BIOCHEM. REGULATION OF E2F TRANSCRIPTION FACTOR FUNCTION

描述(改编自申请人的摘要)：理解 转录调控是阐明细胞 正常生长和变形期的对照。独一无二的特点 E2F转录因子的活性是由两种病毒调节的 和手机信号。E2F活性显著增加，以响应 腺病毒感染。此外，E2F还参与转录 对细胞分化和增殖信号的反应，以及E2F 在几个癌基因和其他对细胞至关重要的基因中都存在一些位点 成长。了解E2F功能的分子基础可以提供 对细胞和病毒转录调控的新见解。 E2F的腺病毒调控提供了一个有趣的系统，在这个系统中 单个转录因子的活性可以由两个不同的 机械装置。E2F受腺病毒E1a和E4蛋白调节， 同时利用翻译后修饰和蛋白质-蛋白质 相互作用，分别。该方案中的实验是设计的 探讨E2F的功能和腺病毒对其分子和功能的调控。 生化水平。第一个目标是基因解剖 E2F功能和调控所需的蛋白质结构域。一位酋长 E2F的优势是在体外可利用几种具有良好特性的 快速评分DNA结合、转录激活、 蛋白质之间的相互作用和磷酸化。第二个目标是 磷酸化在调节E2F活性中的作用 活着。已经证明E2F需要磷酸化。 在体外激活，但在体内的功能作用尚不清楚。活体内 将通过免疫沉淀法确定两者的E2F的磷酸化 ~(32)P标记的HeLa细胞和后续分析在核中的比较 未感染和感染的HeLa细胞的提取物。与 突变分析--磷酸化位点的功能意义 可以在体内和体外进行评估。

DESCRIPTION (Adapted from applicant's abstract): An understanding of transcriptional regulation is fundamental to elucidation of cellular controls during normal growth and during transformation. A unique feature of the E2F transcription factor is the regulation of activity by both viral and cellular signals. E2F activity is markedly increased in response to adenovirus infection. In addition, E2F is involved in the transcriptional response to cellular differentiation and proliferative signals, and E2F sites are present in several oncogenes and in other genes critical for cell growth. Understanding the molecular basis of E2F function could provide new insights into both cellular and viral transcriptional regulation. The adenovirus regulation of E2F provides an intriguing system in which the activity of a single transcription factor can be regulated by two distinct mechanisms. E2F is regulated by the adenovirus E1A and E4 proteins and utilizes both post-translational modification and protein-protein interactions, respectively. The experiments in this proposal are designed to probe the function and adenovirus regulation of E2F on the molecular and biochemical level. A first objective is a genetic dissection of the protein domains required for E2F function and regulation. A chief advantage of E2F is the availability of several well-characterized in vitro assays to rapidly score DNA binding, transcriptional activation, protein-protein interactions, and phosphorylation. A second objective is an assessment of the role of phosphorylation in modulating E2F activity in vivo. It has been demonstrated that phosphorylation is required for E2F activation in vitro, but the functional role in vivo is unknown. In vivo phosphorylation will be determined by immunoprecipitation of E2F from both 32P-labelled HeLa cells and then subsequent analysis be compared in nuclear extracts from both uninfected and infected HeLa cells. In conjunction with mutational analysis, the functional significance of phosphorylation sites can be assessed both in vivo and in vitro.