Cell cycle by ORC1 and other E2F targets

ORC1 和其他 E2F 靶标的细胞周期

• 批准号: 6365251
• 负责人: MAKI ASANO
• 金额: $ 23.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6365251
• 关键词: DNA binding protein; Drosophilidae; apoptosis; cell cycle; cell growth regulation; cell proliferation; cyclins; gel electrophoresis; gel mobility shift assay; gene expression; genetically modified animals; imaginal disc; immunoprecipitation; in situ hybridization; microarray technology; northern blottings; nucleic acid probes; phosphorylation; polymerase chain reaction; protein degradation; transcription factor; western blottings

Regulation of the cell cycle is essential in all eukaryotes. The E2F transcription factor plays a central role in cell cycle regulation; it also regulates apoptosis and cell type- specific differentiation. The long-term objective of this proposal is to understand the mechanisms by which E2F coordinately regulates these fundamental biological events. The general strategy of the proposal is to study the E2F pathway in Drosophila melanogaster because: 1) the E2F pathway is remarkably conserved in flies and mammals; 2) there are unique opportunities to study cell cycle progression in unperturbed tissue in flies; 3) transgenic flies offer the opportunities to study aspects of the E2F pathway in the intact organism. Three specific aims will be pursued. (1) Identification of the mechanisms underlying ORC1 degradation. We have shown previously that maintaining the proper level of ORC1 is essential for normal cell cycle progression and viability. ORC1 levels rise as a result of E2F-dependent transcription and fall at the end of S phase. We will perform a mutational analysis to identify residues in ORC1 that mediate this degradation, and test whether Cyclin-dependent phosphorylation plays a role in this process. (2) Investigation of E2F-dependent regulation of G2/M progression. Recent experiments suggest that, in addition to its long-appreciated role in regulating G1/S, E2F also regulates the expression of genes involved in controlling G2/M. Using transgenic flies, we will test the role of E2F in regulating the expression of G2/M target genes in vivo. (3) Identification of novel E2F target genes using cDNA microarrays. E2F activity appears to coordinately regulate cell cycle progression, apoptosis, and differentiation, at least in some cell types. However, only a few of the E2F-regulated genes involved in these diverse processes have been identified. We will identify mRNAs that are up-regulated in response to ectopic E2F in imaginal disc cells using cDNA microarrays, with the goal of better understanding how the E2F pathway coordinately regulates proliferation, apoptosis, and terminal differentiation.

细胞周期的调控在所有真核生物中都是必不可少的。 E2 F转录因子在细胞周期调控中起核心作用;它还调节细胞凋亡和细胞类型特异性分化。 这项建议的长期目标是了解E2 F协调调节这些基本生物事件的机制。 该提案的总体策略是研究黑腹果蝇中的E2 F途径，因为：1）E2 F途径在果蝇和哺乳动物中非常保守; 2）在果蝇未受干扰的组织中研究细胞周期进程有独特的机会; 3）转基因果蝇提供了在完整生物体中研究E2 F途径的机会。将追求三个具体目标。(1)确定ORC 1降解的潜在机制。 我们之前已经表明，维持适当水平的ORC 1对于正常的细胞周期进程和活力是必不可少的。 ORC 1水平的上升是E2 F依赖性转录的结果，并在S期结束时下降。 我们将进行突变分析，以确定ORC 1中介导这种降解的残基，并测试细胞周期蛋白依赖性磷酸化是否在这一过程中发挥作用。(2)研究E2 F依赖性调节G2/M进程。 最近的实验表明，除了其在调节G1/S中的长期作用外，E2 F还调节参与控制G2/M的基因的表达。 使用转基因果蝇，我们将测试E2 F在体内调节G2/M靶基因表达中的作用。(3)使用cDNA微阵列鉴定新的E2 F靶基因。 E2 F活性似乎协调调节细胞周期进程、凋亡和分化，至少在某些细胞类型中是这样。 然而，只有少数的E2 F调节基因参与这些不同的过程已被确定。 我们将使用cDNA微阵列鉴定在成虫椎间盘细胞中异位E2 F反应中上调的mRNA，目的是更好地理解E2 F通路如何协调调节增殖、凋亡和终末分化。