Genetic Analysis of E2F Function During Development

描述(由申请人提供)：本研究项目的长期目标是了解在发育中的生物体中用于控制G1-S细胞周期转变的调控机制。研究的重点是E2F转录因子家族，它们对G1-S转换所需基因的表达进行正向和负向调节，包括参与核苷酸生物合成、复制起点识别、DNA复制和细胞周期进程控制的那些基因。E2F的过度表达足以使静止的细胞进入S期，而E2F依赖的转录的遗传抑制会减弱DNA的合成，导致细胞在G1期积累。相应地，E2F基因在过度表达时表现为癌基因，在许多癌症中发现了破坏E2F活性的突变。然而，E2F活性的全部范围尚不明确，最近的研究表明，E2F可能调控许多基因的表达，这些基因除参与G1-S调控外，还参与细胞功能的多个方面。此外，E2F转录因子在整个动物环境中受发育信号调控的机制还不完全清楚。将利用果蝇强大的分子遗传学、细胞生物学和转基因技术来研究E2F的调节和功能。果蝇含有两种E2F转录因子，分别称为E2F1和E2F2。目前的工作模型认为，E2F2作为一个专用的阻遏因子，而E2F1既是一个激活因子，又是一个抑制因子。如何调节这些活性以控制不同发育阶段的细胞周期将在三个方面得到解决：1)利用遗传和生化策略确定E2F1和E2F2在发育过程中相互作用的机制；2)通过基因表达微阵列确定E2F依赖的转录对DNA复制的贡献，以鉴定新的E2F靶基因；以及3)通过使用正向遗传筛选来确定E2F在胚胎发生过程中的新调节因子，以阐明调控E2F活性的发育途径。

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the regulatory mechanisms used to control the G1-S cell cycle transition in a developing organism. The focus of the research is the E2F family of transcription factors, which both positively and negatively regulate the expression of genes required for the G1-S transition, including those involved in nucleotide biosynthesis, recognition of origins of replication, DNA replication, and control of cell cycle progression. The overexpression of E2F is sufficient to drive quiescent cells into S-phase, and genetic inhibition of E2F dependent transcription attenuates DNA synthesis and leads to the accumulation of cells in G1. Correspondingly, E2F genes behave as oncogenes when overexpressed, and mutations that deregulate E2F activity are found in many cancers. However, the full scope of E2F activity is undefined, and recent studies suggest that E2F may regulate the expression of many genes that are involved in diverse aspects of cellular function in addition to G1-S control. Moreover, the mechanisms by which E2F transcription factors are regulated by developmental signals in the context of a whole animal are not completely understood. The powerful molecular genetics, cell biology, and transgenic techniques of Drosophila melanogaster will be utilized to study E2F regulation and function. Drosophila contains two E2F transcription factors, termed E2F1 and E2F2. The current working model holds that E2F2 acts as a dedicated repressor while E2F1 functions as both an activator and a repressor. How these activities are regulated in order to control the cell cycle at different stages of development will be addressed in three aims: 1) Determine the mechanism of interplay between E2F1 and E2F2 during development using genetic and biochemical strategies, 2) Determine the contribution of E2F-dependent transcription to DNA replication through the use of gene expression microarrays to identify and characterize novel E2F target genes, and 3) Elucidate the developmental pathways that regulate E2F activity by employing a forward genetic screen to identify novel regulators of E2F during embryogenesis.