Mutation P291fsinsC in the Transcription Factor ...

Mutation P291fsinsC in the Transcription Factor Hepatocyte Nuclear Factor-1 is Dominant Negative Kazuya Yamagata, Qin Yang, Koji Yamamoto, Hiromi Iwahashi, Jun-ichiro Miyagawa, Kohei Okita, Issei Yoshiuchi, Jun-ichi Miyazaki, Tamio Noguchi, Hiromu Nakajima, Mitsuyoshi Namba, Toshiaki Hanafusa, and Yuji Matsuzawa

The type 3 form of maturity-onset diabetes of the young (MODY3) results from mutations in the gene encoding the transcription factor, hepatocyte nuclear factor-1 (HNF-1 ). The mechanism by which mutations in only one allele of the HNF-1 gene impair pancreatic -cell function is unclear. The functional form of HNF-1 is a dimer—either a homodimer or a heterodimer with the structurally related protein HNF-1 —that binds to and activates transcription of the genes whose expression it regulates. HNF-1 is composed of three functional domains: an amino-terminal dimerization domain (amino acids 1–32), a DNA-binding domain with POU-like and homeodomain-like motifs (amino acids 150–280), and a COOH-terminal transactivation domain (amino acids 281–631). Because the dimerization domain is intact in many of the mutant forms of HNF-1 found in MODY subjects, these mutant proteins may impair pancreatic -cell function by forming nonproductive dimers with wild-type protein, thereby inhibiting its activity; that is, they are dominant-negative mutations. This hypothesis was tested by comparing the functional properties of the frameshift mutation P291fsinsC, the most common mutation identified to date in MODY3 patients, and wild-type HNF-1 . P291fsinsC-HNF-1 showed no transcriptional transactivation activity in HeLa cells, which lack endogenous HNF-1 . Overexpression of P291fsinsC-HNF-1 in MIN6 cells, a mouse -cell line, resulted in an ~40% inhibition of the endogenous HNF1 activity in a dosage-dependent manner. Furthermore, heterodimer formation between wild-type and P291fsinsC mutant proteins were observed by electrophoretic mobility shift assay. These data suggest that the P291fsinsC mutation in HNF-1 functions as a dominant-negative mutation. However, other mutations, such as those in the promoter region and dimerization domain, may represent loss of function mutations. Thus mutations in the HNF-1 gene may lead to -cell dysfunction by two different mechanisms. Diabetes 47:1231–1235, 1998 From the Second Department of Internal Medicine (K.Yamag., Q.Y. , K.Yamam., H.I., J.Miyag., K.O., I.Y., H.N., M.N., T.H., Y.M.), and the Department of Nutrition and Physiological Chemistry (J.Miyaz.), Osaka University Medical School, Osaka; and the Department of Biochemistry (T.N.), Fukui Medical School, Fukui, Japan. Address correspondence and reprint requests to Dr. Kazuya Yamagata, Second Department of Internal Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan. E-mail: kazu@imed2. med.osaka-u.ac.jp. Received for publication 2 February 1998 and accepted in revised form 28 April 1998. DAPI, 4 ,6-diamidino-2-phenylindole dihydrochloride; EMSA, electrophoretic mobility shift assay; HNF, hepatocyte nuclear factor; MODY, maturity-onset diabetes of the young; PKL, L-type pyruvate kinase; WT, wild-type. DIABETES, VOL. 47, AUGUST 1998

