transcription factors [16,17]. The mammalian Mediator complex is composed of at least 28–30 subunits of varying sizes and compositions [13,17] which display a dynamic nature of associ- ation and dissociation. These subunits are organized as a tightly associated core sub-complex, and associate with several groups of subunits that are believed to constitute distinct modules. Although the complex seems to be universally required for all genes, specific subunits are dedicated to regulation of distinct expression programs via interactions with relevant gene-specific transcrip- tional activators. In this work, 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. This interaction is disrupted by the two MODY mutations at the LXXLL motif binding pocket, and normal insulin secretion is impaired as a result. These observations suggest a key role for MED25 as an active Mediator component in HNF4a signaling, which can be exploited for more effective therapeutic intervention. Materials and Methods Yeast Two-hybrid The initial screening using the lexA system was carried out through the Yeast Model System Genomics (YMSG) facility at the Duke University. HNF4a-ligand binding domain (LBD) (142–368) was cloned into the bait vector pGBKT7short (a modified version of pGBKT7 (Clontech) with tags between Gal4BD and bait removed) and the mouse pancreatic library (Clontech) was used as prey. Self-activation was tested with an empty Gal4BD bait vector before the screening against the library. Two hybrid screens were performed with a standard method using the yeast strain S. cerevisiae AH109. Primary isolates were re-streaked on trp2/leu2/ his2/3 mM 3-AT plates and grown several days for lacZ assays. Positive colonies that showed a color change in LacZ assays were picked for colony PCR or for isolation of DNA. Approximately 66106 independent transformations were screened, of which 27 clones were positive for lacZ assays. Each positive hit was retested for one to one interaction by examining the growth of the transformant and performing lacZ assays. Subsequently, the positive clones were sequenced using automated DNA sequence analysis (ABI) and homologies were identified using BLASTN/ BLASTX (National Center for Biotechnology Information). Construction of expression vectors The luciferase reporter plasmids, pCMV Sport6 MED25 harboring the full length cDNA of Human MED25 (or PGC- 1a), pcDNA3 HNF4a FL containing the full length of human HNF4a, and firefly reporter vector pGL3 (BA1)3 were constructed as described previously [18]. The same vectors were used for transfection and insulin secretion assays. For in vitro binding studies, recombinant HNF4a-LBD proteins with a TEV cleavage site were cloned into pET41a (Novagen) with a GST tag, while the full-length PGC-1a or MED25 proteins containing the LXXLL motif (wt or the NR mutant) were also cloned into pET41a (Novagen) vector. MED25 NR and HNF4a MODY Mutant Generation The ‘Quick Change Multi Site-Directed Mutagenesis’ kit (Stratagene) was used to generate the MED25-NR (LXXLL) and HNF4a MODY mutant constructs. The plasmid templates used in the mutagenesis protocol were pCMV Sport6 MED25 and pcDNA3 HNF4a-FL. All of the generated constructs with the mutated sequences were verified with DNA sequencing. HNF4a-MED25 Interactions in Beta-Cells Cell Culture For transcriptional assays, HeLa cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin G, 50 mg/ml streptomycin (Sigma), and 0.1 mM non- essential amino acids (Invitrogen). For the remaining cell culture experiments, mouse insulinoma 6 (MIN6) cells of passage 28 through 30 were cultured in Dulbecco’s modified Eagle’s media (DMEM) containing 5 mM glucose, 10% (vol/vol) fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM glutamine, and 100 mM b-mercaptoethanol [19]. MIN6 cells were a kind gift from Dr. Sabire O¨zcan at the University of Kentucky [20]. Transient Transfection and Transcription Assays (Luciferase Reporter Assays) The full length cDNA of human HNF4a wt or the mutant were subcloned into the pcDNA3(+)/Neo vector (Invitrogen), and the reporter vector pGL3-(BA)3 containing three copies of the HNF4a response element within the promoter of human Apolipoprotein B was constructed and used for luciferase assays in the absence or presence of transfected coactivators (MED25 or PGC-1a). HeLa cells were transfected using Opti-MEM and LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Briefly, a total of 30 ng of pcDNA3 HNF4a and 150 ng of pCMV Sport6 PGC-1a, 50 ng of pCMV Sport6 MED25, 50 ng of pGL3 (HNF1a)1 and 10 ng of pRL-TK (control renilla luciferase vector) were used for transfection of 16105 cells seeded on 24-well plate one day before transfection. For RNA interference experiments, 20 pmol of siRNA of MED25, HNF4a, and PGC-1a were used for inhibition. For ERa and PPARc luciferase assays, 6 hrs after transfection, cells were treated with 10 nM of estradiol or 1 mM of Troglitazone for 48 hrs. These additional luciferase vectors were kind gifts from Dr. Dan Noonan. After transfection and incubation, cells were washed with 16 PBS and lysed with luciferase lysis buffer supplied with the Luciferase assay kit (Promega). Luciferase activity was measured using the Dual Luciferase assay system (Promega) and Lmax Luminometer (Molecular Devices). All values were normalized by the relative ratio of firefly luciferase activity and renilla luciferase activity. At least four independent transfections were performed in dupli- cate. Glutathione S-transferase (GST) Pull-down Assays HNF4a-LBD GST fusion proteins were produced in Escherichia coli, while [35S]-labeled MED25 (wt and the NR mutant), and PGC-1a wt were produced with a TNT reticulocyte lysate in vitro transcription and translation kit (Promega). About 1 mg of fusion proteins bound on beads were resuspended in 50 ml of GST pull down buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.1% NP-40), and mixed with 50 ml of the in vitro translate before being allowed for binding at 4uC for 4 hrs. Immobilized GST fusion protein was detected by SDS-PAGE and Western blotting using Coomassie blue staining and probing with a GST specific antibody (GE Healthcare). The beads were then washed 3 times with 1 ml of GST pull down buffer and resuspended in SDS–PAGE sample buffer. After electrophoresis, the [35S]-labeled proteins were detected by autoradiography. Co-immunoprecipitation MIN6 cells were transfected with pCMV Sport6 MED25 and pcDNA3 HNF4a using Metafectene pro reagent (Biontex). 48 hours after transfection, cells were washed with 16 PBS and lysed with 1 ml of IP lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e44007