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.
Highlights
HNF4␣ is one of the master regulators of pancreatic -cell development and function, and mutations in the HNF4␣ gene are well-known monogenic causes of diabetes
We present the evidence that EBP1 acts as a repressor of HNF4␣-mediated transcription through interacting with the LXXLL motif– binding pocket of HNF4␣, which has a direct link to glucose-stimulated insulin secretion in -cells that is impaired in the HNF4␣ mutationdriven diabetes
These findings are in line with the initial yeast two-hybrid screening outcomes in which the protein products corresponding to the amino acid 197–373 or 286 –367 region of EBP1 containing the LXXLL motif showed interactions with both HNF4␣–ligand-binding domain (LBD) alone and the full-length HNF4␣
Summary
ErbB3-binding protein 1 (EBP1) represses HNF4␣-mediated transcription and insulin secretion in pancreatic -cells. The details of the structure suggested that EBP1’s LXXLL motif competes with HNF4␣ 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. HNF4␣ (hepatocyte nuclear factor 4␣) is a unique member of the nuclear receptor (NR) superfamily and plays a critical role in early vertebrate development and metabolic regulation [1] It is highly expressed in the liver, kidney, intestine, and pancreas, and its crucial role in these vital organs has been proven by a genome-wide expression profiling study [2] and conditional inactivation of its gene in mice [3,4,5]. We present the evidence that EBP1 acts as a repressor of HNF4␣-mediated transcription through interacting with the LXXLL motif– binding pocket of HNF4␣, which has a direct link to glucose-stimulated insulin secretion in -cells that is impaired in the HNF4␣ mutationdriven diabetes
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