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aturity-onset diabetes of the young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, early onset (usually before age 25 years), and impaired glucose-stimulated insulin secretion (1–3). The type 3 form of MODY, MODY3, results from mutations in the liver-enriched, but not liver-restricted, homeodomain-containing transcription factor, hepatocyte nuclear factor-1 (HNF-1 ) (4). In the liver, HNF-1 plays a role in regulating the expression of proteins involved in carbohydrate and lipid metabolism, clotting factors, and serum proteins (5). The function of HNF-1 in the pancreatic -cell is less clear, although it has been reported to have a small effect on in vitro transcription of the rat insulin I gene (6). It may also regulate the expression of several genes involved in the uptake and metabolism of glucose by the -cell: the GLUT 2 gene has an HNF-1 binding site in its promoter (7) and the expression of the L-type pyruvate kinase (PKL), which is also present in pancreatic islets and insulinoma cells, is regulated in part by HNF-1 (8–10). Thus the -cell dysfunction that predates the development of diabetes in MODY3 subjects (2) may be due to a reduction in the levels of key proteins involved in the control of glucosestimulated insulin secretion. Human HNF-1 is a protein of 631 amino acids composed of three functional domains: an amino-terminal dimerization domain (amino acids 1–32), a DNA-binding domain with POU-like and homeodomain-like motifs (amino acids 150–280), and a COOH-terminal transactivation domain (amino acids 281–631) (5). Mutations associated with MODY3 have been found in all regions of the gene, including the promoter (11–15). HNF-1 binds DNA as a homodimer or a heterodimer with the structurally related protein HNF-1 , suggesting that the mutant proteins may be capable of inhibiting the wild-type protein in the cell by the formation of nonfunctional mutant/wild-type protein dimers (i.e., they function as dominant-negative mutations) (16). However, this cannot be the only mechanism, since mutations associated with MODY have been found in the promoter and dimerization domain of HNF-1 (14,15). These mutations, which are examples of loss of function mutations, suggest that the level of HNF-1 per se plays a critical role in determining normal -cell function. The most common mutation in HNF-1 results from insertion of a C in a polyC tract centered around codon 290 (designated as Pro291fsinsC or P291fsinsC) occurring in ~20% of the families identified to date as having MODY3 (4,11–13). This frameshift mutation leads to the synthesis of a protein of 1231

