Genetic evidence that HNF-1alpha-dependent transcriptional control of HNF-4alpha is essential for human pancreatic beta cell function

Abstract

Mutations in the genes encoding hepatocyte nuclear factor 4alpha (HNF-4alpha) and HNF-1alpha impair insulin secretion and cause maturity onset diabetes of the young (MODY). HNF-4alpha is known to be an essential positive regulator of HNF-1alpha. More recent data demonstrates that HNF-4alpha expression is dependent on HNF-1alpha in mouse pancreatic islets and exocrine cells. This effect is mediated by binding of HNF-1alpha to a tissue-specific promoter (P2) located 45.6 kb upstream from the previously characterized Hnf4alpha promoter (P1). Here we report that the expression of HNF-4alpha in human islets and exocrine cells is primarily mediated by the P2 promoter. Furthermore, we describe a G --> A mutation in a conserved nucleotide position of the HNF-1alpha binding site of the P2 promoter, which cosegregates with MODY. The mutation results in decreased affinity for HNF-1alpha, and consequently in reduced HNF-1alpha-dependent activation. These findings provide genetic evidence that HNF-1alpha serves as an upstream regulator of HNF-4alpha and interacts directly with the P2 promoter in human pancreatic cells. Furthermore, they indicate that this regulation is essential to maintain normal pancreatic function.

Figure 1

Expression of HNF-4α transcripts in human tissues. (a) Schematic representation of possible combinations of HNF-4α splice variations (adapted from ref. 27). Numbers indicate exons. Arrows indicate oligonucleotides used for RT-PCR. (b) RT-PCR analysis of HNF-4α exon 1A (transcribed from the P1 promoter) versus exon 1D (transcribed from the P2 promoter) in pancreatic tissues and liver. β-actin is used as internal control for the RT-PCR procedure. Only one band is amplified using primers designed to amplify HNF-4α exon 1A+2, indicating that transcripts originating in either tissue do not contain exon 1B. (c) RT-PCR analysis of HNF-4α 3′ end splice variations in human islets and liver. The 8F+10R primer set amplifies two fragments containing or lacking an extended exon 9 (9+) insertion. According to these results, liver contains predominantly HNF-4α1, -4α2, and -4α3 transcripts, whereas pancreatic cells contain HNF-4α7, -4α8, and -4α9 variants.

Figure 2

Pedigree of the MODY family with the –181G→A mutation in the P2 promoter of HNF-4α. Squares, male; circles, female; unfilled symbols are normal glucose tolerant; filled symbols are diabetic. Arrowhead indicates proband. The text below each individual represents the following: ID no., genotype (N, normal; M, mutant), age at diagnosis, body mass index (kg/m2), treatment (OHA, oral hypoglycemic agents). Subject 144 was treated with insulin during her second pregnancy.

Figure 3

HNF-1α binding affinity in wild-type and mutant human P2 promoter oligonucleotides. (a) Schematic illustration of oligonucleotides containing the human HNF-4α P2 promoter HNF1 site (bold), and sites containing either the –181G→A mutation (P2 HNF1G→Α) or an artificial mutation intended to completely disrupt HNF1 binding (P2 HNF1→SAC). Mutated bases are in lower case. (b) EMSA of radiolabeled wild-type HNF1 probe. Lanes 1 and 2: incubation with translation reactions using empty vector and pCMVTag-HNF1α, respectively. Lane 2 represents the maximal binding obtained in the absence of any cold competitor. Lanes 3–11: same as 2, except for preincubation with the indicated unlabeled probes at 1×, 10×, and 100× excess relative to the labeled probe. Lanes 12 and 13: same as 2, except for preincubation with either anti–HNF-1α or preimmune antisera, respectively. Similar results were obtained with mouse pancreatic nuclear extracts (not shown). (c) Results from two experiments such as the one shown in b were used to calculate oligonucleotide concentrations required for half-maximal displacement (HNF1G→A, 9.67 ± 1.45 nM; HNF1, 1.36 ± 0.22 nM). *P < 0.05; **P < 0.01.

Figure 4

Effect of P2 promoter mutations on HNF-1α–dependent activation. (a) Schematic representation of the P2 promoter plasmids used in the transfection experiments described. Positions of the HNF1 site and the previously reported TAAT box are depicted. (b) Cotransfection of fibroblasts with indicated reporter constructs plus increasing amounts (0.05–10 ng) of pBJ5-HNF1α. Data are expressed as percentages of transfections performed with empty pBJ5. (c) Effect of DCoH on HNF-1α–dependent activation of indicated constructs. (d) Effect of HNF1G→A and HNF1→SAC mutations in cell lines expressing endogenous HNF-1α. Data are expressed as percentages of results obtained with P2.371. *P < 0.05; **P < 0.01.

Figure 5

Summary of human genetic findings supporting positive cross regulation between HNF-1α and HNF-4α in human pancreatic cells. MODY has been found in subjects with loss-of-function mutations in the coding region of HNF-1α (a), HNF-1α binding site in the P2 promoter of the HNF-4α gene (b), HNF-4α coding region (c), and the HNF-4α binding site (DR1) in the promoter of the HNF-1α gene (d). References are provided in the text.