TRANSCRIPTION FACTOR TARGET MAPPING IN MAMMALIAN GENOME

哺乳动物基因组转录因子目标图谱

• 批准号: 7117495
• 负责人: Bing Ren
• 金额: $ 34.97万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-16 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7117495
• 关键词: Burkitt' s lymphoma; DNA; animal genetic material tag; binding sites; carcinogenesis; crosslink; formaldehyde; functional /structural genomics; genetic mapping; genetic promoter element; genetic transcription; genome; immunoprecipitation; intermolecular interaction; microarray technology; neoplasm /cancer genetics; neoplastic cell; nucleic acid structure; protein binding; protein structure function; protooncogene; technology /technique development; tissue /cell culture; tissue /cell preparation; transcription factor

A large number of transcription factors have been impliacted in tumorigenesis, yet little is known about their genomic binding sites under normal and pathological conditions. In order to understand the molecular mechanisms whereby abnormal functions of these factors lead to cancer, we propose to develop a genome wide location analysis (GWLA) technique that allows the rapid identification of direct in vivo targets for transcription factors. This approach involves formaldehyde fixation of cells, immunoprecipitation of crosslinked chromatin DNA fragments, and detection of enriched transcription factor binding sites with DNA microarray technologies. The GWLA technique has several distinct advantages over existing ones: First, the method directly examines the in vivo protein-DNA interactions throughout the genome, and can reveal functions of a transcription factor under both normal and diseased states. Second, this unbiased approach : does not require prior knowledge of a transcription factor's function, therefore can uncover its never biological properties. Third, the method has broad applications and can also be applied to discovery of DNA methylation patterns or mapping of other functional elements in the genome relevant to tumorigenesis. Through extensive preliminary experiments, we have verified the utility and exquisite sensitivity of this method with many transcription factors in both yeast and human cells. In the R21 phase studies, we will develop quantitative measures to assess the robustness and reliability of this method. In addition, we will demonstrate that this method can be used to map transcription factor binding sites in mouse genome, in the R33 phase, we will further develop and fully implement a GWLA system to identify targets for human and mouse transcription factors. Because a vast majority of known transcription factors bind close to gene promoters, our GWLA system will be focused on examination of promoter occupancy by specific : transcription factors in cells. We will first annotate gene promoters in the human or mouse genome, and then build DNA microarrays to represent these regions. We will also establish a standard protocol for target identification, and validate the performance of our system using a number of cancer-related transcription factors. This system should prove to be a powerful tool in mechanistic studies as welt as cancer diagnosis.

在肿瘤发生中，已经暗示了大量的转录因子，但对于正常和病理条件下的基因组结合位点知之甚少。为了了解这些因素异常功能导致癌症的分子机制，我们建议开发一种基因组广泛的位置分析（GWLA）技术，该技术允许快速鉴定转录因子的体内直接靶标。这种方法涉及细胞的甲醛固定，交联染色质DNA片段的免疫沉淀以及使用DNA微阵列技术检测富集转录因子结合位点。 GWLA技术比现有技术具有多个不同的优势：首先，该方法直接研究了整个基因组中的体内蛋白-DNA相互作用，并且可以揭示 在正常状态和患病状态下转录因子的功能。其次，这种公正的方法：不需要先验了解转录因子的功能，因此可以揭示其从未生物学特性。第三，该方法具有广泛的应用，也可以应用于发现与肿瘤发生相关的基因组中其他功能元件的DNA甲基化模式或映射。 通过广泛的初步实验，我们验证了该方法对酵母和人类细胞中许多转录因子的实用性和精致灵敏度。在R21阶段研究中，我们将制定定量措施来评估该方法的鲁棒性和可靠性。此外，我们将证明该方法可用于绘制小鼠基因组中的转录因子结合位点，在R33阶段，我们将进一步开发并完全实施GWLA系统，以识别人类和小鼠转录因子的目标。由于绝大多数已知的转录因子与基因启动子接近结合，因此我们的GWLA系统将集中于通过特定的特定于启动子占用的检查：细胞中的转录因子。我们将首先注释人或小鼠基因组中的基因启动子，然后构建DNA微阵列以代表这些区域。我们还将为目标建立标准协议 使用许多与癌症相关的转录因子识别并验证系统的性能。该系统应被证明是机械研究中的强大工具，例如癌症诊断。