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.

最近与细胞启动子相关的几种蛋白质已成为 发现在控制细胞增殖中具有至关重要的作用 和肿瘤转化。其中之一的重要性， 视网膜母细胞瘤蛋白（RB）被发现为 在每个正常组织中表达，但在肿瘤中突变或删除 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重新引入这些肿瘤时，它们 因此，失去其恶性特征，RB被称为 肿瘤抑制剂。我们已经证明RB是转录 G-O/G-1期间的阻遏物，这可能会限制细胞分裂 因为它压抑了一组基因的表达 DNA合成。为了支持RB的这种角色，我们发现 Rb（ - ）细胞DNA合成持续存在于G-O/G-1截止性细胞中。这是 然后可以理解RB功能的损失如何导致损失 细胞周期控制。在静态细胞（G-O/G-1）中，RB被束缚在 E2F的基因启动子，一种与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，并且 它被由107 kDa蛋白（PLO7）组成的复合物替换为E2F。 Cyclin A和CDK2激酶。我们已经证明了这个S相络合物 是一种转录激活剂，表明它介导了S期 DNA合成酶的基因的特异性激活。这些结果 建议E2F位点在转录抑制剂和 在细胞周期中激活剂，并且该活动负责 防止G-0/G-1中DNA合成酶的基因表达 在S期激活这些基因。我们将检查这些功能 与E2F关联的蛋白质。为了分析RB的功能，我们有 构建了一个融合基因，其中RB与DNA结合区域相关 酵母蛋白Gal-4。 GAL-4 DNA结合结构域已被广泛使用 将转录的活性蛋白作为启动子作为链接的蛋白质作为工具 检查他们的活动。在转染测定中，RB-GAL-4抑制 来自包含GAL-4结合位点的启动子的转录，表明 该RB是转录阻遏物，E2F仅用于系绳 RB向发起人。 RB中的突变和删除将被制成并融合到 GAL-4。将分析所得融合蛋白的活性 转染测定法以识别RB的阻遏域。相似的 技术将用于识别和表征转录 S相特异性E2F复合物中的激活剂。另外， E2F将在体内检查。我们将通过功能使E2F站点灭活 隔离细胞E2F。这将通过转染 含有E2F结合位点的竞争者质粒。我们预计E2F是 细胞增殖需要，这些实验应提供 第一个直接证据表明是这样。拟议的研究应导致 进一步了解细胞周期控制和机制 在肿瘤发生中发生这种控制的损失的基础。