HNF4a-MED25 Interactions in Beta-Cells Figure 2. Effects of MED25 on HNF4a and other NR-mediated transcription. (A) Overall transcriptional activity measured by standard luciferase-based transcriptional reporter assays on a HNF4a-responsive element. The left four lanes are without HNF4a transfection (thus single transfection of the indicated protein) and the right five lanes correspond to the ones with HNF4a transfection (thus double transfections of HNF4a- and the respective protein expressing vectors). HNF4a serves as a negative control, while PGC-1a serves as a positive control. MED25 NR refers to the LXXLL mutant. CTL in the first lane of each half refers to an empty vector, and all data have been normalized against firefly Renilla luciferase activity. Data represent the mean6SE (n = 4). Asterisk (*) indicates a significant difference between MED25 plus HNF4a and control (CTL) while (**) indicates a significant difference between MED25 plus HNF4a and MED25 NR plus HNF4a. (B) Gene knockdown experiments to test the specific protein effects. Luciferase assays for HNF4a-mediated transcription after transfections with the indicated proteins and the respective siRNA. CTL siRNA refers to scrambled RNA as a negative control while PGC-1a serves as a comparative positive control. (C) Effects of MED25 on ERa-mediated transcription. Luciferase assays of single transfection or double transfections with the indicated proteins in the presence of 10 nM Estradiol, a potent ligand for ERa. MED25 has a strong impact on ERa-mediated transactivation. (D) The same set of experiments as (C) for PPARc in the presence of 1 mM Troglitazone, an agonist for PPARc. MED25 shows no significant influence on PPARc-mediated transactivation. doi:10.1371/journal.pone.0044007.g002 sion in the reporter assays, suggesting the direct involvement of MED25 in HNF4a-mediated transactivation. In order to investigate whether MED25 is also recruited by other NRs, a selective set of NRs have been tested by similar overexpression and transcription assays. We chose PPARc, ERa, PR, RARa, and RXR for the studies, among which only ERa showed a positive response to the overexpression of MED25 (Figure 2C). Other NRs showed no significant responses (Figure 2D and Figure S1), while they all showed positive response to the overexpression of PGC-1a. These results indicate that PGC-1a is recruited by many NRs as a general coactivator, while a specific subunit of the Mediator complex (or sometimes more than one) is recruited by each NR [13,20] including MED25 as a specific Mediator component for HNF4a and ERa. Like the MED25-HNF4a interactions, the positive response on ERa-mediated transcription by MED25 was attenuated by the overexpression of the MED25 NR mutant (Figure 2C), again suggesting the involvement of the LXXLL motif in this interaction and activation. MED25 Enhances HNF4a Target Gene Expression Leading to Glucose-stimulated Insulin Secretion in ß-cells MODY patients are mainly characterized by a severe impairment of insulin secretion [28,29], and MODY gene products, including HNF4a, are monogenic causes of an insulin secretion defect resulting in diabetes. To probe the involvement of MED25 in HNF4a subtype specific target gene expression and insulin secretion in ß-cells, we next tested whether MED25 is required for HNF4a transcriptional activation of previously known HNF4a target genes directly involved in ß-cell insulin secretion such as PPARa [3], L- pyruvate kinase (L-PK) [30,31], GLUT2 [30,31], and Kir6.2 [3,4]. These proteins are involved in the insulin secretion signalling pathway at certain stages such as glucose sensing and transport (GLUT2), TCA or Krebs cycle, i.e. ATP production by mitochondrial enzymes (L- PK), ATP-dependent potassium channel (Kir6.2), and transcrip- tional regulation of additional gene products along the pathway (PPARa). Target gene expression levels were measured by means of transient transfection in MIN6 cells followed by quantification of enriched DNA by real time PCR and Q-PCR. Our results showed that MED25 was necessary for full activation of the majority of HNF4a subtype specific target genes involved in insulin secretion. As shown in Figures 3A and B, expressions of aforementioned HNF4a target genes were mostly increased upon transfections of HNF4a and MED25. PPARa displayed the highest response to double transfection (12-fold increase) followed by L-PK and GLUT2 (both 8-fold increases), while Kir6.2 showed no response. Although the ATP-dependent potassium channel is one of the central players in glucose- stimulated insulin secretion, and altered potassium channel activity is related to the impaired insulin secretion in MODY patients [32,33], there are contradicting data on whether or not one of its subunits, Kir6.2, is a direct target of HNF4a. While one group reported that Kir6.2 is a target gene of HNF4a in ß- PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e44007