- Research Article
- Open Access
Differential Effects of HNF-1α Mutations Associated with Familial Young-Onset Diabetes on Target Gene Regulation
© The Feinstein Institute for Medical Research 2011
- Received: 5 July 2010
- Accepted: 9 December 2010
- Published: 15 December 2010
Hepatocyte nuclear factor 1-α (HNF-1α) is a homeodomain transcription factor expressed in a variety of tissues (including liver and pancreas) that regulates a wide range of genes. Heterozygous mutations in the gene encoding HNF-1α (HNF1A) cause familial young-onset diabetes, also known as maturity-onset diabetes of the young, type 3 (MODY3). The variability of the MODY3 clinical phenotype can be due to environmental and genetic factors as well as to the type and position of mutations. Thus, functional characterization of HNF1A mutations might provide insight into the molecular defects explaining the variability of the MODY3 phenotype. We have functionally characterized six HNF1A mutations identified in diabetic patients: two novel ones, p.Glu235Gly and c-57-64delCACGCGGT;c-55G>C; and four previously described, p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg. The effects of mutations on transcriptional activity have been measured by reporter assays on a subset of HNF-1α target promoters in Cos7 and Min6 cells. Target DNA binding affinities have been quantified by electrophoretic mobility shift assay using bacterially expressed glutathione-S-transferase (GST)-HNF-1α fusion proteins and nuclear extracts of transfected Cos7 cells. Our functional studies revealed that mutation c-57-64delCACGCGGT;c-55G>C reduces HNF1A promoter activity in Min6 cells and that missense mutations have variable effects. Mutation p.Arg271Trp impairs HNF-1α activity in all conditions tested, whereas mutations p.Val133Met, p.Glu235Gly and p.Pro379Arg exert differential effects depending on the target promoter. In contrast, substitution p.Thr196Ala does not appear to alter HNF-1α function. Our results suggest that HNF1A mutations may have differential effects on the regulation of specific target genes, which could contribute to the variability of the MODY3 clinical phenotype.
Heterozygous mutations in the gene encoding hepatocyte nuclear factor 1-α (HNF-1α) are the cause of familial young-onset diabetes, also known as maturity-onset diabetes of the young (MODY)-3 (1). This monogenic β-cell diabetes is characterized by autosomal-dominant inheritance, early age of onset and a progressive β-cell failure resulting in increasing hyperglycemia. Patients with mutations in HNF-1α show also a decreased renal threshold for glucose reabsorption and increased sensitivity to sulfonylureas.
In this work, we analyze the functional effect of six HNF-1α variants located at different gene positions that we have identified in an HNF-1α mutation screening among a group of patients with familial, young-onset diabetes. Two mutations are considered novel: c-57-64delCACGCGGT;c-55G>C, located in the 5′ untranslated region (5′UTR) of HNF1A, and p.Glu235Gly, located in the DNA binding domain. Other mutations, located in the DNA binding and transactivation domains, have been previously reported (p.Val133Met, p.Thr196Ala, p.Arg271Trp and p.Pro379Arg) (8,9). Here, we report the effect of the 5′UTR mutation on HNF1A expression as well as the differential effects of the missense mutations on the ability of HNF-1α to bind and activate a subset of target genes.
Patients and Mutation Screening
The diabetic patients involved in this study were referred to our laboratory for a molecular diagnosis of monogenic diabetes. The probands of families P36 and P56 were referred to us by author MG, family P20 by author J-JG, family P30 by author SA, family P37 by author MD and family P52 by author V-MA. The clinical diagnosis was made using classical criteria (1). All families were of Spanish Caucasian descent except family P56, who was Hispanic. Informed consent was obtained from the subjects or their parents. The studies were performed according to the Declaration of Helsinki as revised in 2000 and approved by the corresponding ethical committees.
