ived from the same gene, encompassing the 407 744 region of MED25 containing the LXXLL motif. This mouse sequence shares 91% identity with its human counterpart. interacts with HNF4a in living cells and present MED25 as a potential candidate for anchoring the Mediator complex to HNF4a-responsive promoters. MED25 Mediates HNF4a Transactivation and MED25 MedChemExpress Pyrroloquinolinequinone disodium salt involvement is Specific to a Selective Set of Nuclear Receptors Since MED25 is a 
component of the eukaryotic transcriptional Mediator complex, we first tested its involvement in HNF4a-mediated transcription by over-expressing both proteins and measuring the changes in the reporter gene expression level by HNF4a luciferase assays. As shown in Physical Interaction between HNF4a and MED25 To confirm their physical interactions, GST pull-down assays were carried out using a GST fusion protein of HNF4a-LBD and in vitro-translated MED25 along with PGC-1a as positive control. As shown in 4 HNF4a-MED25 Interactions in Beta-Cells 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. Other NRs showed no significant responses, 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 is recruited by each NR 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, 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 -cells MODY patients are mainly characterized by a severe impairment of insulin secretion, 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 -cells, we next tested whether MED25 is required for HNF4a transcriptional activation of previously known HNF4a target genes directly involved in -cell insulin secretion such as PPARa, L- pyruvate kinase , GLUT2, and Kir6.2. These proteins are involved in the insulin secretion signalling pathway at certain stages such as glucose sensing and transport, TCA or Krebs cycle, i.e. ATP production by mitochondrial enzymes, ATP-dependent potassium channel, and transcriptional regulation of additional gene products along the pathway. 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 HNF4a-MED25 Interactions in Beta-Cells cells through conditional HNF4a knockout experiments, another group reported that the expression level of Kir6.2 in HNF4a knockout mice was unchanged as compared with control mice. Our findings partially support the l