library as prey. MED25, among others, including the well-known nuclear receptor coactivator 2 (NCoA2) [25], was identified as a putative binding partner when the HNF4a-ligand binding domain (LBD) was used as bait, and was positively retested for one to one interaction through examining the growth of the transformant (Figure 1A). DNA sequence analysis of the positive clones revealed three cDNAs derived from the same gene, encompassing the 407– 744 region of MED25 (AAH21333) containing the LXXLL motif. This mouse sequence shares 91% identity with its human counterpart. 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 Figure 1B, [35S]-labeled MED25 wt showed evidence for interaction with the HNF4a-LBD in the absence of additional external ligand (although much weaker than HNF4a/PGC-1a interactions) while the MED25 mutant (MED25 NR) in which the sole LXXLL sequence (646–650) was mutated to LXXAA failed to interact, suggesting its involvement in this interaction. The previous studies with MED1 and NRs noted that a polyamine such as spermine significantly enhances the binding [26]; however, no such effect was observed for the interaction between HNF4a and MED25 (Figure 1B). To further confirm their interactions in vivo, co-immunoprecip- itation experiments were carried out with whole-cell extracts derived from MIN6 (mouse insulinoma 6) cells. Full-length HNF4a and MED25 (or MED25 NR) were overexpressed, and the cell lysates were subject to immunoprecipitation with HNF4a polyclonal antibody and control IgG antibody before running on a 10% SDS-PAGE gel and performing Western blot analysis with MED25 antibody. As shown in Figure 1C, MED25 was efficiently coprecipitated in MIN6 cells with HNF4a and MED25 NR was only weakly coprecipitated while the control IgG was not. The weak in vivo residual interaction by MED25 NR suggests a possible involvement of other regions of MED25 for HNF4a interactions although the LXXLL motif plays a major role. Taken together, these binding analysis results prove that MED25 physically HNF4a-MED25 Interactions in Beta-Cells 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 Involvement is Specific to a Selective Set of Nuclear Receptors Since MED25 is a component of the eukaryotic transcrip- tional 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 Figure 2A, MED25 substantially enhanced the expression of the reporter gene while two other controls (empty vector (CTL) and another MODY gene product HNF1a) showed no increase. The increase in transcription promoted by the addition of MED25 was even greater than that by the addition of PGC-1a, despite its weaker in vitro binding (Figure 1B), implying that strong in vitro interactions between HNF4a and its transcriptional regulators do not necessarily reflect functionality on endogenous promot- ers. This may also imply that the Mediator complex plays a more dominant role in overall transactivation than coactivators [27], although we cannot rule out the possibility that these weaker transcription factor-Mediator interactions are further supported by additional protein-protein interactions in vivo. This involvement of MED25 in HNF4a-mediated transcription was substantially attenuated by mutations in the LXXLL motif (MED25 NR), indicating that this activation is MED25-specific and once again the C-terminal LXXLL motif in MED25 is important for this interaction. The involvement of MED25 in HNF4a-mediated transcription was next tested by knockdown of individual proteins using siRNA prior to forced expression of proteins of interest including PGC-1a as a control (Figure 2B). Knockdown of each protein (HNF4a, PGC-1a, or MED25) followed by overexpression of HNF4a alone or HNF4a with a coactivator/Mediator partner resulted in knockdown-specific reduction of HNF4a-mediated gene expres- Figure 1. A single subunit, MED25, binds directly to HNF4a in a ligand-independent manner. (A) Yeast-two-hybrid interaction of MED25 and HNF4a-LBD (positive colony in the sector at the 11 O’Clock direction) shown together with negative interacting candidates. One-to-one co- transformed yeasts were selected for growth on Trp2/Leu- plates, and streaked onto Trp2/Leu2/His2/3 mM 3-AT plates. (B) GST pull-down evidence of the interactions. In vitro translated and 35S labeled MED25 (wt or the LXXLL mutant) was incubated with immobilized GST-tagged HNF4a- LBD in the absence of any external ligand. PGC-1a was used as a positive control while GST alone was used as a negative control (lanes #2 and 4). The MED25 LXXLL mutant (MED25 NR) failed to interact, and the addition of 1 mM spermine did not affect the interactions. (C) Immunoblotting evidence of the interactions. Nuclear extracts of MIN6 cells transfected with HNF4a and MED25 were subjected to immunoprecipitation with either IgG (negative control) or HNF4a antibodies followed by Western blot of the immunoprecipitates with MED25 subunit antibodies (input amount on the left). doi:10.1371/journal.pone.0044007.g001 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e44007