To screen for HNF-1α mutations, the exons, their flanking intron regions and the minimal promoter of the HNF1A gene were polymerase chain reaction (PCR) amplified, and their products were sequenced. The upstream and downstream primers used were as follows: exon 1, 5′-gcttggcctagtggggttttg-3′ and 5′-cctctaggctctcctgggag-3′; exon 2, 5′-tgggctccataactgctttc-3′ and 5′-ccagggaagatgcggggtag-3′; exon 3, 5′-agaatcaagggcaaggtcag-3′ and 5′-gaccaaaccagcatgtttc-3′; exon 4, 5′-tcagaaccctccccttcatg-3′ and 5′-cacgtgtcccttgtccccac-3′; exon 5, 5′-caatg gagtttgaagtgctg-3′ and 5′-gccaaggaaagatgaggttg-3′; exon 6 5′-agggagattctggagcagtc-3′ and 5′-cacccaggtgcccacccatc-3′; exon 7, 5′-cagctgattccctccccttc-3′ and 5′-atccattgacagccaacctc-3′; exon 8, 5′-gaggcctgggactagggctg-3′ and 5′-tctgtcactggccgagggag-3′; exon 9, 5′-cctgtgacagagcccctcac-3′ and 5′-ggacagcaacagaaggggtg-3′; exon 10, 5′-gtacccctagggacaggcag-3′ and 5′-cccccaagcaggcagtacag-3′; and promoter, 5′-agcactgttcttggcacatg and 5′-ggcagacacaaaccaaactc-3′. Screening for the presence of novel mutations on a panel of 54 healthy control individuals was done by single-strand conformation polymorphism of PCR fragments under two conditions (14).
Site-Directed Mutagenesis and Construction of HNF-1α Mutants
A construct encoding the mouse HNF-1α cDNA (pcDNA3-mHNF1A) was prepared by cloning a 2.2-Kb EcoRI-EcoRV fragment from pBJ5mHNF (15) into pcDNA3 (Invitrogen, Carlsbad, CA, USA).
To generate plasmid pGEX5.2-HNF1A(1–281), expressing GST-HNF-1α(1–281) fusion protein, a DNA fragment containing amino acids 1–281 of HNF-1α was amplified from pcDNA3-mHNF1A using oligonucleotides GST1AF: 5′-gggatccccatggtttctaagctg-3′ and GST1AR: 5′-gggatcctcacaacttgtgccggaag-3′ and inserted into the BamHI site of the polylinker of pGEX-5X-2 (GE Healthcare, Barcelona, Spain).
Mutations were introduced into the pcDNA3-mHNF1A or into the pGEX5.2-HNF1A(1–281) constructs by PCR using a kit (QuikChange II Site Directed Mutagenesis Kit; Stratagene, La Jolla, CA, USA). To generate missense mutations p.Val133Met, p.Thr196Ala, p.Glu235Gly, p.Arg271Trp and p.Pro379Arg (note that residue Pro379 corresponds to residue Pro378 in the mouse protein), the following oligonucleotides were used, respectively: 5′-ccagcgggagatggtggacaccacgggtc-3′, 5′-gagcccacaggcgatgagctcccagctaagaagg-3′, 5′-gaagagcgagagaccttggtggaggggtgtaatag-3′, 5′-ctacaactggtttgccaattggcgcaaggagg-3′ and 5′-gtcccctgccgcgggtcagcaccctg-3′. Constructs carrying mutations were checked by sequencing and digestion with SacI, MfeI and KpsI in case of mutants mHNF1A(T196A), mHNF1A(R271W) and mHNF1A(P378R), respectively.
Plasmids pGL3-1AP and pGL3-1APm (which contain the proximal promoter and, wild-type or mutant, human HNF1A 5′UTR, driving the luciferase gene) were generated by amplifying a DNA fragment containing bases from −504 to +9 of HNF1A (positions 211–723 of GenBank® accession number U73499) from genomic DNA of patient P37 with primers 1AP2F: 5′-tctcctgtctcagcatatg-3′ and 1A1-1R: 5′-cttctccagccaggtag-3′ and cloned into pGEMTeasy (Promega, Madison, WI, USA). After sequencing, the inserts were cut off with SmaI and SacI and introduced into pGL3basic (Promega).
