all P < 0.00001), corresponding to the age at which half the subjects had become diabetic. Each dose of G319S accelerated median disease onset by approximately 7 years. Thus, the transactivation-deficient HNF1A G319S mutation affects the dynamics of disease onset. The demonstration of a functional consequence for HNF1A G319S provides a mechanistic basis for its strong association with Oji-Cree type 2 diabetes and its unparalleled specificity for diabetes prediction in these people, in whom diabetes presents a significant public health dilemma. The findings also show that HNF1A mutations can be associated with typical adult-onset insulin-resistant obesity-related diabetes in addition to maturity-onset diabetes of the young.

Defects in pancreatic beta-cell function contribute to the development of type 2 diabetes, a polygenic disease that is characterized by insulin resistance and compromised insulin secretion. Hepatocyte nuclear factors (HNFs) -1alpha, -3beta, -4alpha, and Pdx-1 contribute in the complex transcriptional circuits within the pancreas that are involved in beta-cell development and function. In mice, a heterozygous mutation in Pdx-1 alone, but not Hnf-1alpha(+/-), Hnf-3beta(+/-), or Hnf-4alpha(+/-), causes impaired glucose-stimulated insulin secretion in mice. To investigate the possible functional relationships between these transcription factors on beta-cell activity in vivo, we generated mice with the following combined heterozygous mutations: Pdx-1(+/-)/Hnf-1alpha(+/-), Pdx-1(+/-)/Hnf-3beta(+/-), Pdx-1(+/-)/Hnf-4alpha(+/-), Hnf-1alpha(+/-)/Hnf-4alpha(+/-), and Hnf-3beta(+/-)/Hnf-4alpha(+/-). The greatest loss in function was in combined heterozygous null alleles of Pdx-1 and Hnf-1alpha (Pdx-1(+/-)/Hnf-1alpha(+/-)), or Pdx-1 and Hnf-3beta (Pdx-1(+/-)/Hnf-3beta(+/-)). Both double mutants develop progressively impaired glucose tolerance and acquire a compromised first- and second-phase insulin secretion profile in response to glucose compared with Pdx-1(+/-) mice alone. The loss in beta-cell function in Pdx-1(+/-)/Hnf-3beta(+/-) mice was associated with decreased expression of Nkx-6.1, glucokinase (Gck), aldolase B (aldo-B), and insulin, whereas Nkx2.2, Nkx-6.1, Glut-2, Gck, aldo-B, the liver isoform of pyruvate kinase, and insulin expression was reduced in Pdx-1(+/-)/Hnf-1alpha(+/-) mice. The islet cell architecture was also abnormal in Pdx-1(+/-)/Hnf-3beta(+/-) and Pdx-1(+/-)/Hnf-1alpha(+/-) mice, with glucagon-expressing cells scattered throughout the islet, a defect that may be connected to decreased E-cadherin expression. Our data suggest that functional interactions between key islet regulatory factors play an important role in maintaining islet architecture and beta-cell function. These studies also established polygenic mouse models for investigating the mechanisms contributing to beta-cell dysfunction in diabetes.

Mutations in the human genes encoding hepatocyte nuclear factors (HNF) 1alpha, 1beta, 4alpha, and IPF1(PDX1/IDX1/STF1) result in pancreatic beta cell dysfunction and diabetes mellitus. In hepatocytes, hnf4alpha controls the transcription of hnf1alpha, suggesting that this same interaction may operate in beta cells and thus account for the common diabetic phenotype. We show that, in pancreatic islet and exocrine cells, hnf4alpha expression unexpectedly depends on hnf1alpha. This effect is tissue-specific and mediated through direct occupation by hnf1alpha of an alternate promoter located 45.6 kb from the previously characterized hnf4alpha promoter. Hnf1alpha also exerts direct control of pancreatic-specific expression of hnf4gamma and hnf3gamma. Hnf1alpha dependence of hnf4alpha, hnf4gamma, hnf3gamma, and two previously characterized distal targets (glut2 and pklr) is established only after differentiated cells arise during pancreatic embryonic development. These studies define an unexpected hierarchical regulatory relationship between two genes involved in human monogenic diabetes in the cells, which are relevant to its pathophysiology. Furthermore, they indicate that hnf1alpha is an essential component of a transcription factor circuit whose role may be to maintain differentiated functions of pancreatic cells.

The transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1beta gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the Xenopus embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in Xenopus the wild-type HNF1beta, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1beta mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in Xenopus. Thus, the overexpression of both human mutants has a different effect on renal development in Xenopus, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1beta can be functionally characterized in Xenopus. Our findings imply that HNF1beta not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.

Maturity-onset diabetes of the young, a monogenic form of Type II diabetes mellitus, is most commonly caused by mutations in hepatic nuclear factor 1alpha (HNF-1alpha). Here, the dimerization motif of HNF-1alpha is shown to form an intermolecular four-helix bundle. One face contains an antiparallel coiled coil whereas the other contains splayed alpha-helices. The "mini-zipper" is complementary in structure and symmetry to the top surface of a transcriptional coactivator (dimerization cofactor of homeodomains). The bundle is destabilized by a subset of mutations associated with maturity-onset diabetes of the young. Impaired dimerization of a beta-cell transcription factor thus provides a molecular mechanism of metabolic deregulation in diabetes mellitus.

Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1.

Glucose-6-phosphatase catalyzes the terminal step in the gluconeogenic and glycogenolytic pathways. Transcription of the gene encoding the glucose-6-phosphatase catalytic subunit (G6Pase) is stimulated by cAMP and glucocorticoids whereas insulin strongly inhibits both this induction and basal G6Pase gene transcription. Previously, we have demonstrated that the maximum repression of basal G6Pase gene transcription by insulin requires two distinct promoter regions, designated A (from -271 to -199) and B (from -198 to -159). Region B contains an insulin response sequence because it can confer an inhibitory effect of insulin on the expression of a heterologous fusion gene. By contrast, region A fails to mediate an insulin response in a heterologous context, and the mutation of region B within an otherwise intact promoter almost completely abolishes the effect of insulin on basal G6Pase gene transcription. Therefore, region A is acting as an accessory element to enhance the effect of insulin, mediated through region B, on G6Pase gene transcription. Such an arrangement is a common feature of cAMP and glucocorticoid-regulated genes but has not been previously described for insulin. A combination of fusion gene and protein-binding analyses revealed that the accessory factor binding region A is hepatocyte nuclear factor-1. Thus, despite the usually antagonistic effects of cAMP/glucocorticoids and insulin, all three agents are able to use the same factor to enhance their action on gene transcription. The potential role of G6Pase overexpression in the pathophysiology of MODY3 and 5, rare forms of diabetes caused by hepatocyte nuclear factor-1 mutations, is discussed.

Phenylalanine hydroxylase stimulator (PHS) is a component of the phenylalanine hydroxylation system that is involved in the regeneration of the cofactor tetrahydrobiopterin. It is also identical to the dimerization cofactor of hepatocyte nuclear factor 1 (HNF1) (DCoH) that is able to enhance the transcriptional activity of HNF1. Moreover, it has the structural potential for binding macromolecules such as proteins and nucleic acids, consistent with its involvement in gene expression. We investigated whether PHS/DCoH could enhance the expression of phenylalanine hydroxylase (PAH). Cotransfection assays showed that DCoH itself could not transactivate the 9-kb human PAH 5' flanking fragment. However, this 9-kb fragment was transactivated by HNF1 in a dose-dependent manner with a maximum of nearly 8-fold activation; DCoH potentiated this transactivation by another 1.6-fold. The HNF1 binding sites were located at -3.5 kb in a region that is 77.5% identical to the mouse liver-specific hormone-inducible PAH gene enhancer. This study suggests a possible dual function of PHS in vivo in the human phenylalanine hydroxylation system: it is involved in the regeneration of the cofactor tetrahydrobiopterin and can also enhance the expression of the human PAH gene.

