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用于控制发育生物体中G1-S细胞周期转变的调节机制。本研究的重点是转录因子E2F家族，该家族转录因子可对G1-S过渡所需基因的表达进行正向和负向调控，包括参与核苷酸生物合成、复制起源识别、DNA复制和细胞周期进程控制的基因。过度表达E2F足以驱动静止细胞进入s期，基因抑制E2F依赖性转录会减弱DNA合成，导致G1期细胞聚集。相应地，E2F基因在过度表达时表现为致癌基因，并且在许多癌症中发现了E2F活性失调的突变。然而，E2F活性的全部范围尚不清楚，最近的研究表明，除了G1-S控制外，E2F可能调节许多涉及细胞功能各个方面的基因的表达。此外，E2F转录因子受整个动物发育信号调控的机制尚不完全清楚。利用黑腹果蝇强大的分子遗传学、细胞生物学和转基因技术，研究E2F的调控和功能。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作为一个专用的抑制因子，而E2F1同时作为激活因子和抑制因子。如何调节这些活动以控制不同发育阶段的细胞周期将在三个目标中解决：1)利用遗传和生化策略确定E2F1和E2F2在发育过程中的相互作用机制；2)利用基因表达微阵列鉴定和表征新的E2F靶基因，确定E2F依赖性转录对DNA复制的贡献；3)利用正向遗传筛选鉴定胚胎发生过程中E2F的新调控因子，阐明调节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.