Tissue Culture, Transient Transfections, Immunoblot and Luciferase Assays
Cos7 and Min6 cells were grown as described elsewhere (16,17). Transient transfections were performed with lipofectAMINE Reagent (Invitrogen), in 80% confluent 12-well culture dishes, including the corresponding luciferase reporter construct, HNF1A expression vectors and pCMVβ (Clontech) to control for efficiency of transfection. The amounts of each plasmid are indicated in the figures. Luciferase activity was measured after 24 h in cell lysates using the Luciferase Assay System (Promega) and normalized for transfection efficiency by the β-galactosidase activity of pCMVβ. Transfection efficiency, measured by β-galactosidase staining with X-gal was in a range of 16–24% from experiment to experiment. Western blot analysis was carried out using the polyclonal antibody HNF-1α C19 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Reporter plasmid pGL3-HNF4AP2 contains the human HNF-4α P2 promoter, which includes one HNF-1α binding site, cloned into the SmaI site of pGL3 basic (Promega). The HNF-4α P2 promoter was cloned from human genomic DNA with primers HNF4AP2F: 5′-gacacacccatagtttggag-3′ and HNF4AP2R: 5′-tcacgctgaccatggccaag-3′ amplifying bases −418 to +13 of the P2 promoter (bases 8036943 to 8037373 of the Homo sapien chromosome 20 genomic contig, reference assembly, GeneBank® accession number NT011362.9). Reporter plasmid pGL3-GLUT2 contains base pairs −1308 to +46 of the mouse GLUT2 promoter (18). Reporter plasmid β28-Luc contains three copies of the β-fibrinogen HNF1 binding site (15). Reporter plasmid pGL3-RIP, containing base pairs −872 to −169 of the rat insulin 2 gene promoter, was provided by Dr. A. Barberá (Institut d’investigations Biomediques August Pi i Sunyer [IDIBAPS], Barcelona, Spain).
Production of Recombinant Wild-Type and Mutant GST-HNF-1α(1–281) Fusion Proteins
The Escherichia coli BL21 strain was transformed with the indicated pGEX5.2-HNF1A(1–281) plasmids and grown at 37°C to mid-log phase. Expression of fusion proteins was induced by adding isopropyl-β-d-thiogalactoside (IPTG) to a final concentration of 0.2 mmol/L, and cultures were incubated with orbital shaking for 16 h at 22°C. GST-HNF-1α(1–281) fusion proteins were purified from crude E. coli extracts by single-step affinity chromatography using glutathione-agarose beads (Sigma-Aldrich Chemie, GmbH, Steinheim, Germany), essentially as described by Galan et al. (19). Fusion proteins were stored at a concentration of about 1 mg/mL at −80°C with 30% glycerol. Protein concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories GmbH, Munich, Germany). GST-HNF-1α(1–281) purification resulted in a single band of approximately 56 kDa on Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
DNA Binding Studies
Purified GST-fusion proteins or nuclear cell extracts were incubated with doublestrand 32P-labeled oligonucleotides (1A: 5′-tcgacttggttaataattcaccagag-3′, based on the 21-mer primer described by Chi et al. , and 4AP2: 5′-tcgaagtgactggttactctttaacgtatccac-3′, based on the oligonucleotide P2 described by Boj et al. ) containing high-affinity HNF-1α binding sites in a 15-µL reaction mixture containing 20 mmol/L HEPES buffer, pH 7.9, 40 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L EGTA, 0.5 mmol/L DTT, 4% Ficoll and 1 µg poly(dIdC) at 25°C for 20 min. Supershift analysis was performed by incubating 0.2 µg of the polyclonal antibody HNF-1α C-19 with the nuclear extracts for 5 min on ice before adding the probe. The reaction mixture was loaded on a 6% nondenaturating polyacrylamide gel containing 0.25 × TBE buffer (23 mmol/L Tris-borate, 0.5 mmol/L EDTA) and run at 4°C. Nuclear extracts were prepared as described by Navas et al. (16) and the relative amounts of HNF-1α expressed proteins were determined by Western blotting. To determine relative binding affinity (Ka) and maximal binding (Bmax), mobility shift analysis was performed with increasing amounts of 32P-labeled oligonucleotides and set levels of HNF-1α proteins as indicated. To quantify the bound and free oligonucleotides, radioactive bands were analyzed with a Phosphorimager (Bio-Rad Laboratories GmbH). Specific counts were determined after subtracting the background, and the data were analyzed by the Scatchard method (21). Statistical significance was analyzed by the two-tailed Student t test.