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315 amino acids lacking most of the transactivation domain. In the present study, we examined the functional properties of the P291fsinsC protein and showed that it can inhibit the activity of wild-type HNF-1 , i.e., that it functions as a dominant-negative mutation. RESEARCH DESIGN AND METHODS Expression of wild-type and P291fsinsC forms of human HNF-1 in cultured cells. A human HNF-1 cDNA clone including the entire coding region with 33 and 455 nucleotides of the 5 - and 3 -untranslated region (Gene Bank accession number M57732), respectively, was isolated from a liver cDNA library (Clontech, Palo Alto, CA) and subcloned into pBluescript SK(+) (Stratagene, La Jolla, CA). The coding region and 455 nucleotides of the 3 -untranslated region were cloned in frame into the mammalian expression vector pcDNA3.1/HisC (Invitrogen, Carlsbad, CA). The recombinant HNF-1 expressed from this vector included amino acids 1–631 preceded by the amino terminal Met-(His)6Xpress-Enterokinase peptide (in single-letter abbreviations for the amino acids, the amino terminal sequence of recombinant HNF-1 produced in this system was MGGSHHHHHHGMASMTGGQQMGRDLYDDDDKVPG-HNF-1 ). The P291fsinsC mutation was introduced into HNF-1 by in vitro mutagenesis using a Chameleon Double-Stranded Site-Directed Mutagenesis Kit (Stratagene). The sequences of all constructs were verified before expression studies. Wild-type (WT) HNF-1 and P291fsinsC-HNF-1 (33 nucleotides of the 5 -untranslated and coding regions and 455 nucleotides of the 3 -untranslated region) were also expressed using the RIP expression vector (17), which has the rat insulin II promoter and the 3 -untranslated region of the mouse major histocompatibility complex E gene. Preparation of rat PKL promoter–luciferase reporter construct. HNF-1 is one of the proteins involved in regulating the expression of the rat PKL gene in liver and insulinoma cells (8–10). A 3-kb fragment of the 5 -flanking region of the rat PKL gene (nucleotides –3000 to +37 relative to the cap site), which was used to generate transgenic mice carrying the rat PKL/chloramphenicol acetyltransferase fusion gene (18), was subcloned into the HindIII site of pGL3-basic luciferase reporter (Promega, Madison, WI). This fragment contains all cis-acting elements necessary for tissue-specific expression, including the HNF-1 binding site (19). Expression of WT-HNF-1 and mutant HNF-1 in COS-7 cells. COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and transfected with 2 µg WT-HNF-1 -pcDNA3.1/HisC, P291fsinsC-HNF-1 -pcDNA3.1/HisC, or pcDNA3.1/HisC vector using LipofectAMINE (Life Technologies/Gibco, Gaithersburg, MD) according to the manufacturer’s protocol. After 48 h, cells were lysed in extraction buffer (100 mmol/l NaCl, 50 mmol/l Tris-HCl [pH 8.0], 20 mmol/l EDTA, and 1% SDS). Then 20 µg of protein were subjected to 10% SDS-PAGE and transferred by electroblotting to an Immobilon-P membrane (Millipore, Bedford, MA). The membranes were blocked for 1 h with phosphate-buffered saline containing 5% skim milk (DIFCO, Detroit, MI) and then incubated overnight at 4°C with Anti-Xpress antibody (Invitrogen). After being washed and reblocked, the membrane was incubated at room temperature for 1 h in the presence of horseradish peroxidase–conjugated anti-mouse IgG antibody (Promega). The membranes were finally washed for 2 h with gentle shaking, and the antibody binding was visualized using enhanced chemiluminescence Western blotting detection reagents (Amersham, Little Chalfont, U.K.). Expression of WT-HNF-1 and mutant HNF-1 in MIN6 cells. MIN6 cells were cultured as previously described (20) and transfected for 6 h using TRANSFECTAM (BioSepra, Marlborough, MA) with 5 µg WT-HNF-1 -pcDNA3.1/HisC or P291fsinsC-HNF-1 -pcDNA3.1/HisC on Lab-Tek chamber slides (Nalge Nunc, Naperville, IL). After 48 h, cells were fixed with 4% paraformaldehyde (buffered at pH 7.4 with 0.1 mol/l phosphate buffer) for 10 min at 4°C and blocked with Block Ace (Yukijirushi, Sapporo, Japan) for 40 min at room temperature. Mouse AntiXpress antibody was applied for 12 h at 4°C, after which an indirect immunofluorescence method was applied using biotinylated horse anti-mouse IgG (Vector, Burlingame, CA) and Fluorescein Avidin D (Vector). Nuclei were visualized by staining with 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI). Transactivation of rat PKL promoter–luciferase reporter gene by WT-HNF1 and mutant HNF-1 . HeLa and MIN6 cells were transfected using TRANSFECTAM with the indicated amounts of expression and reporter vectors together with 100 ng of pRL-SV40 (Promega) as an internal control. The transactivation activity of WT-HNF-1 and P291fsinsC-HNF-1 was measured after 48 h using the Dual Luciferase Reporter Assay System (Promega) and Lumat LB9501 Measuring System for Bio- and Chemiluminescence (Berthold Japan, Osaka, Japan). Each study was repeated three to six times. In vitro translation and electrophoretic mobility shift assay. WT-HNF-1 and mutant HNF-1 proteins were synthesized using the TNT T7 Quick Coupled Transcription/Translation System (Promega). In vitro translated proteins were incubated with the 32P-labeled oligonucleotide containing the rat PKL HNF-1 1232

binding site sequence (9) in a 20-µl reaction mixture containing 20 mmol/l HEPES (pH 7.9), 75 mmol/l KCl, 3% Ficoll, 1 mmol/l MgCl2, 0.1 mmol/l EDTA, 1 mmol/l dithiothreitol, 2 µg poly (dI-dC), and radiolabeled probe at 25°C for 30 min. The DNA-protein complexes were analyzed on 5% polyacrylamide gels using 0.5 Tris-borate/EDTA buffer (0.045 mol/l Tris-borate, 0.001 mol/l EDTA).

