BIOCHEM. REGULATION OF E2F TRANSCRIPTION FACTOR FUNCTION

描述（改编自申请人摘要）：理解 转录调控是阐明细胞内 在正常生长和转化过程中进行控制。 一个独特的功能 E2 F转录因子的主要作用是调节病毒 和蜂窝信号。 E2 F活性显著增加， 腺病毒感染 此外，E2 F还参与转录调控。 对细胞分化和增殖信号的反应，以及E2 F 在几个癌基因和其他对细胞增殖至关重要的基因中， 增长 了解E2 F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。 腺病毒对E2 F的调节提供了一个有趣的系统，其中， 单个转录因子的活性可以由两种不同的转录因子调节。 机制等 E2 F受腺病毒E1 A和E4蛋白调节， 利用翻译后修饰和蛋白质-蛋白质 互动，分别。 该方案中的实验设计 探讨E2 F在腺病毒介导下的分子生物学功能及腺病毒对E2 F的调控作用， 生化水平。 第一个目标是对 E2 F功能和调节所需的蛋白质结构域。 一名首席 E2 F的优势是几种良好表征的体外 对DNA结合，转录激活， 蛋白质-蛋白质相互作用和磷酸化。 第二个目标是 评估磷酸化在调节E2 F活性中的作用， vivo. 已经证明E2 F需要磷酸化 在体外活化，但在体内的功能作用是未知的。 体内 磷酸化将通过免疫沉淀E2 F来确定， 32 P标记的HeLa细胞，然后在细胞核中比较随后的分析。 从未感染和感染的HeLa细胞中提取。 结合 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.