Development of a universal tagging method for genome wide ChIP analyses

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）相结合的技术在更大的科学界中得到了广泛的普及。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。为了克服这一限制，我们开发了一种方法，利用重组腺相关病毒（rAAV）通过同源重组介导的“敲入”将表位标签编码的DNA引入内源性位点。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位点。通过ChIP-chip分析，我们发现FLAG标签可以通过市售的抗FLAG抗体促进STAT3和CHD7结合位点的全基因组鉴定。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一般适用性。在目标2中，我们将评估3xFLAG标记的转录因子在全基因组芯片分析中的保真度。在Aim 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。这些研究将深刻地影响我们对转录网络及其控制的生物过程的认识。

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.