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

基本信息

批准号：
7506798
负责人：
Peter Christopher Scacheri
金额：
$ 39.25万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7506798
关键词：
Achievement Address Affinity Antibodies Binding Binding Sites Biological Process Blood Breast Cardiovascular Diseases Cell Line Cloning Colorectal Cancer Communities Condition Coupled DNA DNA Microarray Chip DNA Microarray format Data Development Disease Elements Embryo Enhancers Epitopes Frequencies Gene Expression Regulation Genes Genome Goals Human Genome Kidney Knock-in Mouse Knowledge Lead Length Liver Lung Malignant Neoplasms Measures Mediating Methods Muscle Mutation Nerve Nerve Degeneration Numbers Pancreas Pathogenesis Performance Process Prostate Proteins Public Health Range 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 homologous recombination human disease improved insight pluripotency promoter research study stem success transcription factor vector

项目摘要

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）：染色质免疫沉淀与 DNA 微阵列（ChIP-chip）和测序（ChIP-seq）相结合的技术已在更大的科学界广泛流行。此类分析产生的数据已开始为基因调控、疾病发病机制、胚胎干（ES）细胞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上的应用受到阻碍。为了克服这一限制，我们开发了一种方法，通过同源重组介导的“敲入”，使用重组腺相关病毒（rAAV）将编码表位标签的DNA引入内源基因座。作为原理证明，我们使用这种策略将编码三重 FLAG 表位 (3xFLAG) 的序列敲入结直肠癌细胞的 STAT3 和 CHD7 基因座中。使用 ChIP 芯片分析，我们表明 FLAG 标签有助于使用市售抗 FLAG 抗体在全基因组范围内识别 STAT3 和 CHD7 结合位点。在这个为期3年的R01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。这些目标的实现应有助于对多种转录因子进行大规模 ChIP 分析。此类研究将深刻影响我们对转录网络及其控制的生物过程的了解。公共卫生相关性：转录调控是一个高度协调的过程。导致转录调控异常的基因突变可能导致许多人类疾病，包括发育障碍、神经退行性疾病、心血管疾病和癌症。我们提议在此开发的技术旨在加速人类基因组内功能 DNA 元件的识别。反过来，这应该会加速我们对转录调控和人类疾病潜在机制的理解。