HNF-4 and coactivators in apoAl gene regulation

描述(由申请人提供)：本提案旨在获得载脂蛋白AI(ApoAI)基因在肝细胞中如何转录调控的详细分子理解。载脂蛋白AI是高密度脂蛋白的主要蛋白质成分，其血浆水平与动脉粥样硬化呈负相关，动脉粥样硬化是发达国家死亡和发病的主要原因。该项目的一个目的也是为了了解孤儿核受体HNF-4通过RNA聚合酶II激活转录的机制，该受体先前参与了载脂蛋白AI的转录控制。除了载脂蛋白AI之外，HNF-4还调节许多与“代谢综合征”相关的基因[包括高血压和非胰岛素依赖型糖尿病(NIDDM)]。此外，它的基因被独立地分离出来，作为青年成熟期糖尿病的致病因素(MODY1)。此外，HNF-4是乙肝病毒基因表达的主要转录因子，而乙肝病毒基因表达是肝癌的病因。因此，预计该项目将具有广泛的相关性。将使用细胞和体外方法来鉴定HNF-4介导的载脂蛋白AI基因肝脏特异性增强子激活所需的转录因子。正在研究中的转录因子将包括另一种肝脏丰富因子，HNF-3(已知与HNF-4协同作用)，介体(最近被证明是HNF-4功能所必需的全球共激活因子)和关键染色质辅助因子(组蛋白乙酰转移酶，ATP重塑)。细胞分析(染色质免疫沉淀[CHIP])将描绘活细胞中转录因子的相互作用。将监测ApoAI基因在成熟肝细胞中的稳定表达，以及导致ApoAI基因在发育中的小鼠类胚体中最初表达的途径(在培养中)。体外方法将补充这些体内研究。因此，还将进行功能分析(从染色质模板体外转录，以概括载脂蛋白AI基因调控的核心特征)和结构分析(蛋白质-蛋白质和蛋白质-DNA相互作用)。总之，拟议的研究将使我们能够理解相关转录因子协同作用的机制基础，这些转录因子调节体内ApoAI基因的表达，并有助于其动态平衡。它们还可能揭示代谢和心血管领域未来治疗发展的目标。

DESCRIPTION (provided by applicant): This proposal aims to obtain a detailed molecular understanding of how the gene for apolipoprotein AI (apoAI) is transcriptionally regulated in liver cells. ApoAI is the major protein component of high-density lipoprotein, HDL, whose plasma levels are inversely correlated with atherosclerosis, a leading cause of mortality and morbidity in developed countries. An aim of the project is also to understand the mechanism by which the orphan nuclear receptor HNF-4, previously implicated in apoAI transcriptional control, activates transcription by RNA polymerase II. In addition to apoAI, HNF-4 regulates many genes involved in the "metabolic syndrome" [including hypertension and non-insulin dependent diabetes mellitus (NIDDM)]. Also, its gene was independently isolated as the causative factor for maturity onset diabetes of the young (MODY1). Furthermore, HNF-4 is a major transcription factor for hepatitis B viral gene expression, an etiologic agent for liver cancer. The project is therefore expected to have broad relevance. Cellular and in vitro methods will be employed to identify transcription factors that are required for HNF-4-mediated activation at the liver-specific enhancer of the apoAI gene. Transcription factors under study will include another liver-enriched factor, HNF-3 (known to synergistically function with HNF-4), Mediator (the global coactivator recently shown to be necessary for HNF-4 function) and key chromatin cofactors (histone acetyl transferases, ATP-remodeling). Cellular analyses (chromatin immunoprecipitation [ChIP]) will delineate transcription factor interactions in living cells. Both steady-state expression of the apoAI gene in mature hepatocytes, as well as the pathway leading to the initial onset of apoAI gene expression in developing mouse embryoid bodies (in culture) will be monitored. In vitro approaches will complement these in vivo studies. Thus, functional (in vitro transcription from chromatin templates to recapitulate core features of apoAI gene regulation) and structural (protein-protein and protein-DNA interactions) analyses will also be undertaken. Together, the proposed studies will permit understanding of the mechanistic basis for the synergistic function of the relevant transcription factors, which regulate apoAI gene expression in vivo and contribute to its homeostasis. They will also potentially reveal targets for future therapeutic developments in the metabolic and cardiovascular areas.