HNF4a-MED25 Interactions in Beta-Cells Figure 4. Lack of synergistic activation by MED25 and MED1 on HNF4a target genes and insulin secretion. (A) Overall transcriptional activity measured by standard luciferase-based transcriptional reporter assays on a HNF4a-responsive element. Data represent the mean6SE (n = 4). Compared with HNF4a alone, *p,0.01. (B) The RNA amounts were quantified by real-time PCR with the primers against each HNF4a target genes following transfection of the indicated expression vectors. Data represent the mean6SE (n = 3). Compared with HNF4a alone, *p,0.01. (C) Q-PCR results of the same RNA quantifications showing similar effects. (D) Effects of both MED25 and MED1 on insulin secretion. These data indicate that each Mediator component individually act on HNF4a, and lack synergistic activation. Data represent the mean6SE (n = 3). Compared with HNF4a alone, *p,0.01. doi:10.1371/journal.pone.0044007.g004 indicating the linear relationship between the HNF4a target gene expression and insulin secretion in ß-cells. It is interesting to point out that the effects by double transfection are intermediate values of the two individual transfections, possibly reflecting the fact that the functional unit of HNF4a is a dimer and each monomer has a roughly equal access to either MED25 or MED1, resulting in mixed complex formation and intermediate transactivation values. Since the LXXLL motifs of both MED25 and MED1 are essential for the physical interaction with HNF4a, both Mediator subunits are likely to compete for the same binding site on HNF4a and disallow synergistic activation. This lack of synergistic activation is consistent with the findings on MED14 and MED1 towards Glucocorticoid Receptor-mediated transcription [35], and MED25 and MED1 towards Retinoid Receptor-mediated tran- scription [21], although it is quite contrary to other Mediator subunit combinations such as MED19/MED26 or MED16/ MED23 towards non-NR transcription factors [36,37]. For NR activation, each Mediator components appear to act indepen- dently and display distinct protein recruitment patterns [21,35] although we cannot rule out the possible involvement of additional Mediator components through LXXLL motif-independent inter- actions. Disruptive Effects by MODY Mutations Near the LXXLL Motif Binding Pocket Several mutations have been identified from the MODY patients and point mutations can be very instructive site-specific indicators of protein function and structure. Thus, we probed the effects of the two MODY point mutations (D206Y and M364R) found near the LXXLL motif binding site for MED25 recruitment, target gene expression and insulin secretion (Figure 5A). The selected mutations are proximal to the LXXLL motif binding pocket, and any substitutions at these positions are believed to influence coactivator/Mediator recruitment. While the D206 residue is located on the first turn of helix H4 at the fringe of the LXXLL motif binding pocket, M364 is located at the rim of the LXXLL motif binding groove (Figure 5A). Previously, the mutational effects of these two MODY point mutations have been thoroughly investigated by us through a series of experiments on their structural integrity and specific functional roles such as coactivator recruitment, overall transcription, ligand selectivity, and target gene recognition [38]. For protein interaction assays, PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e44007