The relationship between the enzymatic and the transcriptional activity of the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor 1 (DCoH) has been elucidated by site-directed mutagenesis. DCoH dimers harbor a binding site for hepatocyte nuclear factor 1 (HNF1), two active centers that bind pterins, and a saddle-shaped surface that resembles nucleic acid binding domains. Two domains of the protein have been selectively targeted to determine if a change in one activity affects the other. No strong correlation has been found, supporting the idea that carbinolamine dehydratase activity is not required for HNF1 binding in vitro or transcriptional coactivation in vivo. Double mutations in the active center, however, influence the in vivo transcriptional activity but not HNF1 binding. This finding suggests that some active center residues also are used during transcription, possibly for binding of another (macro)molecule. Several mutations in the saddle led to a surprising increase in transcription, therefore linking this domain to transcriptional regulation as well. The transcriptional function of DCoH therefore is composed of two parts, HNF1 binding and another contributing effect that involves the active site and, indirectly, the saddle.

Previous studies have shown that in vitro expression of the neural cell adhesion molecule (N-CAM) can be regulated by the products of homeobox genes HoxB9, -B8, and -C6. N-CAM is a Ca(2+)-independent immunoglobulin-related CAM that plays an important role in neural development. In the present study, we investigated whether the liver cell adhesion molecule (L-CAM) a member of the Ca(2+)-dependent CAM family (cadherins) is also regulated by homeobox-containing genes. In transient cotransfection experiments of NIH 3T3 cells, we observed that both HoxD9 and liver-enriched POU-homeodomain transcription factor, HNF-1, activated chloramphenicol acetyltransferase gene reporter constructs containing the L-CAM promoter and an enhancer present in the second intron of the chicken L-CAM gene. Using electrophoretic mobility-shift assays, we found that components of cell extracts from NIH 3T3 cells transfected with HoxD9 bound to a small region of the L-CAM enhancer having a consensus sequence that is a putative binding site for HNF-1. Components of extracts from the chicken hepatoma cell line LMH that had been transfected with an HNF-1 expression vector also bound to this same site. In nuclear run-on experiments with nuclei from LMH cells that were transfected with expression vectors for HoxD9 or HNF-1, L-CAM RNA levels were increased 33-fold and 4-fold respectively. Using the same run-on procedure, it was confirmed that nuclei prepared from normal embryonic chicken liver cells expressed the RNAs for HoxD9, HNF-1, and L-CAM. Taken together with previous observations, these data raise the possibility that homeobox-containing genes will have a widespread role in the place-dependent expression of CAMs belonging both to immunoglobulin-related and to cadherin families.

Serum colloid osmotic pressure is believed to control the hepatic output of plasma proteins, including albumin. The present study was aimed at identifying the molecular basis for feedback control of albumin gene expression in highly differentiated hepatoma cells. The steady-state level of albumin mRNA and the activity of a 282-bp albumin promoter-chloramphenicol acetyltransferase reporter gene in cells incubated in the presence of increasing amounts of serum albumin or dextran were significantly and selectively decreased. When nuclear extracts from cells exposed to 5% (wt/vol) serum albumin were tested in a gel-retardation assay with six oligonucleotide probes containing DNA elements of the albumin promoter, only the element B-retarded band, which contains the nucleotide recognition sequence for hepatocyte nuclear transcription factor 1 alpha (HNF-1 alpha), was consistently decreased as compared to nuclear extract from cells not exposed to serum albumin. Moreover, the activity of a reporter gene with a basal TATA-promoter driven by multiple HNF-1 alpha recognition elements was selectively inhibited in cells incubated in the presence of 5% serum albumin. A reduction of HNF-1 alpha mRNA appears to be responsible for this response to a change in the level of macromolecules in the incubation medium. These results indicate that activity of a dominant liver transcription factor, HNF-1 alpha, controlling the transcription of several liver-specific genes, is modulated by a fluctuation in the level of oncotically active macromolecules.

