Regulatory mechanisms of HBV X gene Transcription

描述（由申请人提供）：慢性B型肝炎病毒（HBV）感染仍然是一个主要问题，世界上有4亿慢性携带者，迄今为止，没有可靠的治疗方法。HBV X基因是肝癌发生的一个主要因素，对病毒在体内的复制和感染至关重要。X基因产物使多种细胞功能失调，包括Ca 2+依赖的信号转导途径、转录和凋亡途径，并最终导致肝细胞的转化。 然而，X基因表达的检测在技术上是困难的，因此X基因转录的调控机制一直不清楚。 本项目的目标是鉴定和表征X基因转录的重要转录因子。 最近的研究揭示了一般转录因子（GTF）的多样性和核心启动子-增强子特异性在转录调控中的重要性。 因此，X基因核心启动子的功能，可以解释X基因转录的独特模式将特别研究。 X mRNA转录起始于多个位点，启动子区不含常用的核心启动子元件，如TATA盒。 X基因启动子可能使用了以前未知的核心启动子元件、核心启动子结合因子和一组非传统的GTF。 X核心启动子可以优先与特定类别的转录调节子和辅助调节子相互作用以建立独特的表达模式。在这项工作中，核心启动子元件将通过广泛的诱变来定义，并且它们的结合蛋白将通过使用DNA-蛋白质相互作用来纯化（具体目标1）。 X基因转录所需的GTF将通过免疫耗竭和体外重建转录来确定（特定目标2）。 X基因启动子的调控将通过瞬时转染、体外转录和各种生化相互作用试验进行研究（特异性目的3）。 通过彻底表征X基因转录中涉及的转录因子，这项研究将为确定控制HBV基因表达的治疗和策略的潜在靶点奠定基础。 它还将有助于对TATA-less启动子转录的一般理解，TATA-less启动子涵盖了人类约三分之二的蛋白质编码基因。 结合X蛋白的功能研究，我们的研究将有助于进一步了解X基因在肝癌发生中的作用。

DESCRIPTION (provided by applicant): Chronic hepatitis B virus (HBV) infection remains a major problem, with 400 million chronic carriers in the world for whom, to date, there is no reliable treatment. The HBV X gene is a major factor in hepato-carcinogenesis and is essential for viral replication and infection in vivo. The X gene product deregulates various cellular functions, including the Ca2+dependent signal transduction pathways, transcription and apoptosis pathways, and eventually leads to transformation of liver cells. However, detection of X gene expression is technically difficult and thus the regulatory mechanisms of X gene transcription have been unclear. The goal of this project is to identify and characterize the transcription factors important for X gene transcription. Recent investigations revealed the importance of general transcription factor (GTF) diversity and core promoter-enhancer specificity in transcriptional regulation. Therefore, features of the X gene core promoter that may explain the unique pattern of the X gene transcription will be particularly studied. X mRNA transcription starts at multiple sites, and the promoter region contains no commonly used core promoter elements such as the TATA box. The X gene promoter may use previously unknown core promoter elements, core promoter-binding factors, and a nontraditional set of GTFs. The X core promoters may preferentially interact with particular classes of transcriptional regulators and coregulators to establish unique expression patterns. In this work, the core promoter elements will be defined by extensive mutagenesis and their binding proteins will be purified by using DNA-protein interaction (Specific Aim 1). The GTFs required for X gene transcription will be determined by immunodepletion and in vitro reconstituted transcription (Specific Aim 2). Regulation of the X gene promoter will be investigated by transient transfection, in vitro transcription, and various biochemical interaction assays (Specific Aim 3). By thoroughly characterizing the transcription factors involved in X gene transcription, this research will serve as a foundation for identifying potential targets for therapeutics and strategies to control HBV gene expression. It will also contribute to the general understanding of transcription from TATA-less promoters, which covers about two thirds of protein-coding genes in humans. Combined with the functional studies of the X protein, our research will improve understanding of the role that the X gene plays in hepatocarcinogenesis.