TRANSCRIPTION FACTOR E2F AND CELL CYCLE CONTROL

转录因子 E2F 和细胞周期控制

• 批准号: 2101457
• 负责人: Steven Jay Weintraub
• 金额: $ 7.25万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2101457
• 关键词: DNA replication; cell cycle proteins; cell growth regulation; chemical binding; chimeric proteins; gene induction /repression; genetic regulatory element; intermolecular interaction; molecular cloning; molecular site; neoplasm /cancer genetics; nucleic acid sequence; phosphorylation; protein structure function; retinoblastoma; site directed mutagenesis; tissue /cell culture; transcription factor; tumor suppressor genes; western blottings

Several proteins that associate with cellular promoters have recently been found to have an essential role in the control of cellular proliferation and neoplastic transformation. The importance of one of these, the retinoblastoma protein (Rb), was recognized when it was found to be expressed in every normal tissue, but mutated or deleted in tumors such as retinoblastoma, osteosarcoma, cervical cancer, small cell lung cancer, and bladder cancer. When wild-type Rb is reintroduced into these tumors, they lose their malignant characteristics, therefore, Rb has been called a tumor suppressor. We have demonstrated that Rb is a transcriptional repressor during G-O/G-1 and that is likely to constrain cell division because it represses the expression of a set of genes that is required for DNA synthesis. In support of such a role for Rb, we have found that in Rb(-) cells DNA synthesis persists in G-O/G-1-arrested cells. It is understandable then how the loss of Rb function could lead to a loss of cell cycle control. In quiescent cells (G-O/G-1) Rb is tethered to the promoters of genes by E2F, a protein that binds to a specific site in the promoters. In proliferating cells, Rb is released from E2F in late G-1 and it is replaced on E2F by a complex consisting of a 107 kDa protein (plO7), cyclin A, and cdk2 kinase. We have demonstrated that this S phase complex is a transcriptional activator, suggesting that it mediates the S phase- specific activation of the genes for DNA synthesis enzymes. These results suggest that E2F sites alternate between transcriptional inhibitors and activators during the cell cycle and that this activity is responsible for preventing expression of genes for DNA synthesis enzymes in G-0/G-1 and activating these genes in S phase. We will examine the functions of these proteins that associate with E2F. To analyze the function of Rb, we have constructed a fusion gene where Rb is linked to the DNA binding region of the yeast protein Gal-4. The Gal-4 DNA binding domain has been widely used to tether transcriptionally active proteins to promoters as a tool to examine their activities. In transfection assays, Rb-Gal-4 inhibited transcription from promoters containing Gal-4 binding sites, suggesting that Rb is a transcriptional repressor and that E2F only serves to tether Rb to promoters. Mutations and deletions in Rb will be made and fused to Gal-4. The activity of the resulting fusion proteins will be analyzed in transfection assays to identify the repressor domain of Rb. Similar technology will be used to identify and characterize the transcriptional activator in the S phase-specific E2F complex. Additionally, the role of E2F will be examined in vivo. We will functionally inactivate E2F sites by sequestering cellular E2F. This will be done by transfection of a competitor plasmid containing E2F binding sites. We anticipate that E2F is required for cell proliferation, and these experiments should provide the first direct evidence that this is so. The proposed studies should lead to a further understanding of cell cycle control and the mechanisms underlying the loss of this control that occurs in tumorogenesis.

最近发现了几种与细胞启动子相关的蛋白质 发现在细胞增殖的控制中具有重要作用 和肿瘤转化。其中之一的重要性， 视网膜母细胞瘤蛋白（Rb），当它被发现 在每个正常组织中表达，但在肿瘤中突变或缺失，例如 视网膜母细胞瘤、骨肉瘤、宫颈癌、小细胞肺癌和 膀胱癌。当野生型 Rb 被重新引入这些肿瘤时，它们 失去了其恶性特征，因此，Rb被称为 肿瘤抑制剂。我们已经证明 Rb 是一种转录因子 G-O/G-1 期间的阻遏物，可能会限制细胞分裂 因为它抑制了一组基因的表达，而这些基因是 DNA合成。为了支持 Rb 的这种作用，我们发现 Rb(-) 细胞 DNA 合成在 G-O/G-1 停滞的细胞中持续存在。这是 那么 Rb 功能的丧失如何导致 细胞周期控制。在静止细胞 (G-O/G-1) 中，Rb 与 E2F 是一种与基因中特定位点结合的蛋白质 发起人。在增殖细胞中，Rb 在 G-1 晚期从 E2F 释放， 它在 E2F 上被由 107 kDa 蛋白质 (plO7) 组成的复合物取代， 细胞周期蛋白 A 和 cdk2 激酶。我们已经证明了这个 S 相复合体 是一种转录激活剂，表明它介导 S 期- DNA合成酶基因的特异性激活。这些结果 表明 E2F 位点在转录抑制剂和 细胞周期中的激活剂，并且该活动负责 阻止 G-0/G-1 中 DNA 合成酶基因的表达，以及 在S期激活这些基因。我们将检查这些功能 与 E2F 相关的蛋白质。为了分析Rb的功能，我们有 构建了一个融合基因，其中 Rb 连接到 DNA 结合区 酵母蛋白 Gal-4。 Gal-4 DNA结合域已被广泛应用 将转录活性蛋白与启动子连接作为工具 检查他们的活动。在转染测定中，Rb-Gal-4 抑制 从含有 Gal-4 结合位点的启动子转录，表明 Rb 是转录抑制因子，E2F 仅起到束缚作用 Rb 给启动子。 Rb 中的突变和缺失将被产生并融合到 Gal-4。所得融合蛋白的活性将在 转染测定以鉴定 Rb 的阻遏结构域。相似的 技术将用于识别和表征转录 S 期特异性 E2F 复合体中的激活剂。此外，角色 E2F将在体内进行检查。我们将通过以下方式在功能上停用 E2F 站点 隔离细胞 E2F。这将通过转染 含有 E2F 结合位点的竞争质粒。我们预计 E2F 是 细胞增殖所需的，这些实验应该提供 第一个直接证据表明情况确实如此。拟议的研究应该导致 进一步了解细胞周期控制及其机制 肿瘤发生过程中这种控制的丧失是潜在的。