Molecular Functions of the Adenovirus E1A Oncogene

描述（申请人提供）：人腺病毒E1 A 243 R癌蛋白 编码转录抑制功能，定位于N-末端 结构域（E1 A 1-80），并且是诱导细胞周期进展所必需的， 肿瘤细胞转化我们的目标是了解 E1 A阻遏的分子细节，并确定天然细胞 在腺病毒感染期间，E1 A阻遏结构域靶向启动子。 第一个具体目标是通过以下方式来定义E1 A阻遏的机制： 使用蛋白质-蛋白质相互作用和转录抑制的体外研究 系统将对EIA单氨基酸取代突变体的大面板进行分析。 用于定义E1 A、p300/CBP和TBP之间的相互作用，并确定其 与E1 A抑制功能相关。E1 A的3D结构 N-末端阻遏结构域将通过NMR光谱测定，以帮助 了解E1 A与其细胞伴侣之间的相互作用。我们的工作 一种模型是E1 A进入参与生长的特定细胞启动子 通过p300/CBP作为分子支架进行调节，”然后它可以破坏 TBP和TATA盒之间的相互作用。为了测试这个模型， 将在体外组装并负载已知量的p300/CBP的复合物， 分析E1 A抑制性。第二个具体目标是确定 体内E1 A阻遏的机制。瞬时表达将用于（i） 确定E1 A是否可以利用启动子结合的p300/CBP来访问特定的 基因，（ii）定义E1 A阻遏型启动子的分子决定簇 通过染色质免疫沉淀（CHIP），和（iii）分析分子 非阻遏型启动子对E1 A阻遏的抗性基础。提供 TBP作为E1 A抑制的最终靶点的作用的遗传学证据， TBP单氨基酸取代突变体的详细突变分析将 在体内和体外进行。总的来说，来自体外和 体内研究将允许开发详细的分子模型， E1 A阻遏的机制。第三个具体目标将由CHIP确定 分析E1 A阻遏结构域靶向的天然细胞启动子 感染静止的人类细胞。的功能性后果 E1 A和特异性细胞启动子之间的相互作用将通过 基因特异性mRNA和蛋白产物的动力学研究。

DESCRIPTION (provided by applicant): The human adenovirus E1A 243R oncoprotein encodes a transcription repression function that localizes to the N-terminal domain (E1A 1-80) and is required for induction of cell cycle progression and neoplastic cell transformation. Our goals are to understand the mechanism of E1A repression in molecular detail and to identify the natural cellular promoters targeted by the E1A repression domain during adenovirus infection. The first specific aim is to define mechanism(s) of E1A repression through in vitro studies using protein-protein interaction and a transcription-repression system. A large panel of EIA single amino acid substitution mutants will be used to define interactions among E1A, p300/CBP, and TBP and to establish their relevance to the E1A repression function. The 3D structure of the E1A N-terminal repression domain will be determined by NMR spectroscopy to help understand the interactions between E1A and its cellular partners. Our working model is that E1A accesses specific cellular promoters involved in growth regulation through p300/CBP as a molecular scaffold," where it then can disrupt interaction between TBP and the TATA box. To test this model, preinitiation complexes assembled in vitro and loaded with known amounts of p300/CBP will be analyzed for E1A repressibility. The second specific aim is to define the mechanism of E1A repression in vivo. Transient expression will be used (i) to establish whether E1A can utilize promoter-bound p300/CBP to access specific genes, (ii) to define the molecular determinants of E1A-repressible promoters by chromatin immunoprecipitation (CHIP), and (iii) to analyze the molecular basis of resistance to E1A repression by non-repressible promoters. To provide genetic proof for the role of TBP as an ultimate target of E1A repression, detailed mutational analysis of TBP single amino acid substitution mutants will be performed in vivo and in vitro. Collectively, the findings from in vitro and in vivo studies will allow the development of a detailed molecular model(s) for the mechanism of E1A repression. The third specific aim will identify by CHIP analysis the natural cellular promoters targeted by the E1A repression domain during infection of quiescent human cells. The functional consequences of interaction between E1A and specific cellular promoters will be established by kinetic studies of the gene specific mRNA and protein products.