Regulation of Liver-Specific Gene Expression

项目描述（申请人提供）：本项目的长期目标是阐明肝脏特异性基因表达的分子机制。当前的目标是确定一个关键的肝富集转录因子，肝细胞核因子4a （HNF4a）的作用机制。HNF4a主要存在于肝脏、肾脏、肠道和胰腺中，是配体依赖转录因子（核受体）超家族的一个高度保守的成员，它调节许多对基础代谢以及外源和药物代谢至关重要的靶基因。尽管HNF4a的配体尚未被明确鉴定，但它已通过调节乙型肝炎病毒基因和P450 （cyp）基因与多种人类疾病相关，包括血友病、糖尿病、动脉粥样硬化和癌症。HNF4a也被认为是从昆虫到人类等生物体的重要基因，对成人肝脏表型至关重要。尽管HNF4a具有明显的重要性，但其作用机制仍有待阐明。为了解决这一问题，将追求以下三个具体目标：1)研究异常大的F结构域在HNF4a功能中的作用。虽然我们目前的结果表明F结构域在募集共调控分子中起作用，但对这一独特结构域的结构或功能知之甚少。我们将使用多种体内和体外方法来研究F结构域与HNF4a的其他区域以及其他蛋白质之间的相互作用；2) HNF4a相互作用蛋白的鉴定与分析。将继续利用生物化学和分子生物学技术研究HNF4a与已知的共调节因子之间的相互作用。新发现的相互作用蛋白也将被研究；3) HNF4a磷酸化位点的鉴定与分析。我们将完成HNF4a中超过13个磷酸化位点的定位，并通过基于细胞的实验阐明它们在体外和体内的功能。

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular mechanism of liver-specific gene expression. The immediate goal is to determine the mechanism of action of one critical liver-enriched transcription factor, hepatocyte nuclear factor 4a (HNF4a). Found primarily in the liver, kidney, intestine and pancreas, HNF4a is a highly conserved member of the superfamily of ligand-dependent transcription factors (nuclear receptors) that regulates many target genes essential to basic metabolism as well as xenobiotic and drug metabolism. Whereas a ligand has not yet been definitively identified for HNF4a, it has been linked to several human diseases including hemophilia, diabetes, atherosclerosis and cancer via regulation of hepatitis B viral genes and P450 (cyp) genes. HNF4a is also known to be an essential gene in organisms ranging from insect to man and to be essential for the adult liver phenotype. Despite the obvious importance of HNF4a, much remains to be elucidated about its mechanism of action. To address this, the following three Specific Aims will be pursued: 1) Investigation of the role of the unusually large F domain in HNF4a function. While our current results indicate that the F domain plays a role in recruiting co-regulatory molecules, little else is known about the structure or function of this unique domain. We will use a variety of in vivo and in vitro methods to investigate the interaction between the F domain and other regions of HNF4a as well as other proteins; 2) Identification and analysis of HNF4a interacting proteins. Studies will be continued on the interaction between HNF4a and known co-regulators using biochemical and molecular biological techniques. Newly identified interacting proteins will also be investigated; 3) Identification and analysis of HNF4a phosphorylation sites. We will finish mapping the more than 13 phosphorylation sites in HNF4a and elucidate their function in vitro and in vivo in cell-based assays.