Development of a universal tagging method for genome wide ChIP analyses

描述(申请人提供)：染色质免疫沉淀与DNA微阵列(芯片-芯片)和测序(芯片-序列)相结合的技术在更大的科学界中得到了广泛的欢迎。这些分析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，由于缺乏芯片级抗体，该技术在大多数转录因子上的应用受到阻碍。为了克服这一局限性，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导入内源基因座。作为原理的证明，我们使用这一策略将编码三重标志表位(3xFLAG)的序列插入到结直肠癌细胞的STAT3和CHD7基因座中。利用芯片分析，我们发现FLAG标签有助于用商业上可用的抗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标记的转录因子用于全基因组芯片分析的保真度。在目标3中，我们将开发高通量标记大量转录因子的方法。这些目标的实现应有助于对多种转录因子进行大规模芯片分析。这样的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知识。 与公共卫生相关：转录调控是一个高度协调的过程。导致转录异常调控的基因突变可导致一系列人类疾病，包括发育障碍、神经退行性疾病、心血管疾病和癌症。我们在这里建议开发的技术旨在加速识别人类基因组中的功能DNA元件。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。

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.