RESULTS

Expression of WT-HNF-1 and P291fsinsC-HNF-1 in COS-7 and MIN6 cells. Recombinant Xpress epitopetagged WT-HNF-1 and P291fsinsC-HNF-1 were efficiently expressed in COS-7 cells, a monkey kidney–derived cell line; the sizes of the recombinant proteins are those predicted from their sequences, 73 and 36 kDa, respectively (Fig. 1). The intracellular distribution of these proteins was studied in MIN6 cells. MIN6 cells were transfected with epitopetagged WT-HNF-1 and P291fsinsC-HNF-1 ; 20 of 550 cells (3.6%) and 19 of 455 cells (4.2%), respectively, were positive for staining. Immunostaining localized epitope-tagged WTHNF-1 to the nucleus (Fig. 2A and B). A different staining pattern was observed in cells expressing P291fsinsC-HNF-1 ; 13 of 19 cells (68%) showed the same localization as WTHNF-1 . However, signals were detected only in the cytoplasm in 6 of 19 (32%) P291fsinsC-HNF-1 –expressing cells (Fig. 2C and D). Cells stained in both nucleus and cytoplasm were not observed in our method. Transactivation activity of WT-HNF-1 and P291fsinsCHNF-1 . Functional properties of the P291fsinsC-HNF-1 were tested using human cervical carcinoma HeLa cells, which do not have endogenous HNF-1 (21). HeLa cells were transfected with constructs encoding WT-HNF-1 and P291fsinsCHNF-1 together with the rat PKL promoter–luciferase reporter gene. The mutant HNF-1 did not stimulate transcription of the PKL-luciferase reporter, whereas WT-HNF-1 stimulated transcription and generated a significant increase in reporter gene activity (Fig. 3A). To test whether P291fsinsC-HNF-1 acted as a dominant-negative regulator, increasing amounts (molar ratio 1:0.1 to 1:5) of P291fsinsC-

FIG. 1. Expression of Anti-Xpress-Epitope–tagged WT-HNF-1 and P291fsinsC human HNF-1 in COS-7 cells. COS-7 cells were transfected with WT-HNF-1 -pcDNA3.1/HisC, pcDNA3.1/HisC empty control, or P291fsinsC-HNF-1 -pcDNA3.1/HisC DNA. After 48 h, cell extracts were prepared and proteins were fractionated by SDS-PAGE. The gel was electroblotted to an Immobilon-P membrane, and the membrane was probed with the Anti-Xpress antibody to identify epitope-tagged WT-HNF-1 and P291fsinsC HNF-1 . DIABETES, VOL. 47, AUGUST 1998

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FIG. 2. Intracellular localization of Anti-Xpress-Epitope–tagged WTHNF-1 and P291fsinsC human HNF-1 in MIN6 cells. MIN6 cells were transfected with WT-HNF-1 -pcDNA3.1/HisC (A,B) or P291fsinsCHNF-1 -pcDNA3.1/HisC DNA (C,D). Cells were stained with AntiXpress antibody (A), DAPI (B), Anti-Xpress antibody and DAPI (400 ) (C), or Anti-Xpress antibody and DAPI (1,000 ) (D).

