ErbB3-binding protein 1 (EBP1) represses HNF4α-mediated transcription and insulin secretion in pancreatic β-cells

Abstract

HNF4α (hepatocyte nuclear factor 4α) is one of the master regulators of pancreatic β-cell development and function, and mutations in the HNF4α gene are well-known monogenic causes of diabetes. As a member of the nuclear receptor family, HNF4α exerts its gene regulatory function through various molecular interactions; however, there is a paucity of knowledge of the different functional complexes in which HNF4α participates. Here, to find HNF4α-binding proteins in pancreatic β-cells, we used yeast two-hybrid screening, a mammalian two-hybrid assay, and glutathione S-transferase pulldown approaches, which identified EBP1 (ErbB3-binding protein 1) as a factor that binds HNF4α in a LXXLL motif-mediated manner. In the β-cells, EBP1 suppressed the expression of HNF4α target genes that are implicated in insulin secretion, which is impaired in HNF4α mutation-driven diabetes. The crystal structure of the HNF4α ligand-binding domain in complex with a peptide harboring the EBP1 LXXLL motif at 3.15Å resolution hinted at the molecular basis of the repression. The details of the structure suggested that EBP1's LXXLL motif competes with HNF4α coactivators for the same binding pocket and thereby prevents recruitment of additional transcriptional coactivators. These findings provide further evidence that EBP1 plays multiple cellular roles and is involved in nuclear receptor-mediated gene regulation. Selective disruption of the HNF4α-EBP1 interaction or tissue-specific EBP1 inactivation can enhance HNF4α activities and thereby improve insulin secretion in β-cells, potentially representing a new strategy for managing diabetes and related metabolic disorders.

Keywords: EBP1; HNF4alpha; corepressor; diabetes; gene regulation; insulin secretion; metabolic regulation; nuclear receptor; pancreatic beta-cell; protein complex; protein–protein interaction.

Figure 1.

EBP1 binds directly to HNF4α in a LXXLL motif–mediated manner. A, initial yeast two-hybrid findings were confirmed by mammalian two-hybrid assay. This interaction was greatly reduced when the EBP1–NR mutant (a mutation of the LXXLL motif of EBP1 to LXXAA) was used. Interaction between the two proteins, as GAL4 and VP16 fusion constructs, results in an increase in firefly luciferase expression over the negative controls. B, crude minimal interaction domain mapping by the same experiment with a series of HNF4α truncation mutants when EBP1 full-length was used. HNF4α LBD (positions 151–377) is sufficient for the interaction with EBP1, whereas the AF1 domain (positions 1–46), DNA binding domain (DBD, positions 46–133), and F-domain (FD, positions 377–465) all failed to interact. These findings confirm the initial interaction domain mapping results by GST pulldown experiments (Fig. S1). Protein expression levels of individual constructs have been confirmed by Western blotting before the experiments.

Figure 2.

Effects of EBP1 on HNF4α and other NR-mediated transcription. A, overall transcriptional activity measured by standard luciferase-based transcriptional reporter assays on HNF4α-responsive elements. The LXXLL motif mutant (EBP1–NR) and gene knockdown experiment with shRNA were also performed to test the mutational and specific protein effects, respectively (lanes 4 and 5 and lanes 8 and 9). The left five lanes are without HNF4α transfection (thus empty vector, single transfection of EBP1-expressing vectors, or with shEBP1), and the right four lanes correspond to the ones with HNF4α transfection (thus single or double transfections of HNF4α- and EBP1-expressing vectors or with shEBP1). The scatter plots with individual data points (n = 6) are shown. The midlines indicate the average (or mean) values, and the vertical lines indicate the S.E. * indicates a p value <0.001 with respect to each other. ns, nonsignificant. All data have been normalized against firefly Renilla luciferase activity. B–F, overall transcriptional activity measured by standard luciferase-based transcriptional reporter assays on other representative NR-responsive elements (PPARγ, ERα, PR, RARα, and RXRα). For ERα and PPARγ, 10 nm estradiol, a potent ligand for ERα, and 1 μm troglitazone, an agonist for PPARγ, were added, respectively. EBP1 showed repressive effects on these NR-mediated transactivations except ERα (C).

Figure 3.

Effects of EBP1 on HNF4α target gene expression implicated in insulin secretion from pancreatic β-cells. A, target gene RNA amounts were measured by Q-PCR following transfection of the indicated expression vectors. The experimental results are shown in triplicate, whereas actin was used as a loading control in the bottom panel. Indicated molecular weights are predicted values as markers were not included during gel running. Expression levels of PPARα, l-PK, and GLU2 went down, whereas Kir6.2 showed no response. B, real-time PCR quantification with the primers against responsive HNF4α target genes. The scatter plots with individual data points (n = 6) are shown. The midlines indicate the average (or mean) values, and the vertical lines indicate the S.E. * indicates a p value <0.001 with respect to each other. They all equally went down, and these results are in good agreement with the Q-PCR data shown in A. C, ChIP assay using the anti-HNF4α antibody was performed on chromatin extracted from HNF4α and/or EBP1 overexpression vector-treated MIN6 cells, and the specific GLUT2 promoter region containing the HNF4α recognition site was amplified using PCR. Lanes 2 and 5 represent endogenous HNF4α binding to GLUT2 promoter. EBP1 partially inhibited HNF4α binding to the GLUT2 promoter region. D, effects of EBP1 WT, EBP1–NR mutant, and shEBP1 on insulin secretion shown by color change (right panel) and its quantification by absorption at 450 nm (left panel). * indicates a p value <0.001 with respect to each other. ns, nonsignificant. Darker colors (right panel) indicate more insulin secretion as shown by higher data points in their actual numerical value plots. These data show a direct correlation between HNF4α target gene expression and insulin secretion.

Figure 4.

EBP1 LXXLL motif interaction with HNF4α and its effect on HNF4α–LBD overall conformation. A, overall structure of the complex and the close-up view of the LXXLL motif–mediated EBP1 binding to HNF4α represented by the final 2F_o − F_c map contoured at 1 σ (inset). The backbone of the bound peptide is shown as a purple wire in the inset, whereas the overall structure is shown as a ribbon diagram. The bound fatty acid (structural ligand) is also shown as sticks in the overall structure. B, HNF4α-apo protein conformation (PDB access code 1M7W), coactivator-bound activated conformation (PDB access code 1PZL or 3FS1), and competitive-repressor-bound conformation (PDB access code 6CHT), which display two different states of dimeric assembly. Only the coactivators are capable of fully inducing active conformation in which the helix 12 (H12) of both monomers fold in. C, sequence alignment of the LXXLL motifs that can bind to HNF4α and used in the structure determinations (B). Among three LXXLL motifs of PGC-1α, only the middle one with the highest binding affinity is included in the figure. D, normalized melting curves depicting shifted thermal stability of HNF4α–LBD when forming complexes with the LXXLL motif–containing peptide of each protein (colored lines) from that of the HNF4α–LBD apo protein (black line). A melting temperature (T_m) for apo protein and ΔT_m values for peptide-bound proteins are indicated in parentheses. PGC-1α peptide shows the best binding followed by SRC-1 and EBP1 peptides.