The MODY1 gene for hepatocyte nuclear factor 4alpha and a feedback loop control COUP-TFII expression in pancreatic beta cells

Abstract

Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4alpha (HNF4alpha) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. The dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4alpha mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4alpha by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4alpha mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.

FIG. 1.

COUP-TFII expression in adult mouse pancreas and in the 832/13 INS-1 cell line. (A to F) Immunostaining of pancreatic sections from adult mice by using mouse monoclonal antibodies against COUP-TFII (A), insulin (B and D), and COUP-TFI (C) and a secondary antibody as a control for specificity (E and F). The sections shown are from the same experiment with the same exposure times. Original magnification, ×200. (G and H) 832/13 INS-1 cells were subjected to immunofluorescence staining for COUP-TFII (G) and superimposed nuclear counterstaining using DAPI (H). Original magnification, ×1,000.

FIG. 2.

Mapping the COUP-TFII promoter and its transactivation by HNF4α. (A) 832/13 INS-1 cells were transiently cotransfected with a luciferase reporter (luc) gene driven by various lengths of the mouse COUP-TFII promoter, designated by their 5′-end positions relative to the defined COUP-TFII gene transcription initiation site (20), and a control vector expressing Renilla luciferase (0.45 μg of DNA per million cells). The ratio of firefly luciferase activity to Renilla luciferase activity is expressed in arbitrary units as the level of induction (n-fold) relative to the activity of the promoterless pGL3 expression vector. RSV, Rous sarcoma virus promoter; *, P < 0.01 compared to values for the −48 and −688 constructs; **, P < 0.01 compared to the value for the −328 construct. (B) The DNA sequence of the bp −328 region of the COUP-TFII promoter contains an HRE motif. The HRE is a direct repeat (DR-1; arrows) with a 1-nucleotide spacer and is conserved in mice, rats, and humans. (C) Effect of transfecting 832/13 INS-1 cells with a vector expressing either wild-type HNF4α (WT-HNF4α; bars 2, 7, 8, 10, and 11) or DN-HNF4α (bars 3 and 12) or with both vectors (bars 4 and 5) at the concentrations indicated on reporter plasmids carrying the luciferase gene controlled by the bp −328, DR-1-mutated bp −328, and bp −48 COUP-TFII promoter constructs. Bars 1, 6, and 9, transfections with 100 ng of expression vector with no cDNA; $, P < 0.01 compared to the value for bar 2; *, P < 0.05 compared to the value for bar 3; §, P < 0.05 compared to the value for bar 6; **, P < 0.01 compared to the values for bars 4 and 5; −, absent. Means ± standard errors of the means (SEM) of results obtained from at least three separate transfections performed in triplicate are shown.

FIG. 3.

HNF4α binds to the COUP-TFII DR-1 site in vitro and in vivo. (A) Results of EMSA using the COUP-TFII DR-1 probe are shown. The probe was incubated with 832/13 INS-1 cell nuclear extracts (lanes 1 to 6) and mouse liver cell nuclear extracts (lanes 7 to 9). Competition experiments were performed with 50 ng of unlabeled oligonucleotides. Lanes: 1, 832/13 INS-1 cell nuclear extracts; 2, same as lane 1 plus 1 μl of anti-HNF4α antibody; 3, same as lane 1 plus 1 μl of anti-USF antibody; 4, same as lane 1 plus M competitor (COUP-TFII DR-1 mutant oligonucleotide); 5, same as lane 1 plus COUP-TFII DR-1 competitor; 6, same as lane 1 plus MLP competitor; 7, liver cell nuclear extracts; 8, same as lane 7 plus 1 μl of anti-USF antibody; and 9, same as lane 7 plus 1 μl of anti-HNF4α antibody. Positions of HNF4α-specific retarded bands are indicated by arrows. The asterisk indicates the position of the supershifted complexes. MLP, major late promoter; F, free probe; ns, nonspecific; −, absent. (B) Results of ChIP from 832/13 INS-1 cells. Targets for RT-QPCR amplifications were the proximal COUP-TFII promoter containing the DR-1 DNA binding site and a downstream coding region (negative control). The panel shows the results of three independent experiments measuring the relative amounts of target chromatin precipitated by the antibody against HNF4α compared to those precipitated by control IgG, presented as the average signal normalized to the IgG coding region control (± SEM; *, P = 0.03).

FIG. 4.

COUP-TFII mRNA expression in INS-1 DN-HNF4α-expressing cells. Cells were cultured in the presence (+) or absence (−) of 500 ng of doxycycline/ml for 24 h. (A) The upper panel shows DN-HNF4α expression as demonstrated by Western blot analysis with antibodies against the Myc tag (30 μg/lane). The lower panel shows the same blot hybridized with antibodies against cyclophilin as the loading control. (B) RT-QPCR analysis of COUP-TFII mRNA in INS-1 DN-HNF4α-26 cells. The results shown are the means ± SEM of results obtained in three independent experiments.

FIG. 5.

HNF4α mRNA expression in 832/13 INS-1 cells transfected with COUP-TFII siRNA. (A and C) 832/13 INS-1 cells were transfected with unrelated (control) or COUP-TFII-specific siRNA by electroporation and cultured for 48 h in INS-1 medium. Total RNA was extracted and subjected to RT-QPCR to measure mRNA levels for COUP-TFII and controls Neuro-D1 and Pdx1 (A) and for HNF4α (C). The results shown are the means ± SEM of results obtained in eight independent experiments. (B) Western blot of nuclear extracts (20 μg/lane) probed with antibodies against COUP-TFII (upper panel) and against GAPDH as a loading control (lower panel).

FIG. 6.

Effects of adenovirus-mediated overexpression of COUP-TFII in 832/13 INS-1 and mouse pancreatic beta cells. 832/13 INS-1 cells and islets were cultured in INS-1 medium and infected (at 2 or 5 PFU/cell and 200 PFU/cell, respectively) with adenovirus expressing GFP (Ad-GFP) or Ad-hCOUP-TFII as indicated. (A, left panel) For EMSA, the oligonucleotide duplex corresponding to the COUP-TFII DR-1 site was used as a probe. Lanes: 1, Ad-GFP-infected 832/13 INS-1 cell nuclear extracts; 2, Ad-hCOUP-TFII-infected 832/13 INS-1 cell nuclear extracts; 3, same as lane 2 plus 1 μl of anti-COUP-TFII antibody. The position of the COUP-TFII-specific retarded band is indicated on the left. The asterisk indicates the position of the supershifted complexes. (A, right panel) Representative Western blot analysis of COUP-TFII expression. The upper image shows COUP-TFII protein; the lower image shows the same blot incubated with antibodies against GAPDH as a loading control. (B, C, and D) Total RNA was extracted and subjected to RT-QPCR to measure mRNA levels for HNF4α (B), PPARα (C), and endogenous rat COUP-TFII (D). −, absent. (E) ChIP from 832/13 INS-1 cells. Targets for amplifications were the proximal COUP-TFII promoter containing the DR-1 DNA binding site and a downstream coding region as a negative control. The panel shows the results of three independent experiments measuring the relative amounts of target chromatin precipitated by the antibody against COUP-TFII compared to those precipitated by control IgG. The results are presented as the average of the percentage of input (± SEM; *, P = 0.003).

FIG. 7.

Schematic representation of the positive (arrows) and negative (flat-headed symbol) effects of COUP-TFII integrated in a complex regulatory network. Adapted from Médecine Sciences (23) with permission of the publisher.