The maturity-onset diabetes of the young (MODY1) transcription factor HNF4a regulates expression of genes required for glucose transport and metabolism (aldolase By2,3 glyceraldehyde 3-dehydrogenaseyliver pyruvate kinaseyembryonic stem cellsyvisceral endoderm)

Hepatocyte nuclear factor 4a (HNF4a) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4a gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4a protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4a function. The effect of loss of function on HNF4a target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic b-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4a. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4a. These data provide direct evidence that HNF4a is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis. Hepatocyte nuclear factor 4a (HNF4a) belongs to the steroidy thyroid hormone receptor superfamily of transcription factors and first was identified by its interaction with a cis-regulatory sequence of liver specific gene promoters (1). Like other members of the family, HNF4a contains a zinc finger regions and binds DNA as a dimer. The carboxyterminal region contains a large hydrophobic domain (amino acids 133–373) reminiscent of the dimerization and ligand binding domain of other steroid hormone receptors (2). Although HNF4a displays significant sequence similarities to the mammalian retinoic-x receptor a, it does not heterodimerize with any of the other nuclear receptors identified and exists as stable homodimers in solution (3). Targeted disruption of the HNF4a gene results in early embryonic death caused by dysfunction of the visceral endoderm (VE) in which it is expressed (4, 5). In the adult HNF4a is located primarily in the liver, gut, kidney, and pancreatic islets (1, 6). HNF4a interacts with regulatory elements in promoters and enhancers of genes whose products are involved in diverse function, including cholesterol, fatty acid, amino acid, and glucose metabolism, as well as liver development and differentiation (5, 7, 8). Furthermore it has been demonstrated that HNF4a is critical for regulating expression of numerous genes in vivo (9). We recently have shown that a mutation in HNF4a can cause a form of early-onset type 2 diabetes (maturity-onset diabetes of the young; gene named MODY1) (10). The molecular mechanisms by which mutations in HNF4a cause an autosomal dominant form of type 2 diabetes are unknown. Clinical studies suggest that MODY1 is characterized by a defect in glucose-stimulated insulin secretion, suggesting that abnormal gene expression in the pancreatic b-cell is responsible for this disorder (11). The present study was designed to determine the biological function of the MODY1 HNF4(Q268X) mutation and the effect on the transactivation of downstream target genes. We demonstrate that the mutant truncated HNF4a protein has lost its transcriptional transactivation function and does not bind to HNF4 binding sites in vivo. Furthermore, we show that loss of function of HNF4a leads to impaired expression of genes involved in glucose transport and glycolysis, two steps known to be important for glucose uptake from the enterohepatic circulation into hepatocytes and for insulin secretion of pancreatic b-cells. MATERIALS AND METHODS Expression of Proteins and Immunoblot Analysis. cDNAs for HNF4a and the deletion mutants cloned into Bluescript were transcribed by using T3 RNA polymerase, and the transcripts were translated in the rabbit reticulocyte lysate TNT system (Promega) as described (12). The relative amounts of the translated proteins were determined by SDSy PAGE. Electrophoretic Mobility-Shift Assay (EMSA). In vitrotranslated receptor protein was incubated with the 32P-labeled oligonucleotide in a 15-ml reaction mixture containing 10 mM Hepes buffer, 50 mM KCl, 1.0 mM DTT, 2.5 mM MgCl2, 10% glycerol, and 1 mg of poly(dI-dC) at 25°C for 20 min. The reaction mixture then was loaded on a 6% nondenaturing The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9413209-6$2.00y0 PNAS is available online at http:yywww.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: MODY, maturity-onset diabetes of the young; HNF, hepatocyte nuclear factor; EMSA, electrophoretic mobility-shift assay; ES, embryonic stem; CMV, cytomegalovirus; VE, visceral endoderm; EB, embryoid body; HPRT, hypoxanthine phosphoribosyltransferase; L-PK, liver pyruvate kinase; aldoB, aldolase B. *To whom reprint requests should be addressed at: Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Avenue, Box 292, New York, NY 10021. e-mail: stoffel@rockvax. rockefeller.edu.