Identification of HNF1A Mutations in Patients with Young-Onset Familial Diabetes
Clinical characteristics of probands and HNF1A mutations
Age at diagnosis/actual age (years)
Body mass index (kg/m2)
Fasting plasma glucose (mmol/L)
Basal C-peptide (ng/mL)
Affected family members
c-57 to −64 delCACGCGGT;c-55G>Cc
S, F, 2 pU, pGM
F, S, D
Glimepiride + metformin
M, S, mGM
Metformin + insulin
M, 4 mU, mGM
The available clinical and anthropometric data from the index patients carrying HNF1A changes are given in Table 1. Most of the patients studied as probands for genetic diagnosis had a clinical history of diabetes from at least two consecutive generations. Hyperglycemia values vary from one patient to another as well as hemoglobin (Hb)A1c and treatment at the moment of genetic study.
Effect of Mutation c-57-64delCACGCGGT;c-55G>C on the 5′ UTR of HNF1A
Mutation c-57-64delCACGCGGT;c-55G>C is located within the 5′UTR of HNF1A (Figure 1A). To investigate whether this mutation had an effect on HNF1A expression, we prepared plasmids containing the luciferase reporter gene under the control of the minimal promoter, and wild-type or mutant, 5′UTR of HNF1A (bases c.−504 to +9) and transfected them into insulinoma Min6 cells. We found that this mutation results in an ∼50% decrease in luciferase activity (Figure 1B), thus indicating that transcription of the HNF1A gene is impaired for this mutant allele.
Effect of HNF-1α Missense Mutations on Transcriptional Activation
To characterize the functional consequences of missense mutations p.Val133Met, p.Thr196Ala, p.Glu235Gly, p.Arg271Trp and p.Pro379Arg, direct mutagenesis of the mouse cDNA was performed to prepare the mutants mHNF1A(V133M), mHNF1A(T196A), mHNF1A(E235G), mHNF1A(R271W) and mHNF1A(P378R), respectively. HNF-1α protein is highly conserved between the human and mouse species. The amino acid sequence homology is 94.6% for the entire protein and 99.5% for the DNA binding domain. With the exception of mutation Pro379Arg, which is located in the transactivation domain, all the other missense mutations are located in the DNA binding domain and involve amino acid residues strictly conserved between mouse and human. Pro379 is also conserved in the mouse protein, but its position is 378 because of the insertion of a His residue after position 359 in the human protein.
HNF-1α is an important regulator of gene expression in the β cell as well as in other tissues involved in glucose homeostasis such as the liver. To evaluate the effects of HNF-1α missense mutations, we compared the ability of the mutant proteins to activate transcription from a selected group of tissue-specific target promoters activated in liver (β-fibrinogen), in β -cells (HNF-4α P2, insulin) or in both (GLUT2). Additionally, we tested the effects of these mutations in two different cellular contexts: Min6, an insulinoma-derived cell line that expresses endogenous HNF-1α as well as some of the target genes whose promoters are used, and Cos7, which does not. Although Min6 cells are more comparable to the pancreatic β cells, some of the effects of the exogenous mutant proteins could be masked by the endogenous wild-type. This limitation does not apply to Cos7; however, in this case, the lack of some specific coactivators may also be limiting. The comparison of the results with both cell lines is useful to understand the effect of the HNF-1α mutations on transcriptional activation, and similar approaches have been used previously in the analysis of other MODY3 mutations (22).
Effect of Missense Mutations on HNF-1α DNA Binding
Affinity of HNF-1α wild-type and mutant proteins expressed in Cos7 for 1A and 4AP2 probes.a
Mutations in HNF1A are a common cause of monogenic diabetes characterized by a progressive deficiency in glucose-stimulated insulin secretion. Functional analysis of MODY3 mutations to identify the corresponding molecular defect might contribute to uncover the mechanisms of β-cell impairment in these diabetic patients. In this work, we characterized six different HNF1A variants found in patients diagnosed with familial, young-onset diabetes.
