Development of a universal tagging method for genome wide ChIP analyses

开发用于全基因组 ChIP 分析的通用标记方法

• 批准号: 7916878
• 负责人: Peter Christopher Scacheri
• 金额: $ 19.14万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7916878
• 关键词: Achievement; Address; Affinity; Antibodies; Binding; Binding Sites; Biological Process; Blood; Breast; Cardiovascular Diseases; Cell Line; Cloning; Colorectal Cancer; Communities; Coupled; DNA; DNA Microarray Chip; Data; Development; Disease; Elements; Embryo; Enhancers; Epitopes; Frequencies; Gene Expression Regulation; Genes; Goals; Human Genome; Kidney; Knock-in Mouse; Knowledge; Lead; Length; Liver; Lung; Malignant Neoplasms; Measures; Mediating; Methods; Muscle; Mutation; Nerve; Nerve Degeneration; Pancreas; Pathogenesis; Performance; Process; Prostate; Proteins; Recombinant Proteins; Recombinant adeno-associated virus (rAAV); Regulatory Element; Research; STAT3 gene; Screening procedure; Sequence Analysis; Skin; Solutions; Somatic Cell; Specificity; Techniques; Technology; Testing; Tissues; Transcript; Transcriptional Regulation; Transgenic Organisms; Western Blotting; cancer cell; cell type; chromatin immunoprecipitation; design; developmental disease; embryonic stem cell; genome wide association study; genome-wide; homologous recombination; human disease; improved; insight; pluripotency; promoter; public health relevance; research study; stem; success; transcription factor; vector

DESCRIPTION (provided by applicant): The techniques of chromatin immunoprecipitation coupled with DNA microarray (ChIP-chip) and sequencing (ChIP-seq) have gained widespread popularity among the greater scientific community. The data generated from such analyses have begun to provide valuable insight to the mechanisms of gene regulation, disease pathogenesis, embryonic stem (ES) cell pluripotency, and development. However, application of the technology to most transcription factors is hindered by the lack of ChIP-grade antibodies. To overcome this limitation, we have developed a method whereby recombinant adeno-associated virus (rAAV) is used to introduce epitope tag-encoding DNA into endogenous loci by homologous recombination-mediated "knock-in". As proof of principle, we used this strategy to knock-in sequence encoding a triple FLAG epitope (3xFLAG) into the STAT3 and CHD7 loci in colorectal cancer cells. Using ChIP-chip analyses, we show that the FLAG tag facilitates genome wide identification of STAT3 and CHD7 binding sites with a commercially available anti-FLAG antibody. In this 3-year R01 application, we propose 3 aims. In Aim 1, we will test the general applicability of the targeting approach to cell lines derived from different lineages. In Aim 2, we will assess the fidelity of the 3xFLAG tagged transcription factors for genome wide ChIP analyses. In Aim 3, we will develop methods for high-throughput tagging of a large number of transcription factors. The achievement of these aims should facilitate large-scale ChIP analysis of multiple transcription factors. Such studies will profoundly impact our knowledge of transcriptional networks and the biological processes they control. PUBLIC HEALTH RELEVANCE: Regulation of transcription is a highly coordinated process. Mutations in genes which lead to aberrant regulation of transcription can cause a host of human diseases, including developmental disorders, neurodegenerative conditions, cardiovascular disease, and cancer. The technology we propose to develop here is designed to accelerate the identification of functional DNA elements within the human genome. In turn, this should hasten our understanding of transcriptional regulation and the underlying mechanisms of human disease.

描述（由申请人提供）：染色质免疫沉淀技术与DNA微阵列（ChIP-chip）和测序（ChIP-seq）相结合，在更大的科学界获得了广泛的普及。从这些分析中产生的数据已经开始为基因调控、疾病发病机制、胚胎干细胞（ES）多能性和发育机制提供有价值的见解。然而，由于缺乏ChIP级抗体，该技术在大多数转录因子中的应用受到阻碍。为了克服这种限制，我们已经开发了一种方法，其中重组腺相关病毒（rAAV）用于通过同源重组介导的“敲入”将编码表位标签的DNA引入内源基因座。作为原理的证明，我们使用该策略将编码三重FLAG表位（3xFLAG）的序列敲入结肠直肠癌细胞中的STAT 3和CHD 7基因座。使用ChIP芯片分析，我们表明，FLAG标签有利于全基因组识别STAT 3和CHD 7结合位点与市售的抗FLAG抗体。在这个为期3年的R 01申请中，我们提出了3个目标。在目标1中，我们将测试靶向方法对来自不同谱系的细胞系的普遍适用性。在目标2中，我们将评估3xFLAG标记的转录因子用于全基因组ChIP分析的保真度。在目标3中，我们将开发大量转录因子的高通量标记方法。这些目标的实现将有助于对多种转录因子进行大规模的ChIP分析。这些研究将深刻影响我们对转录网络及其控制的生物过程的认识。 公共卫生相关性：转录调控是一个高度协调的过程。导致转录异常调节的基因突变可引起许多人类疾病，包括发育障碍、神经退行性病症、心血管疾病和癌症。我们在这里提出的技术旨在加速人类基因组中功能性DNA元件的鉴定。反过来，这将加速我们对转录调控和人类疾病潜在机制的理解。