Cell cycle by ORC1 and other E2F targets

ORC1 和其他 E2F 靶标的细胞周期

• 批准号: 6779721
• 负责人: MAKI ASANO
• 金额: $ 24.64万
• 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/6779721
• 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.

细胞周期的调节对于所有真核生物都是至关重要的。 E2F转录因子在细胞周期调控中发挥着核心作用；它还调节细胞凋亡和细胞类型特异性分化。 该提案的长期目标是了解 E2F 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果蝇中的E2F通路，因为：1）E2F通路在果蝇和哺乳动物中非常保守； 2）有独特的机会来研究果蝇未受干扰的组织中的细胞周期进展； 3) 转基因果蝇为研究完整生物体中 E2F 途径的各个方面提供了机会。将追求三个具体目标。 (1) 鉴定 ORC1 降解的机制。 我们之前已经证明，维持适当的 ORC1 水平对于正常的细胞周期进展和活力至关重要。 ORC1 水平由于 E2F 依赖性转录而上升，并在 S 期结束时下降。 我们将进行突变分析，以确定 ORC1 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此过程中发挥作用。 (2) E2F 依赖性 G2/M 进程调节的研究。 最近的实验表明，除了长期以来受到重视的 G1/S 调节作用外，E2F 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 E2F 在体内调节 G2/M 靶基因表达的作用。 (3)利用cDNA微阵列鉴定新的E2F靶基因。 E2F 活性似乎协调调节细胞周期进程、细胞凋亡和分化，至少在某些细胞类型中如此。 然而，只有少数参与这些不同过程的 E2F 调控基因已被识别。 我们将使用 cDNA 微阵列识别成虫盘细胞中因异位 E2F 而上调的 mRNA，目的是更好地了解 E2F 通路如何协调调节增殖、凋亡和终末分化。