BIOCHEMICAL FUNCTIONS OF ADENOVIRUS ONCOGENES

描述(摘自申请者摘要)：腺病毒E1A243R 癌蛋白是一种多功能的转录调节因子，表达 在多个域中编码的各种功能。特别值得关注的是 E1AN-末端蛋白的转录抑制功能 对细胞周期进程和细胞周期的诱导非常重要的序列 转型。我们的主要目标是了解分子机制和 E1a转录抑制的细胞靶点。我们的长期目标是 了解E1a抑制如何调节细胞周期的进展。 我们开发了一种体外转录抑制系统，在该系统中，E1a 1-80是一种含有N-端80个氨基酸的重组蛋白。 用作原型抑制子，专门抑制转录 E1a-可抑制基因。压制需要E1a内的两个小区域 N-末端，似乎不需要启动子元件的上游 塔塔箱。第一个具体目标是将这些发现扩展到其他 E1a-可阻遏启动子，包括那些具有医学意义的启动子 ERBB2原癌基因与HIV-1第二个具体目标是继续保持良好 已有研究强烈表明TBP(TFIID)是一种直接细胞 E1a抑制的靶点，并表明E1A1-80可以阻断相互作用 在TBP和TFIIB之间。我们将：(1)进一步界定TFIID的作用和 TFIIB在E1a抑制中的作用分析 结构域和TBP使用E1a和TBP丙氨酸扫描突变体的集合； (Ii)探讨E1a在阻断TBP与TBP相互作用中的双重作用 TATA-box DNA和TFIIB；(Iii)使用纯化的预引发复合体 (图)询问E1a是否抑制TBP、TFIIB或其他GTF的招募 分析TBP和TFIIB对启动子的抑制能力 体内抑制E1a。第三个具体目标是继续研究 细胞蛋白p300在E1a抑制中的作用。我们会问： (I)E1a在体外可被纯化的p300克服；(Ii)E1a 通过使用E1a，可以在功能上将抑制与p300结合分开 丙氨酸扫描突变体；和(Iii)E1a抑制可以被完全逆转 P300和TBP(或TFIID)过表达或细胞显微注射。这个 第四个具体目标是使用重组的体外转录系统 为了更详细地研究E1a抑制物之间的相互作用 域及其细胞靶标TFIID、TFIIB和p300。这个系统允许 阐明E1a可阻抑和E1a之间差异的实验 不可抑制的促进剂。最后，作为长期目标，我们建议使用 消减文库和mRNA差异显示技术用于鉴定 其表达在表达后早期被调节的细胞基因 静止细胞中的E1a N-末端结构域。功能分析将用于 识别保持静止的基因，即潜在的肿瘤抑制基因 这可能是E1a抑制的靶点，以及(Ii)上调的基因 增长周期中的潜在主开关。

DESCRIPTION (Adapted from Applicant's Abstract): The adenovirus E1A 243R oncoprotein is a multifunctional transcriptional regulator that expresses diverse functions encoded in multiple domains. Of special interest is the transcriptional repression function that maps to E1A N-terminal protein sequences important for induction of cell cycle progression and cellular transformation. Our primary goal is to understand molecular mechanism and cellular targets of E1A transcriptional repression. Our long term goal is to understand how E1A repression regulates progression of the cell cycle. We have developed an in vitro transcription-repression system in which E1A 1-80, a recombinant protein containing the N-terminal 80 amino acids, is used as a prototype repressor to specifically repress transcription of E1A-repressible genes. Repression requires two small regions within the E1A N-terminus and does not appear to require promoter elements upstream of the TATA box. The first specific aim is to extend these findings to other E1A-repressible promoters, including those of the medically significant erbB2 protooncogene and HIV-1. The second specific aim is to continue well developed studies strongly implicating TBP (TFIID) as a direct cellular target of E1A repression and showing that E1A 1-80 can block interaction between TBP and TFIIB. We will: (i) define further the role of TFIID and TFIIB in E1A repression by analyzing interaction between the E1A repression domain and TBP using a collection of E1A and TBP alanine scanning mutants; (ii) probe the dual function of E1A in blocking TBP interaction with TATA-box DNA and with TFIIB; (iii) use purified preinitiation complexes (PICs) to ask whether E1A inhibits recruitment of TBP, TFIIB, or other GTFs to the promoter, and (iv) analyze the ability of TBP and TFIIB to overcome E1A repression in vivo. The third specific aim is to continue studies on the role of cellular protein p300 in E1A repression. We will ask whether: (i) E1A repression in vitro can be overcome by purified p300; (ii) E1A repression can be functionally separated from p300 binding by use of E1A alanine scanning mutants; and (iii) E1A repression can be fully reversed by overexpression or cell microinjection of p300 and TBP (or TFIID). The fourth specific aim is to use a reconstituted in vitro transcription system to study in greater detail the interactions between the E1A repression domain and its cellular targets TFIID, TFIIB, and p300. This system permits experiments to elucidate differences between E1A repressible and nonrepressible promoters. Finally, as long term goal, we propose to use subtractive cDNA libraries and mRNA Differential Display to identify cellular genes whose expression is modulated early after expression of the E1A N-terminal domain in quiescent cells. Functional assays will be used to identify Quiescence maintaining genes, i.e. potential tumor suppressor genes that may be targets for E1A repression, and (ii) up-regulated genes that are potential master switches in the growth cycle.