Regulation of Liver-Specific Gene Expression

描述(申请人提供)：该项目的长期目标是阐明肝脏特异性基因表达的分子机制。目前的目标是确定一种关键的肝脏富集型转录因子--肝细胞核因子4a(HNF4a)的作用机制。HNF4a主要在肝、肾、肠和胰腺中发现，是配体依赖型转录因子(核受体)超家族中高度保守的成员，调节许多基础代谢以及异体和药物代谢所必需的靶基因。虽然HNF4a的配基尚未确定，但它已通过调节乙肝病毒基因和P450(CYP)基因与几种人类疾病有关，包括血友病、糖尿病、动脉粥样硬化和癌症。HNF4a在从昆虫到人类的各种生物中也被认为是一个必不可少的基因，对成人肝脏的表型也是必不可少的。尽管HNF4a具有明显的重要性，但其作用机制仍有许多需要阐明。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结构域在招募共调控分子方面发挥了作用，但对这个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的方法来研究HNF4a的F结构域与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和分析。将利用生化和分子生物学技术继续研究HNF4a与已知的共同调节剂之间的相互作用。新发现的相互作用蛋白也将被研究；3)HNF4a磷酸化位点的鉴定和分析。我们将完成对HNF4a中13个以上磷酸化位点的定位，并通过基于细胞的分析来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.