MED25 was confirmed by in vivo coimmunoprecipitation and in vitro GST pull-down assays. The physiological implications of MED25-mediated transactivation of HNF4a was investigated by reporter gene assays and insulin secretion assays in combination with gene silencing studies. Recently, MED25 was identified as an associated subunit of Mediator with HNF4a also in the liver, in the context of lipid and detoxification gene expression [41]. The eukaryotic Mediator complex is made of multiple subunits and each transcription factor associates with a selective subunit for its transactivation [13,20]. Several potential mechanisms for MED25-dependent Mediator recruitment to NRs have been suggested [21,41]. In this report, we present further supporting evidence that MED25 is functionally recruited by several NRs such as HNF4a and ERa, indicating that MED25 is involved in gene selective activation of target genes by diverse NRs, but not all. Particularly, our findings establish MED25 as a potential anchoring point between the Mediator complex and the HNF4a necessary for full transactivation of HNF4a subtype specific target genes in ß-cells leading to insulin secretion. This interaction occurs in a ligand-independent manner, consistent with the current notion that HNF4a is a constitutively active NR [42,43]. However, we failed to observe any synergistic activation between MED25 and MED1, another Mediator subunit known to be associated with HNF4a [26] towards HNF4a-mediated transcrip- tion. It appears that the synergistic activations between the Mediator subunits are absent in NR-mediated transcription as their LXXLL motifs are likely to compete for the same binding sites on NRs while the synergistic effects are more common in non- NR transcription factors [44]. Several mutations have been identified from MODY patients and previous studies on their mutational effects have shown that subtle disruptions of HNF4a’s molecular function can cause significant effects in afflicted MODY patients [38,45]. Our findings confirm the structural predictions and prove that the principal molecular basis of their reduced transcriptional activities is their impaired ability to recruit the coactivators and the Mediator complex, thus establishing a direct linear linkage between the transcriptional events and the physiological conse- quences. These results further prove that MODY mutations are loss-of- function mutations leading to impaired ß-cell function and demonstrate that the LXXLL motif interaction site, in addition to the more commonly exploited ligand binding pocket, can be an effective drug target site. NRs have been successful drug targets (about 20% of current pharmaceuticals) [46,47], due to their modular structures and multiple key interaction sites (i.e. protein- ligand, protein-protein, and protein-DNA interactions). However, it has been believed that, for therapeutic intervention, complete References 1. Sladek FM, Seidel S (2001) Nuclear Receptors in Genetic Disease; Burris TPaM, E.R.B., editor. New York: Academic Press. 309–361 p. 2. Odom DT, Zizlsperger N, Gordon DB, Bell GW, Rinaldi NJ, et al. (2004) Control of pancreas and liver gene expression by HNF transcription factors. Science 303: 1378–1381. 3. Miura A, Yamagata K, Kakei M, Hatakeyama H, Takahashi N, et al. (2006) Hepatocyte nuclear factor-4alpha is essential for glucose-stimulated insulin secretion by pancreatic beta-cells. J Biol Chem 281: 5246–5257. 4. Gupta RK, Vatamaniuk MZ, Lee CS, Flaschen RC, Fulmer JT, et al. (2005) The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion. J Clin Invest 115: 1006–1015. 5. Parviz F, Matullo C, Garrison WD, Savatski L, Adamson JW, et al. (2003) Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis. Nat Genet 34: 292–296. 6. Yamagata K, Furuta H, Oda N, Kaisaki PJ, Menzel S, et al. (1996) Mutations in the hepatocyte nuclear factor-4alpha gene in maturity- onset diabetes of the young (MODY1). Nature 384: 458–460. HNF4a-MED25 Interactions in Beta-Cells inhibition or drastic modulation of these regulators is not desirable, and only partial agonists or antagonists should be considered [48,49,50]. These partial and selective modulators affect only a subset of functions or act in a cell-type-selective manner, and have substantial benefits by retaining the beneficial therapeutic effects while minimizing undesirable side effects. In order to improve the likelihood of finding desirable selective modulators, various potential target sites need to be explored and the NR-coactivator/Mediator binding pocket has been gaining much attention in recent years [51,52]. We believe our newly obtained results will advance the current understanding of the molecular mechanisms underlying HNF4a function and suggest a better strategy for targeting this protein, especially its interaction with coactivators/Mediator complex, for therapeutic intervention [52,53]. Supporting Information Figure S1 MED25 involvement in other NR-mediated transac- tivations (In Figure 2B, ER and PPARc are shown as representative data. In this supplementary figure, additional data are shown for the remaining NRs): (A) progesterone receptor, PR, (B) retinoic acid receptor a, RARa, and (C) retinoid X receptor, RXR. They all showed negligible responses to MED25, while RARa and RXR showed strong responses to PGC-1a. PGC: PGC-1a, and NR: Med25 LXXLL mutant. (TIF) Figure S2 Knockdown efficiency of MED25 shRNA (shMed25) purchased from Origene tested along with scrambled RNA as a negative control. The protein levels in MIN6 cells were performed by specific MED25 antibody after transfections with MED25 and each shRNA (scrambled RNA or different amounts of shMed25) followed by Immunoblotting. (TIF) Acknowledgments We wish to thank Chandra Tucker at Duke University for her assistance in yeast two-hybrid screenings, Samuel Waxman at the Mount Sinai School of Medicine for their kind gifts of RXR/RAR luciferase constructs, and Dan Noonan at the University of Kentucky for ER, PR and PPARc luciferase constructs. Author Contributions Conceived and designed the experiments: EHH YIC. Performed the experiments: EHH GBR. Analyzed the data: EHH GBR YIC. Wrote the paper: EHH YIC. 7. Wang H, Maechler P, Antinozzi PA, Hagenfeldt KA, Wollheim CB (2000) Hepatocyte nuclear factor 4alpha regulates the expression of pancreatic beta - cell genes implicated in glucose metabolism and nutrient-induced insulin secretion. J Biol Chem 275: 35953–35959. 8. McKenna NJ, Xu J, Nawaz Z, Tsai SY, Tsai MJ, et al. (1999) Nuclear receptor coactivators: multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol 69: 3–12. 9. Wang JC, Stafford JM, Granner DK (1998) SRC-1 and GRIP1 coactivate transcription with hepatocyte nuclear factor 4. J Biol Chem 273: 30847–30850. 10. Torres-Padilla ME, Sladek FM, Weiss MC (2002) Developmentally regulated N- terminal variants of the nuclear receptor hepatocyte nuclear factor 4alpha mediate multiple interactions through coactivator and corepressor-histone deacetylase complexes. J Biol Chem 277: 44677–44687. 11. Dell H, Hadzopoulou-Cladaras M (1999) CREB-binding protein is a transcriptional coactivator for hepatocyte nuclear factor-4 and enhances apolipoprotein gene expression. J Biol Chem 274: 9013–9021. PLOS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e44007