Structural Characterization of Metabolic Gene Regulators

描述（由申请人提供）：该提案侧重于肝细胞核因子4 α（HNF 4a）的结构和功能表征，HNF 4a是代谢基因程序的主要转录调节因子。HNF 4a不响应配体结合，而是似乎依赖于甲基化和磷酸化，以及诱导共激活因子来调节其转录活性。我们最近对HNF 4a的结构表征揭示了意料之外的多个结构域-结构域相互作用。高度互连的结构域排列将亚基二聚化与DNA和辅激活因子结合联系起来。我们希望通过一系列的生物化学和基于细胞的研究来验证这种对受体变构排列的理解，这些研究探索了这些结构域间的连接。我们还发现了几个翻译后修饰，映射到关键结构域连接。因此，第二个目标是了解共价修饰传递到受体的DNA结合结构域的变构机制。第三个目标是扩大我们对共激活剂如何与HNF 4a相互作用的结构和机制的理解。在这里，我们将测试一个新的假设，即包括PGC-1a在内的一些共激活剂可以物理驱动和稳定DNA上HNF 4a的生产性复合物。

DESCRIPTION (provided by applicant): This proposal focuses on the structural and functional characterization of the hepatocyte nuclear factor 4 alpha (HNF4a), a master transcriptional regulator of metabolic gene programs. HNF4a does not respond to ligand binding, but instead appears to rely on methylation and phosphorylation, and on the induction of coactivators for regulating its transcriptional activity. Our recent structural characterization of HNF4a reveals multiple domain-domain interactions that were unanticipated. A highly interconnected domain arrangement links subunit dimerization to both DNA and coactivator binding. We wish to validate this understanding of the receptor's allosteric arrangement using a series of biochemical and cell-based studies that probe these inter-domain connections. We also found several posttranslational modifications that map to key domain-domain junctions. Therefore, a second goal is to understand the allosteric mechanism by which covalent modifications are transmitted to the DNA binding domain of the receptor. The third goal is to expand our structural and mechanistic understanding of how coactivators interact with HNF4a. Here we will test a novel hypothesis that some coactivators, including PGC-1a, can physically drive and stabilize the productive complex of HNF4a on DNA.