TRANSCRIPTION FACTOR TARGET MAPPING IN MAMMALIAN GENOME

大量的转录因子参与了肿瘤的发生，但对它们在正常和病理条件下的基因组结合位点却知之甚少。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，该技术允许快速识别转录因子的直接体内靶点。这种方法包括甲醛固定的细胞，免疫沉淀的交联染色质DNA片段，和检测富集的转录因子结合位点的DNA微阵列技术。与现有技术相比，GWLA技术具有几个明显的优点：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，并且可以揭示 转录因子在正常和疾病状态下的功能。第二，这种无偏的方法：不需要事先知道转录因子的功能，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，并且还可以应用于发现DNA甲基化模式或定位基因组中与肿瘤发生相关的其他功能元件。 通过广泛的初步实验，我们已经验证了这种方法的实用性和灵敏度与许多转录因子在酵母和人类细胞。在R21阶段研究中，我们将开发定量指标来评估该方法的耐用性和可靠性。此外，我们将证明该方法可用于定位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全面实施GWLA系统，以识别人和小鼠转录因子的靶标。由于绝大多数已知的转录因子与基因启动子紧密结合，我们的GWLA系统将集中于检查细胞中特定转录因子的启动子占用。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。我们还将建立一个标准的协议， 鉴定，并使用许多癌症相关的转录因子验证我们的系统的性能。该系统应被证明是一个强大的工具，在机制的研究以及癌症诊断。

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.