HNF4a-MED25 Interactions in Beta-Cells Figure 5. MODY mutational effects on HNF4a-MED25-mediated transactivation and insulin secretion. (A) Close-up view of the LXXLL motif-mediated coactivator binding pocket of HNF4a, showing the locations of the selected MODY mutations for the current studies (D206Y and M364R) highlighted by a ball-and-stick model (gray sticks and labels). The bound SRC-1a LXXLL motif peptide (blue) and PGC-1a LXXLL motif fragment (red) from the known crystal structures (PDB accession codes 1PZL and 3FS1, respectively) are shown as ribbon diagrams. Hydrogen bonds are indicated as dashed lines. The hydrophobic residues lining the LXXLL motif binding groove and the ‘charge-clamp’ residues (K194 and E363) that make hydrogen bonds with the backbone atoms of the bound peptides are also highlighted as yellow and green sticks, respectively. (B) Mutational effects on HNF4a target gene expression implicated in insulin secretion. RNA amounts were quantified by real-time PCR with the primers against each HNF4a target gene. The first set of bars indicates the control experiments where no transfection vectors were added. Each target gene expression is similarly affected by the mutations, and the mutational effects are more pronounced for the M364R mutant in all cases (last set of bars). (C) Q-PCR results of the same mRNA quantification showing similar effects. Data represent the mean6SE (n = 3). Compared with HNF4a alone, *p,0.01. Compared with MED25 wt plus HNF4a, **p,0.01. (D) Effect of MODY mutations on insulin secretion as shown in Figure 3D and 4D. Similar mutational effects are observed, indicating a direct functional linkage between target gene expression and insulin secretion. Data represent the mean6SE (n = 3). Compared with HNF4a alone, *p,0.01. Compared with MED25 wt plus HNF4a, **p,0.01. doi:10.1371/journal.pone.0044007.g005 together with PGC-1a and SRC-1a, MED25 interactions were measured with HNF4a-LBD wt and the MODY mutants [38]. While the D206Y mutation has subtle effect, the M364R mutation significantly impaired MED25 interactions, suggesting that although the neighboring residues such as D206 also play a role, the actual makeup of the hydrophobic groove seem to play a more predominant role in LXXLL motif recognition and coactivator/ Mediator recruitment [39,40]. In agreement with the protein binding assays, insulin-secretion related target gene expression and the subsequent insulin secretion decreased in MODY mutants. As shown in Figures 5B and 5C, expression of each target gene is similarly affected by the mutations, and the mutational effects are more pronounced for the M364R mutant in all cases (last set of bars), as measured by both real time PCR and Q-PCR. Subsequently, insulin secretion was reduced to a similar extent (Figure 5D), implying that these gene products play critical roles in insulin secretion and that there is a linear relationship between the protein levels and insulin secretion. These functional disruptions are believed to be mainly due to their inability to recruit coactivators/Mediator complex to the fullest extent, while their DNA binding activities and ligand selectivities are preserved [38]. Discussion Transcription factors initiate transcription by recognizing their target genes and mediating additional interactions with various proteins including transcriptional coregulators and the Mediator complex to recruit the remainder of the main transcriptional machinery. To gain additional molecular insights into HNF4a function, we performed yeast two-hybrid screens and identified MED25 as a functional interacting partner of HNF4a in ß-cells. Our study was focused on pancreatic ß-cells because HNF4a is one of the culprit gene products for a dominantly inherited form of diabetes, MODY, mainly characterized by the defect in insulin secretion from ß-cells. The interaction between HNF4a and PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e44007