Mutation c-57-64delCACGCGGT;c-55G>C is located within the 5′UTR of HNF1A and was found in a family with a mild clinical phenotype. This mutation was previously reported as a rare polymorphism identified in a patient homozygous for the p.Arg54X mutation (8). However, this mutation cosegregates with diabetes in our patient family and was absent in 55 non-diabetic control subjects. The 5′UTR region of HNF1A, starting at position c-225, could be involved in transcription/translation processes or mRNA stability, as has been shown for other genes (24–26). Other MODY3 mutations have been previously identified in this region. In particular, the substitution c-62C>G, which is contained in the deleted region of our family, has been proposed to modify a Myc/Max binding site (8,27). Interestingly, the c-41-58 sequence is fully conserved among human, mouse and rat species, and the mutation found in family P37 modify three bases within that region. Our results suggest that this mutation affects the transcription of HNF1A in the β cell, although we cannot exclude other additional effects on the posttranscriptional regulation of this gene, nor define its importance with respect to the different mRNA isoforms (13).
The effects of missense mutations on HNF-1α transcriptional activity are heterogeneous and appear to depend on the reporter under study. Mutation p.Pro379Arg is located in a region of the transactivation domain of HNF-1α, which has been proposed to contribute to the transcriptional activity of HNF-1α in a gene- and cell type-dependent manner (28,29). Our results show that mutant mHNF1A(P378R) fails to fully transactivate in a promoter- and cell-specific manner, since it shows a specific reduction of the β-fibrinogen promoter transactivation when tested into Cos7 cells. Consistent with the localization of the mutated residue in the transactivation domain of HNF-1α, the mutant protein binds DNA as well as the wild-type. Thus, our results could be explained by an impairment of the interaction of the mutant protein with a specific coactivator in Cos7 cells. The lack of effect in Min6 cells could be due to the presence of endogenous HNF-1α, as previously proposed for other MODY3 mutations (22).
Mutations pVal133Met, p.Thr196Ala, p.Glu235Gly and p.Arg271Trp are all located in the DNA binding domain. The DNA binding domain of HNF-1α is a bipartite motif containing a Pit 1, Oct 1, and Oct 2, unc-86 (POU)-homeodomain (POUH), involved in DNA recognition, and a POU-specific domain (POUS) that cooperates with the POUH to enhance the binding affinity and specificity (Figure 1A) (20). Residue Arg271 is located in the third α-helix of the POUH domain and is highly conserved in many other homeodomains (30). This residue is required for the conformational stability of the homeodomain and for optimal DNA binding (30). Accordingly, mutation p.Arg271Trp decreased both affinity and maximal DNA binding and, consequently, impaired transactivation in all conditions tested here as well as previously (23). In contrast, mutation p.Thr196Ala does not appear to affect HNF-1α activity in our experimental conditions. The Thr196 residue is conserved in human, mouse, rat and chicken HNF-1α and is localized in the linker between POUS and POUH domains. The mHNF-1A(T196A) mutant shows normal DNA binding and transactivation capacities, such as other missense mutations directed to this flexible interdomain (20). The apparent lack of effect of this mutation is not unexpected, since there is not a clear genotype/phenotype correlation in the P30 family. In particular, the sister of the patient, who was diagnosed at age 31 years, does not bear the p.Thr196Ala change (Table 1). However, this rare polymorphism, which does not appear to be a MODY3 mutation, might be a risk factor for developing diabetes, since it was not detected in 54 healthy subjects studied in our laboratory and has been reported in two other diabetic patients, including one where this change coexists with type 1 diabetes and overweight (9,31).