HNF-1 -pcDNA3.1/HisC DNA were transfected with WTHNF-1 -pcDNA3.1/HisC, PKL-pGL3, or pRL-SV40 DNA. Increasing amounts of P291fsinsC-HNF-1 DNA inhibited luciferase activity up to 30% of the control (Fig. 3B), suggesting that P291fsinsC-HNF-1 has a dominant-negative effect on HNF-1 activity. The functional properties of P291fsinsC-HNF-1 were further investigated using MIN6 cells, an insulin-secreting cell line and model of a normal -cell. The RIP vector gave more efficient expression in MIN6 cells than did pcDNA3.1/HisC. Cells transfected with P291fsinsC-HNF-1 -RIP DNA had reduced luciferase activity compared with cells transfected with empty vector alone, suggesting that the mutant protein was acting to inhibit the activity of the endogenous HNF-1 in the MIN6 cells (Fig. 4A). The dominant-negative effect of P291fsinsC-HNF-1 in MIN6 cells was confirmed by transfecting cells with increasing amounts of P291fsinsC-HNF1 -RIP DNA (molar ratio 0.1:1 to 10:1). Increasing amounts of P291fsinsC-HNF-1 again suppressed the transactivation effect of WT-HNF-1 in a dosage-dependent manner (Fig. 4B). Thus we concluded that P291fsinsC-HNF-1 has a dominant-negative effect on the transactivation of the PKL gene in MIN6 cells. Electrophoretic mobility shift assay analysis. The formation of heterodimers between wild-type and mutant proteins is necessary for a dominant-negative effect (16). The heterodimer formation between WT- HNF-1 and P291fsinsCHNF-1 was tested by electrophoretic mobility shift assay (EMSA). Mutant and WT proteins were prepared by in vitro translation; SDS-PAGE analysis indicated that proteins of the expected sizes were synthesized (data not shown). EMSA showed that both WT-HNF-1 and P291fsinsC-HNF-1 bound to the 32P-labeled oligonucleotide containing the PKLHNF-1 binding site and that the binding could be blocked by the addition of a 50-fold excess of unlabeled oligonucleotide (Fig. 5). Equal amounts of in vitro translated WT-HNF-1 DIABETES, VOL. 47, AUGUST 1998

FIG. 3. Transcriptional activity of PKL in HeLa cells. A: 200 ng of WTHNF-1 -pcDNA3.1/HisC, P291fsinsC-HNF-1 -pcDNA3.1/HisC, or empty pcDNA3.1/HisC DNA were transfected with 500 ng of PKL-reporter gene and 100 ng of pRL-SV40 DNA. B: 200 ng of WT-HNF-1 pcDNA3.1/HisC DNA was transfected alone or with increasing amounts (0.02, 0.2, and 1 µg) of the P291fsinsC-HNF-1 - pcDNA3.1/HisC DNA. The total amount of DNA added was adjusted to 2.2 µg using empty pcDNA3.1/HisC DNA. Luciferase activity was normalized by the activity of pRL-SV40. The luminescence ratio of the pGL3 reporter/control pRL-SV40 was as follows. A: WT-HNF-1 = 0.041, empty pcDNA3.1/HisC = 0.0092; B: 200 ng of WT-HNF-1 only = 0.14, 200 ng of WT-HNF-1 and 1 µg of P291fsinsC-HNF-1 = 0.052. Experiments were repeated three times. SE shown as error bars. *P < 0.05; **P < 0.01.

and P291fsinsC-HNF-1 proteins were mixed and assayed for DNA binding. An additional protein/DNA complex was observed with a mobility between that of the WT and mutant homodimers; in addition, the binding could be blocked by the addition of a 50-fold excess of unlabeled oligonucleotide. These results demonstrated that WT-HNF-1 and P291fsinsC-HNF-1 can form heterodimers in vitro consistent with P291fsinsC-HNF-1 being a dominant-negative regulator of HNF-1 activity. 1233

