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。ApoAI是高密度脂蛋白HDL的主要蛋白组分，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率和发病率的主要原因。该项目的一个目的也是了解孤儿核受体HNF-4，以前参与apoAI转录控制，激活RNA聚合酶II转录的机制。除了apoAI之外，HNF-4还调节参与“代谢综合征”[包括高血压和非胰岛素依赖型糖尿病（NIDDM）]的许多基因。此外，它的基因被独立地分离为年轻人成熟型糖尿病（MODY 1）的致病因素。此外，HNF-4是B型肝炎病毒基因表达的主要转录因子，是肝癌的病原体。因此，预计该项目将具有广泛的相关性。将采用细胞和体外方法来鉴定在apoAI基因的肝脏特异性增强子处HNF-4介导的活化所需的转录因子。研究中的转录因子将包括另一种肝脏富集因子HNF-3（已知与HNF-4协同作用），Mediator（最近显示为HNF-4功能所必需的全局共激活因子）和关键染色质辅因子（组蛋白乙酰基转移酶，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。将监测成熟肝细胞中apoAI基因的稳态表达以及导致发育中小鼠胚状体（培养物中）中apoAI基因表达初始发作的途径。体外方法将补充这些体内研究。因此，还将进行功能（从染色质模板体外转录以概括apoAI基因调控的核心特征）和结构（蛋白质-蛋白质和蛋白质-DNA相互作用）分析。总之，拟议的研究将允许理解相关转录因子的协同功能的机制基础，这些转录因子调节体内apoAI基因表达并有助于其稳态。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.