Discovery of Binding Sites for Transcription Factors

转录因子结合位点的发现

• 批准号: 6952733
• 负责人: ROLAND D GREEN
• 金额: $ 43.18万
• 依托单位: NIMBLEGEN SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6952733
• 关键词: binding sites; biotechnology; clinical research; genes; human genetic material tag; human tissue; immunoprecipitation; microarray technology; technology /technique development; transcription factor

DESCRIPTION (provided by applicant): The overall goals of this proposal are to determine if an oligonucleotide-based microarray can be used for the discovery of in vivo genomic transcription factor binding sites and, if so, to use the arrays to identify all genomic binding sites for specific human transcription factors. In brief, our experiments are based on using chromatin immunoprecipitation to selectively enrich for all the binding sites in the human genome of a particular transcription factor. After immunoprecipitation, the fragments will be labeled and used to probe a genomic microarray (i.e. a ChiP-chip assay). Positive signals will identify genomic regions that contain the binding sites. Sequence comparisons of the identified genomic regions will allow the development of a consensus binding site. Also, our studies can provide information as to which binding sites for different factors are commonly clustered on a genome-wide basis. Our proposal will be divided into three phases; proof of concept, development of a first exon identification method, and discovery of functional elements. The size of the human genome essentially precludes the use of spotted PCR fragments for the development of comprehensive promoter-specific human microarrays. Clearly, the development of high density oligonucleotide arrays are essential if one wishes to perform a comprehensive identification of in vivo binding sites for specific human transcription factors. Therefore, our first Aim focuses on determining that oligonucleotide arrays created using the NimbleGen Maskless Array Synthesis technology can be used in ChiP-chip assays. Our second Aim is focused on the development of a first exon identification method using PromotedExon Discovery Arrays that span the entire ENCODE-selected sequence. The identification of all the first exons utilized in a particular cell type will greatly aid in the interpretation of the ChiP-chip data obtained in Aim 3. Finally, in Aim 3 we propose to use the Promoter-Specific Arrays to globally identify binding sites for numerous human transcription factors. We will then use this information to identify functional sequence elements and common promoter architectures.

描述（由申请人提供）：本提案的总体目标是确定基于多核苷酸的微阵列是否可用于发现体内基因组转录因子结合位点，如果可以，则使用该阵列鉴定特定人转录因子的所有基因组结合位点。简而言之，我们的实验是基于使用染色质免疫沉淀来选择性地富集人类基因组中特定转录因子的所有结合位点。 免疫沉淀后，标记片段并用于探测基因组微阵列（即，芯片芯片测定）。 阳性信号将识别含有结合位点的基因组区域。鉴定的基因组区域的序列比较将允许开发共有结合位点。 此外，我们的研究可以提供信息，不同因素的结合位点通常聚集在全基因组的基础上。 我们的建议将分为三个阶段：概念证明，开发第一个外显子鉴定方法，并发现功能元件。 人类基因组的大小基本上排除了使用点样PCR片段来开发全面的启动子特异性人类微阵列。 显然，如果希望对特异性人转录因子的体内结合位点进行全面鉴定，高密度寡核苷酸阵列的开发是必不可少的。 因此，我们的第一个目标集中在确定使用NimbleGen无掩模阵列合成技术创建的寡核苷酸阵列可用于Chip芯片测定。我们的第二个目标是专注于开发第一个外显子鉴定方法，使用PromotedExon Discovery Arrays，跨越整个ENCODE选择的序列。 在特定细胞类型中使用的所有第一外显子的鉴定将极大地帮助解释在目标3中获得的Chip芯片数据。 最后，在目标3中，我们建议使用启动子特异性阵列来全局识别许多人类转录因子的结合位点。 然后，我们将使用这些信息来识别功能序列元件和共同的启动子结构。