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FIG. 5. EMSA analysis to test for the formation of heterodimers between WT-HNF-1 and P291fsinsC-HNF-1 . We used 1 µg of empty, WT-, or P291fsinsC-HNF-1 - pBluescript SK vector for in vitro translation. We mixed, separately and together, 5 µl of synthesized proteins with 32P-labeled double-stranded oligonucleotide (~1 ng: 2 104 cpm) containing the HNF-1 binding site of the rat PKL promoter. The protein/DNA complexes were separated by polyacrylamide gel electrophoresis. The gel was dried and exposed to X-ray film to visualize the complexes. Then a 50-fold excess (50 ng) of unlabeled oligonucleotide was added to some reactions to confirm the specificity of the protein DNA interactions. Lane 1: synthesized protein from 1 µg of empty pBluescript vector; lane 2: synthesized protein from 1 µg of WTHNF-1 cDNA; lane 3: synthesized protein from 1 µg of WT-HNF-1 cDNA and 50 ng of unlabeled PKL oligonucleotide; lane 4: synthesized protein from 1 µg of P291fsinsC-HNF-1 cDNA; lane 5: synthesized protein from 1 µg of P291fsinsC-HNF-1 cDNA and 50 ng of unlabeled PKL oligonucleotide; lane 6: synthesized protein from 0.5 µg each of WTand P291fsinsC-HNF-1 cDNA; lane 7: synthesized protein from 0.5 µg each of WT- and P291fsinsC-HNF-1 cDNA and 50 ng of unlabeled PKL oligonucleotide. The bands indicate the mobilities of the different protein/DNA complexes.

FIG. 4. Transcriptional activity of PKL in MIN6 cells. A: 50 ng of WTHNF-1 -RIP, P291fsinsC-HNF-1 -RIP, or empty RIP vector DNA were transfected with 500 ng of PKL-reporter gene and 100 ng of pRLSV40. Experiments were repeated six times. B: 50 ng of WT-HNF-1 RIP was transfected alone or with increasing amounts (5, 50, 200, and 500 ng) of the P291fsinsC-HNF-1 RIP expression vector. The total amount of DNA added was adjusted to 1.15 µg using empty RIP vector. Luciferase activity was normalized by the activity of pRL-SV40. The luminescence ratio of pGL3 reporter/control pRL-SV40 was as follows: 50 ng of WT-HNF-1 = 0.247, 50 ng of WT-HNF-1 and 500 ng of P291fsinsC-HNF-1 = 0.102. Experiments were repeated three times. SE shown as error bars. *P < 0.05; **P < 0.01.

DISCUSSION

Mutations in the liver-enriched transcription factor HNF-1 are a common cause of MODY (1). The association of mutations in the promoter of the HNF-1 gene and the amino-terminal dimerization domain with MODY indicates that loss of function of HNF-1 is the basis for MODY in these families (14,15). Because HNF-1 functions as a dimer, some mutations may cause diabetes by a dominant-negative mechanism. The results presented here indicate that the P291fsinsC mutant HNF-1 does indeed have a dominant-negative effect. However, a 10-fold excess of P291fsinsC-HNF-1 could not 1234

completely inhibit HNF-1 –stimulated transcription of the PKL reporter gene (41% of positive control [WT-HNF-1 ]), most likely due to the fact that HNF-1 is not the only transcription factor required for the transactivation of the PKL promoter (22). Immunostaining of P291fsinsC-HNF-1 was found in both the nucleus and cytoplasm of transfected MIN6 cells. Sourdive et al. (23) have shown that COOH-terminal deletions of HNF-1 can change the intracellular distribution of the protein within the cell. Deletion mutants containing 348–416 residues accumulated outside of the nuclear membrane and a mutant HNF-1 with 289 residues were observed in the nucleus and cytoplasm, although predominantly in the nucleus (23). The intracellular localization of P291fsinsCHNF-1 was similar to that of the mutant with 289 residues, suggesting that both mutants are lacking in proper nuclear targeting signals. The nonfunctional dimer between WT-HNF-1 and P291fsinsC-HNF-1 in the nucleus may be unable to activate the target gene, which may explain the cause of the dominant-negative mechanism. Simultaneously, improperly localized P291fsinsC-HNF-1 may dimerize with the normal HNF-1 in the cytoplasm and prevent its entry into the nucleus. Disturbance of the entry of normal HNF-1 could also decrease the transactivation of the target genes. Improper cytoplasmic localization of the truncated insulin promotor factor-1 (IPF-1), which is a pancreatic homeDIABETES, VOL. 47, AUGUST 1998