Tissue-specific expression of insulin-like growth factor binding protein 1 (IGFBP-1) in the liver has been studied using differentiated (H4II and C2Rev7) and dedifferentiated (H5 and C2) rat hepatoma cell lines. Northern blot analysis showed that endogenous IGFBP-1 mRNA was expressed only in the differentiated cell lines. The first 341 base pairs 5' to the transcription initiation site of the human IGFBP-1 gene were inserted upstream of the chloramphenicol acetyltransferase reporter gene (pBP-1(341)). Expression of this gene from the human IGFBP-1 promoter was 10-16 times more efficient in the H4II line than in the other hepatoma cell lines and 40 and approximately 12 times more so than in rat fibroblasts (FR3T3) and a human cervical carcinoma cell line (C33), respectively. Cotransfection of pBP-1(341) and pRSV-HNF1 and/or pRSV-v-HNF1 (eukaryotic expression vectors that drive the synthesis of the liver-enriched trans-acting factor HNF1 or of v-HNF1, a related form) in C33 recipient cells yielded a 6-fold increase in IGFBP-1 promoter activity by HNF1 and a 2-fold increase by v-HNF1. These increases were dependent on the integrity of an HNF1 binding site located 58-74 nucleotides upstream of the cap site. Stimulation of promoter activity by cotransfection of both HNF1 and v-HNF1 fell between these values. Our results indicate that HNF1 is instrumental in human IGFBP-1 promoter activity in vivo and that v-HNF1 modulates this functional role.

The principal pathway for the metabolism of phenylalanine in mammals is via conversion to tyrosine in a tetrahydrobiopterin-dependent hydroxylation reaction occurring predominantly in the liver. Recently, the proposal that certain hyperphenylalaninemic children may have a deficiency of carbinolamine dehydratase, a component of the phenylalanine hydroxylation system, has widened the interest in this area of metabolism. Upon cloning and sequencing the dehydratase, we discovered that this protein is identical to DCoH, the cofactor which regulates the dimerization of hepatic nuclear factor 1 alpha, a homeodomain transcription factor. The identity of the nuclear and cytoplasmic proteins is demonstrated by size, immunoblotting, stimulation of phenylalanine hydroxylase, and dehydratase activity. The evolution of the dual functions of regulation of phenylalanine hydroxylation activity and transcription activation in a single polypeptide is unprecedented.

Systematic mutational analysis previously identified two primary regulatory elements within a minienhancer (-247 to -198) of the rat insulin I promoter that are critical for transcriptional activity. The Far box (-241 to -232) and the FLAT element (-222 to -208) synergistically upregulate transcription and, together, are sufficient to confer tissue-specific and glucose-responsive transcriptional activity on a heterologous promoter. Detailed analysis of the FLAT element further revealed that, in addition to the positive regulatory activity it mediates in tandem with the Far box, it is a site for negative regulatory control. A portion of the FLAT element bears considerable sequence similarity to the consensus binding site for hepatocyte nuclear factor 1 alpha (HNF1 alpha; LF-B1) a liver-enriched homeodomain-containing transcription factor. Here we show that the HNF1-like site within the FLAT element exhibited positive transcriptional activity in both HepG2 and HIT cells and bound similar, but distinguishable, nuclear protein complexes in the respective nuclear extracts. Screening of a hamster insulinoma cDNA library with a PCR-derived probe encompassing the DNA-binding domain of rat HNF1 alpha resulted in isolation of a hamster HNF1 alpha (hHNF1 alpha) cDNA homolog. Specific antiserum identified the HNF1 alpha protein as one component of a specific FLAT-binding complex in HIT nuclear extracts. Expression of the hHNF1 alpha cDNA in COS cells resulted in transactivation of reporter constructs containing multimerized segments of the rat insulin I minienhancer. Thus, HNF1 alpha, one component of a DNA-binding complex involved in transcriptional regulation of the rat insulin I gene, may play a significant role in nonhepatic as well as hepatic gene transcription.

The transcription factors that act in hepatocyte-specific gene expression include proteins that are present mainly in liver cells (HNF-1/LFB1, C/EBP, HNF-3, HNF-4) (HNF, hepatocyte nuclear factor; C/EBP, rat enhancer binding protein) and proteins that are widely distributed (AP-1, NF-1, NF-Y/ACF). We show here that the genes encoding each of these liver-enriched factors exhibit different patterns of transcriptional control in different tissues. In addition, there were several instances in which transcription was detected (e.g., for HNF-1) when no mRNA or specific DNA binding protein was found, suggesting the importance of posttranscriptional control in some instances for these factors. These experiments identify C/EBP, HNF-3, and HNF-4, and perhaps also HNF-1, as targets for the study of cascades of transcriptionally controlled transcription factors in differentiated cells.