Section of Structural Biology, Hormel Institute, University of Minnesota, Austin, Minnesota, United States of America.
PLoS One. 2012;7(8):e44007. doi: 10.1371/journal.pone.0044007. Epub 2012 Aug 30.
Unique nuclear receptor Hepatocyte Nuclear Factor 4α (HNF4α) is an essential transcriptional regulator for early development and proper function of pancreatic ß-cells, and its mutations are monogenic causes of a dominant inherited form of diabetes referred to as Maturity Onset Diabetes of the Young 1 (MODY1). As a gene-specific transcription factor, HNF4α exerts its function through various molecular interactions, but its protein recruiting network has not been fully characterized. Here we report the identification of MED25 as one of the HNF4α binding partners in pancreatic ß-cells leading to insulin secretion which is impaired in MODY patients. MED25 is one of the subunits of the Mediator complex that is required for induction of RNA polymerase II transcription by various transcription factors including nuclear receptors. This HNF4α-MED25 interaction was initially identified by a yeast-two-hybrid method, confirmed by in vivo and in vitro analyses, and proven to be mediated through the MED25-LXXLL motif in a ligand-independent manner. Reporter-gene based transcription assays and siRNA/shRNA-based gene silencing approaches revealed that this interaction is crucial for full activation of HNF4α-mediated transcription, especially expression of target genes implicated in glucose-stimulated insulin secretion. Selected MODY mutations at the LXXLL motif binding pocket disrupt these interactions and cause impaired insulin secretion through a 'loss-of-function' mechanism.
独特的核受体肝细胞核因子 4α（HNF4α）是胰腺β细胞早期发育和正常功能所必需的转录调节因子，其突变是一种称为年轻型成年发病型糖尿病 1 型（MODY1）的显性遗传性糖尿病的单基因原因。作为一种基因特异性转录因子，HNF4α 通过各种分子相互作用发挥其功能，但它的蛋白质募集网络尚未完全表征。在这里，我们报告了 MED25 作为 HNF4α 在胰腺β细胞中的结合伴侣之一的鉴定，导致胰岛素分泌受损，而 MODY 患者的胰岛素分泌受损。MED25 是 Mediator 复合物的一个亚基，是包括核受体在内的各种转录因子诱导 RNA 聚合酶 II 转录所必需的。这种 HNF4α-MED25 相互作用最初是通过酵母双杂交方法鉴定的，通过体内和体外分析得到证实，并被证明以配体非依赖性的方式通过 MED25-LXXLL 基序介导。基于报告基因的转录分析和基于 siRNA/shRNA 的基因沉默方法表明，这种相互作用对于 HNF4α 介导的转录的完全激活至关重要，特别是对涉及葡萄糖刺激的胰岛素分泌的靶基因的表达至关重要。该结合口袋的 LXXLL 基序上的选定 MODY 突变破坏了这些相互作用，并通过“功能丧失”机制导致胰岛素分泌受损。