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odomain transcription factor, and dominant-negative capability have been recently reported (24,25). More investigations will be necessary to clarify the mechanism of the dominantnegative effect of this mutation. The phenotype of patients with MODY due to loss of function mutations (e.g., promoter or dimerization domain mutations) does not appear to differ from that of patients with the dominant-negative P291fsinsC mutation. This suggests that the dominant-negative mutations may not function as such in vivo, possibly due to instability of the mutant mRNA and/or protein (26). Nonetheless, the observation that this mutation can function in a dominant-negative manner in vitro suggests that it may be possible to use such mutations to perturb -cell function in insulinoma cells or transgenic mice and thereby gain a better understanding of how decreased levels of HNF-1 activity lead to -cell dysfunction and diabetes. ACKNOWLEDGMENTS

This work was supported by grants from the Japanese Ministry of Science, Education, and Culture (no. 09877196 and 09671056); the Japan Insulin Study Group; the Ryoichi Naito Foundation for Medical Research; the Mochida Memorial Foundation for Medical and Pharmaceutical Research; and Research for the Future Program of the Japan Society for the Promotion of Science (97L00801). Q.Y. was supported by a research grant from the Japan-China Medical Association. K.Yamag. is a research fellow of the Japan Society for the Promotion of Science. We thank Dr. G. Bell (University of Chicago) for his valuable discussions and critical reading of this manuscript, Dr. R. Palmiter (University of Washington) for providing the RIP expression vector, Dr. Y. Tsujimoto (University of Osaka) for the gift of COS-7 cells, and Dr. Y. Tanizawa (University of Yamaguchi) for the gift of HeLa cells. REFERENCES 1. Fajans SS: Maturity-onset diabetes of the young. Diabetes Metab Rev 5:579–606, 1989 2. Byrne MM, Sturis J, Menzel S, Yamagata K, Fajans SS, Dronsfield MJ, Bain SC, Hattersley AT, Velho G, Froguel P, Bell GI, Polonsky KS: Altered insulin secretory responses to glucose in diabetic and nondiabetic subjects with mutations in the diabetes mellitus susceptibility gene MODY3 on chromosome 12q. Diabetes 45:1503–1510, 1996 3. Lehto M, Tuomi T, Mahtani M, Widen E, Forsblom C, Sarelin L, Gullstrom M, Isomaa B, Lehtovirta M, Hyrkko A, Kanninen T, Orho M, Manley S, Turner R, Brettin T, Kirby A, Thomas J, Duyk G, Lander E, Taskinen MR, Groop L: Characterization of the MODY3 diabetes. J Clin Invest 99:1–7, 1997 4. Yamagata K, Oda N, Kaisaki PJ, Menzel S, Furuta H, Vaxillaire M, Southam L, Cox RD, Lathrop GM, Boriraj VV, Chen X, Cox NJ, Oda Y, Yano H, Le Beau MM, Yamada S, Nishigori H, Takeda J, Fajans SS, Hattersley AT, Iwasaki N, Hansen T, Pedersen O, Polonsky KS, Turner RC, Velho G, Chevre J-C, Froguel P, Bell GI: Mutations in the hepatocyte nuclear factor-1 gene in maturity-onset diabetes of the young (MODY3). Nature 384:455–458, 1996 5. Mendel DB, Crabtree GR: HNF-1, a member of a novel class of dimerizing homeodomain proteins. J Biol Chem 266:677–680, 1991 6. Emens LA, Landers DW, Moss LG: Hepatocyte nuclear factor 1 is expressed in a hamster insulinoma line and transactivates the rat insulin I gene. Proc Natl Acad Sci U S A 89:7300–7304, 1992 7. Takeda J, Kayano T, Fukumoto H, Bell GI: Organization of the human GLUT2 (pan-

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