MED25 Is a Mediator Component of HNF4a-Driven Transcription Leading to Insulin Secretion in Pancreatic Beta-Cells Eun Hee Han, Geun Bae Rha¤, Young-In Chi* Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America Abstract Unique nuclear receptor Hepatocyte Nuclear Factor 4a (HNF4a) 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, HNF4a 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 HNF4a 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 HNF4a-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 HNF4a-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. Citation: Han EH, Rha GB, Chi Y-I (2012) MED25 Is a Mediator Component of HNF4a-Driven Transcription Leading to Insulin Secretion in Pancreatic Beta- Cells. PLoS ONE 7(8): e44007. doi:10.1371/journal.pone.0044007 Editor: Francis C. Lynn, University of British Columbia, Canada Received April 25, 2012; Accepted July 27, 2012; Published August 30, 2012 Copyright: 2012 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the American Diabetes Association (7-08-CD-03) and the Commonwealth of Kentucky Diabetes Research Trust Fund (KDR- PP09-09) to YIC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ychi@hi.umn.edu ¤ Current address: ImClone Systems, New York, New York, United States of America Introduction 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]. HNF4a regulates expression of a wide variety of essential genes, including those involved in liver and pancreatic cell differentiation, embryogenesis and early development, glucose metabolism, lipid homeostasis, and amino acid metabolism. As such, mutations in HNF4a cause a dominantly inherited form of diabetes known as Maturity Onset Diabetes of the Young 1 (MODY1) [6], further underscoring its pivotal role in human pancreatic ß-cell function and metabolic regulation [4,7]. As a member of the NR superfamily, HNF4a is comprised of distinctive modular domains and exerts its function through various molecular interactions via combinatorial recruitment of multi-protein complexes, including transcriptional cofactors and mediators that further regulate the remodeling of chromatin structure at target gene promoters through histone modifications [8]. Well-known transcriptional coregulators of HNF4a include p160/SRC coactivators such as SRC1 and GRIP1 [9], NR corepressors such as NCoR and SMRT [10], CBP [11], and PGC- 1a [12]. However, its entire protein recruiting network is not well characterized, and more key regulators likely remain to be discovered. Thus, 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 (also known as DRIP97, ARC92, p78, ACID1, or PTOV2) as the Mediator component of HNF4a-driven transcrip- tion. Mediator is an evolutionally conserved multi-subunit coactiva- tor complex that transmits gene regulatory signals by serving as a molecular bridge between transcription factors (as well as associated transcriptional coactivators) to the general transcription machinery containing RNA polymerase II [13,14]. Mediator does not possess intrinsic histone/chromatin-modifying activities and is commonly associated with other general coactivators that contain chromatin-modifying/remodeling activities such as p160/SRC coactivators or CBP. In humans, the Mediator complex was first identified as a positive regulator of thyroid hormone receptor (TR)-mediated transcription and characterized as a TR-associated protein (TRAP) complex [15]. Mediator has since been recognized as a broadly functional coactivator for NRs and other gene-specific PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e44007