Abstract
Unique nuclear receptor Hepatocyte Nuclear Factor 4α (HNF4α) is an essential transcriptional regulator for early development and proper function of pancreatic ß-cells, and its mutations are monogenic causes of a dominant inherited form of diabetes referred to as Maturity Onset Diabetes of the Young 1 (MODY1). As a gene-specific transcription factor, HNF4α exerts its function through various molecular interactions, but its protein recruiting network has not been fully characterized. Here we report the identification of MED25 as one of the HNF4α binding partners in pancreatic ß-cells leading to insulin secretion which is impaired in MODY patients. MED25 is one of the subunits of the Mediator complex that is required for induction of RNA polymerase II transcription by various transcription factors including nuclear receptors. This HNF4α-MED25 interaction was initially identified by a yeast-two-hybrid method, confirmed by in vivo and in vitro analyses, and proven to be mediated through the MED25-LXXLL motif in a ligand-independent manner. Reporter-gene based transcription assays and siRNA/shRNA-based gene silencing approaches revealed that this interaction is crucial for full activation of HNF4α-mediated transcription, especially expression of target genes implicated in glucose-stimulated insulin secretion. Selected MODY mutations at the LXXLL motif binding pocket disrupt these interactions and cause impaired insulin secretion through a ‘loss-of-function’ mechanism.
Highlights
Hepatocyte Nuclear Factor 4a (HNF4a) is a unique member of the nuclear receptor (NR) superfamily, and plays a critical role in early vertebrate development and metabolic regulation [1]
We present evidence that MED25 is recruited by HNF4a in a ligand-independent manner, and this interaction is necessary for full HNF4a-mediated transcription leading to insulin secretion in the pancreatic ß-cells
Our results showed that MED25 was necessary for full activation of the majority of HNF4a subtype specific target genes involved in insulin secretion
Summary
Hepatocyte Nuclear Factor 4a (HNF4a) 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 recent genome-wide expression profiling study [2] and conditional inactivation of its gene in mice [3,4,5]. In order to identify additional functional binding partners of HNF4a working in ß-cells, we performed yeast-two-hybrid experiments using various constructs of HNF4a as bait and a ß-cell library as prey, and identified MED25/ TRAP97 ( known as DRIP97, ARC92, p78, ACID1, or PTOV2) as the Mediator component of HNF4a-driven transcription
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