Structural Characterization of Metabolic Gene Regulators

描述（由申请人提供）：本提案侧重于肝细胞核因子4 α (HNF4a)的结构和功能特征，HNF4a是代谢基因程序的主要转录调节因子。HNF4a不响应配体结合，而是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HNF4a的结构表征揭示了意想不到的多个域域相互作用。高度互联的结构域排列将亚基二聚化与DNA和辅激活剂结合联系起来。我们希望通过一系列生化和基于细胞的研究来验证这种对受体变构排列的理解，这些研究探测了这些域间的连接。我们还发现了几个翻译后的修饰，这些修饰映射到关键的结构域连接。因此，第二个目标是了解共价修饰传递到受体DNA结合区域的变构机制。第三个目标是扩展我们对共激活剂如何与HNF4a相互作用的结构和机制的理解。在这里，我们将验证一个新的假设，即一些共激活因子，包括PGC-1a，可以物理驱动和稳定DNA上HNF4a的生产复合体。

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.