Notably, we found that the effect of mutations p.Val133Met and p.Glu235Gly on HNF-1α transactivity depends on the promoter tested. These residues are located in the third α-helix within the POUS and in the second α-helix within the POUH, respectively. Val133 is highly conserved among other POUS and its implication in protein stability has been suggested (20). An impaired protein stability of this mutant could explain the normal level of activation obtained with the β-fibrinogen and insulin promoters when higher amounts of DNA were transfected into Cos7 cells. However, this compensatory effect was not observed with the GLUT2 promoter or when MIN6 cells were used. Additionally, wild-type and mutant HNF-1α levels were similar in nuclear extracts of transfected Cos7 cells. In contrast, our results suggest that these two mutations affect DNA binding of HNF-1α. Although residue Val133 as well as Glu235 are not predicted to directly contact the DNA (20), DNA binding affinity of both mutants for the consensus 1A probe was clearly decreased, suggesting that this defect may be responsible for the reduced HNF-1α-dependent transactivation of most of our reporters. Strikingly, mutations p.Val133Met and p.Glu235Gly did not produce any significant defect in the transactivation of the P2 promoter of HNF-4α, which is the main functional promoter for this gene in the β cell (6). In agreement with these findings, DNA binding affinity of mHNF1A(E235G) to the 4AP2 probe was similar to that of the wild-type protein. Affinity of mHNF1A(V133M) for this probe was slightly decreased, but this effect was smaller than that observed with the consensus 1A binding site. The difference between the binding site sequence of 4AP2 (GTTACTCTTTAAC) and 1A (GTTAATAATTCAC) probes could be responsible for the differential effect of these two mutations on DNA binding affinity. Alternatively, adjacent sequences to the binding site might also contribute to this differential sensibility, as previously shown for the steroidogenic factor-1 (32).
Despite the common progressive β-cell function defect, which characterized the MODY3 phenotype, there is certain variability in the severity of its clinical expression in patients with respect to age of diagnosis, treatment requirements and development of complications. In particular, age of diagnosis has been shown to depend on the type of mutation and HNF-1α isoform affected as well as the presence of additional genetic modifiers (9,12,13). Besides these factors, the individual lifestyle is another important factor that may influence the evolution of the disease. In this regard, for example, it is worth noting the strict control on diet and pharmacological treatment exerted by patient P52 opposed to that exerted by patient P36. The results of our molecular study suggest that the differential effects on the regulation of specific target genes caused by the HNF-1α mutations could also contribute to the differences in the development of the clinical phenotype. HNF-1α is an important transcriptional regulator in liver and in pancreatic islets controlling tissue-specific transcriptional programs (33). In particular, HNF-1α is a major regulator of HNF-4α in pancreas, and there is evidence of a cross-regulatory feedback circuit between HNF-1α and HNF-4α in the β cell (34). Mutations in HNF4A are responsible for another form of monogenic diabetes, MODY1, which shares many clinical features with MODY3. Strikingly, some of the MODY3 missense mutations found in our diabetic patients do not appear to affect HNF4A expression. These results suggest that regulation of HNF4A expression by HNF-1α may not be the simplest explanation for the HNF-1α-deficient phenotype and that regulation of target genes other than HNF4A may also play an important role. Indeed, both HNF-1α and HNF-4α regulate synergistically common target genes involved in β-cell function (35). Considering the key role of HNF-4α in the β-cell function, we would expect that HNF1A mutations resulting in a decrease of HNF-4α expression will have a stronger impact on the regulation of common target genes, thus contributing in a major way to reduced insulin secretion and the progress of diabetes.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
We thank O Vincent (Instituto de Investigaciones Biomedicas CSIC-UAM, Madrid, Spain) for critical reading of the manuscript. This work was supported by the Instituto de Salud Carlos III (grant PI060153) and the Directión General de Universidades e investigation de la Consejería de Educatión de la Comunidad de Madrid-Universidad Complutense de Madrid (grant CCG08-UCM/SAL-3623) to M-A Navas. M Galán was supported by a predoctoral Formación de Personal Investigador (FPI) fellowship from the Comunidad Autónoma de Madrid. C-M García-Herrero was supported by a predoctoral Formación de Profesorado Universitario (FPU) fellowship from the Ministerio de Ciencia e Innovación of Spain.
