E1A Oncoprotein Induced Deregulation of Replication Origin Firing

描述（由申请人提供）：本申请的重点是确定腺病毒（Ad）转化E1A蛋白以p300结合依赖的方式诱导静止细胞进展到S期的机制。E1A与细胞活化的ras或编码E1B19K或55K蛋白的病毒合作，通过结合p300/CBP和Rb家族蛋白，转化啮齿动物细胞。在静止细胞中，E1A与p300/CBP和Rb蛋白相互作用可快速诱导S期。虽然Rb蛋白的失活可以诱导E2F期和S期，但E1A结合p300的作用尚未得到深入研究。先前我们发现p300通过负调控c-Myc来阻止静止细胞过早进入S期。p300与转录因子YY1和上游YY1结合位点的组蛋白去乙酰化酶3合作，使c-Myc处于抑制状态，这有助于维持细胞处于G0期。E1A通过干扰这一机制诱导c-Myc。最近，我们发现在静止细胞中，与DNA合成和细胞周期进展相关的其他一些基因也受到p300的抑制，这些基因是由E1A以p300结合依赖的方式诱导的。shrna下调p300或表达野生型E1A但不表达p300结合缺陷型E1A突变体都会导致c-Myc的诱导，这与快速诱导S期、激活DNA损伤反应和S期阻滞有关。这些细胞含有增加的细胞DNA复制起始活性。我们现在提出研究以确定E1A如何以p300结合依赖的方式解除对起源活性的调节。这些研究有望揭示E1A介导的异常S期进展及其在病毒和细胞转化中的作用。

DESCRIPTION (provided by applicant): The focus of this application is to determine the mechanisms by which adenovirus (Ad) transforming E1A protein induces progression to S phase in quiescent cells, in a p300 binding dependent manner. In cooperation with cellular activated ras or virus encoded E1B19K or 55K proteins and by binding to p300/CBP and Rb family proteins, E1A can transform rodent cells. Interactions of E1A with p300/CBP and Rb proteins in quiescent cells result in rapid induction of S phase. While the inactivation of Rb proteins is well known to induce E2F and S phase, the contribution of E1A binding to p300 has not been thoroughly studied. Previously we showed that p300 prevents premature entry of quiescent cells into S phase by negatively regulating c-Myc. p300 cooperates with transcription factor YY1 and histone deacetylase 3 at an upstream YY1 binding site to keep c-Myc in a repressed state and that this contributes to maintaining cells in G0 phase. E1A induces c-Myc by interfering with this mechanism. Recently, we have discovered that a number of additional genes related to DNA synthesis and cell cycle progression are also repressed by p300 in quiescent cells and these genes are induced by E1A in a p300 binding dependent manner. Either downregulation of p300 by shRNAs or expression of wild type E1A but not p300 binding defective E1A mutants leads to the induction of c-Myc that is linked to rapid induction of S phase, activation of DNA damage response and S phase arrest. These cells contain increased cellular DNA replication origin activity. We now propose studies to determine how the origin activity is deregulated by E1A in a p300 binding dependent manner. These studies are expected to shed new light on E1A mediated aberrant S phase progression and its role in viral and cellular transformation. PUBLIC HEALTH RELEVANCE: Deregulation of cell growth control by a variety of mechanisms is a key step in the development of human cancer. Adenovirus encoded E1A is a time honored model oncoprotein. Understanding its effects on aberrant S phase progression will advance our understanding of how an oncogene by disrupting the growth inhibitory pathways of a cellular protein contributes to neoplastic transformation.