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.

许多转录因子与肿瘤发生有关，但对其在正常和病理条件下的基因组结合位点知之甚少。为了了解这些因子的异常功能导致癌症的分子机制，我们建议开发一种全基因组定位分析（GWLA）技术，该技术可以快速识别转录因子的直接体内靶点。该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利用 DNA 微阵列技术检测富集的转录因子结合位点。与现有技术相比，GWLA 技术具有几个明显的优势：首先，该方法直接检查整个基因组中的体内蛋白质-DNA 相互作用，并可以揭示 转录因子在正常和患病状态下的功能。其次，这种公正的方法：不需要事先了解转录因子的功能，因此可以揭示其从未有过的生物学特性。第三，该方法具有广泛的应用，还可用于发现DNA甲基化模式或绘制基因组中与肿瘤发生相关的其他功能元件。 通过大量的初步实验，我们已经在酵母和人类细胞中验证了该方法对许多转录因子的实用性和灵敏性。在 R21 阶段研究中，我们将制定定量措施来评估该方法的稳健性和可靠性。此外，我们将证明该方法可用于绘制小鼠基因组中转录因子结合位点的图谱，在R33阶段，我们将进一步开发并全面实施GWLA系统来识别人类和小鼠转录因子的靶点。由于绝大多数已知的转录因子与基因启动子紧密结合，因此我们的 GWLA 系统将专注于检查细胞中特定转录因子的启动子占用情况。我们将首先注释人类或小鼠基因组中的基因启动子，然后构建 DNA 微阵列来代表这些区域。我们还将为目标建立一个标准协议 识别，并使用许多与癌症相关的转录因子验证我们系统的性能。该系统应该被证明是机械研究和癌症诊断的强大工具。