- Murphy R, Ellard S, Hattersley AT. (2008) Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes. Nat. Clin. Pract. Endocrinol. Metab. 4:200–13.View ArticleGoogle Scholar
- Mendel DB, Crabtree GR. (1991) HNF-1, a member of a novel class of dimerizing homeodomain proteins. J. Biol. Chem. 266:677–80.PubMedGoogle Scholar
- Wang H, Maechler P, Hagenfeldt KA, Wollheim CB. (1998) Dominant-negative suppression of HNF-1alpha function results in defective insulin gene transcription and impaired metabolism-secretion coupling in a pancreatic beta-cell line. EMBO J 17:6701–13.View ArticleGoogle Scholar
- Shih DQ, et al. (2001) Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism. Diabetes 50:2472–80.View ArticleGoogle Scholar
- Boj SF, Parrizas M, Maestro MA, Ferrer J. (2001) A transcription factor regulatory circuit in differentiated 265 pancreatic cells. Proc. Natl. Acad. Sci. U. S. A. 98:14481–6.View ArticleGoogle Scholar
- Hansen SK, et al. (2002) Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta cell function. J. Clin. Invest. 110:827–33.View ArticleGoogle Scholar
- Mendel DB, Hansen LP, Graves MK, Conley PB, Crabtree GR. (1991) HNF-1 alpha and HNF-1 beta (vHNF-1) share dimerization and homeo domains, but not activation domains, and form heterodimers in vitro. Genes Dev. 5:1042–56.View ArticleGoogle Scholar
- Ellard S, Colclough K. (2006) Mutations in the genes encoding the transcription factors hepatocyte nuclear factor 1 alpha (HNF1A) and 4 alpha (HNF4A) in maturity-onset diabetes of the young. Hum. Mutat. 27:854–69.View ArticleGoogle Scholar
- Bellanne-Chantelot C, et al. (2008) The type and the position of HNF1A mutation modulate age at diagnosis of diabetes in patients with maturity-onset diabetes of the young (MODY)-3. Diabetes 57:503–8.View ArticleGoogle Scholar
- Fajans SS, Bell GI. (2006) Phenotypic heterogeneity between different mutations of MODY subtypes and within MODY pedigrees. Diabetologia 49:1106–8.View ArticleGoogle Scholar
- Stride A, Shepherd M, Frayling TM, Bulman MP, Ellard S, Hattersley AT. (2002) Intrauterine hyperglycemia is associated with an earlier diagnosis of diabetes in HNF-1alpha gene mutation carriers. Diabetes Care 25:2287–91.View ArticleGoogle Scholar
- Kim SH, et al. (2003) Genetic modifiers of the age at diagnosis of diabetes (MODY3) in carriers of hepatocyte nuclear factor-1alpha mutations map to chromosomes 5p15, 9q22, and 14q24. Diabetes 52:2182–6.View ArticleGoogle Scholar
- Harries LW, Ellard S, Stride A, Morgan NG, Hattersley AT. (2006) Isomers of the TCF1 gene encoding hepatocyte nuclear factor-1 alpha show differential expression in the pancreas and define the relationship between mutation position and clinical phenotype in monogenic diabetes. Hum. Mol. Genet. 15:2216–24.View ArticleGoogle Scholar
- Navas MA, Vaisse C, Boger S, Heimesaat M, Kollee LA, Stoffel M. (2000) The human HNF-3 genes: cloning, partial sequence and mutation screening in patients with impaired glucose homeostasis. Hum. Hered. 50:370–81.View ArticleGoogle Scholar
- Kuo CJ, Conley PB, Hsieh CL, Francke U, Crabtree GR. (1990) Molecular cloning, functional expression, and chromosomal localization of mouse hepatocyte nuclear factor 1. Proc. Natl. Acad. Sci. U. S. A. 87:9838–42.View ArticleGoogle Scholar
- Navas MA, Munoz-Elias EJ, Kim J, Shih D, Stoffel M. (1999) Functional characterization of the MODY1 gene mutations HNF4(R127W), HNF4(V255M), and HNF4(E276Q). Diabetes 48:1459–65.View ArticleGoogle Scholar
- Mirasierra M, Vallejo M. (2006) The homeoprotein Alx3 expressed in pancreatic beta-cells regulates insulin gene transcription by interacting with the basic helix-loop-helix protein E47. Mol. Endocrinol. 20:2876–89.View ArticleGoogle Scholar
- Bonny C, Thompson N, Nicod P, Waeber G. (1995) Pancreatic-specific expression of the glucose transporter type 2 gene: identification of ciselements and islet-specific trans-acting factors. Mol. Endocrinol. 9:1413–26.PubMedGoogle Scholar
- Galan M, et al. (2006) Effects of novel maturityonset diabetes of the young (MODY)-associated mutations on glucokinase activity and protein stability. Biochem. J. 393:389–96.View ArticleGoogle Scholar
- Chi YI, Frantz JD, Oh BC, Hansen L, Dhe-Paganon S, Shoelson SE. (2002) Diabetes mutations delineate an atypical POU domain in HNF-1alpha. Mol. Cell 10:1129–37.View ArticleGoogle Scholar
- Scatchard G. (1949). The attractions of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51:660–72.View ArticleGoogle Scholar
- Cervin C, et al. (2002) Characterization of a naturally occurring mutation (L107I) in the HNF1 alpha (MODY3) gene. Diabetologia 45:1703–8.View ArticleGoogle Scholar
- Bjorkhaug L, Sagen JV, Thorsby P, Sovik O, Molven A, Njolstad PR. (2003) Hepatocyte nuclear factor-1 alpha gene mutations and diabetes in Norway. J. Clin. Endocrinol. Metab 88:920–31.View ArticleGoogle Scholar
- Dix DJ, Lin PN, Kimata Y, Theil EC. (1992) The iron regulatory region of ferritin mRNA is also a positive control element for iron-independent translation. Biochemistry 31:2818–22.View ArticleGoogle Scholar
- Wilhide CC, et al. (1997) The human integrin beta3 gene is 63 kb and contains a 5′-UTR sequence regulating expression. Blood 90:3951–61.PubMedGoogle Scholar
- Mendell JT, Dietz HC. (2001) When the message goes awry: disease-producing mutations that influence mRNA content and performance. Cell 107:411–14.View ArticleGoogle Scholar
- Godart F, et al. (2000) Identification of seven novel nucleotide variants in the hepatocyte nuclear factor-1alpha (TCF1) promoter region in MODY patients. Hum. Mutat. 15:173–80.View ArticleGoogle Scholar
- Soutoglou E, Papafotiou G, Katrakili N, Talianidis I. (2000) Transcriptional activation by hepatocyte nuclear factor-1 requires synergism between multiple coactivator proteins. J. Biol. Chem. 275:12515–20.View ArticleGoogle Scholar
- Wu GD, Chen L, Forslund K, Traber PG. (1994) Hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and HNF-1 beta regulate transcription via two elements in an intestine-specific promoter. J. Biol. Chem. 269:17080–5.PubMedGoogle Scholar
- Chi YI. (2005) Homeodomain revisited: a lesson from disease-causing mutations. Hum. Genet. 116:433–44.View ArticleGoogle Scholar
- Bowden SA, Hoffman RP. (2008) Triple diabetes: coexistence of type 1 diabetes mellitus and a novel mutation in the gene responsible for MODY3 in an overweight adolescent. Pediatr. Diabetes 9:162–4.View ArticleGoogle Scholar
- Ito M, Achermann JC, Jameson JL. (2000) A naturally occurring steroidogenic factor-1 mutation exhibits differential binding and activation of target genes. J. Biol. Chem. 275:31708–14.View ArticleGoogle Scholar
- Servitja JM, et al. (2009) Hnf1alpha (MODY3) controls tissue-specific transcriptional programs and exerts opposed effects on cell growth in pancreatic islets and liver. Mol. Cell. Biol. 29:2945–59.View ArticleGoogle Scholar
- Ferrer J. (2002) A genetic switch in pancreatic beta-cells: implications for differentiation and haploinsufficiency. Diabetes 51:2355–62.View ArticleGoogle Scholar
- Boj SF, Petrov D, Ferrer J. (2010) Epistasis of transcriptomes reveals synergism between transcriptional activators Hnf-1alpha and Hnf-4alpha. PLoS Genet. 6:e1000970.View ArticleGoogle Scholar