Visualizing the effect of epigenetic modifications on transcription and mutation

项目总结/摘要 组蛋白修饰和胞嘧啶-5 DNA甲基化代表了两个重要的 表观遗传标记这两个非常不同的修改工作密切合作，以控制 转录潜力，从而影响生物过程的广泛多样性。过去 近十年来，人们对这些表观遗传修饰的作用产生了极大的兴趣， 人类健康和疾病。拟议应用程序的目标是推进我们的 理解这两种修饰，通过开发和应用两种新的体内 成像技术能够可视化的表观遗传，甚至遗传的后果， 这些修改。 第一个具体目标是开发一种体内成像系统，以评估因果关系。 个体表观遗传修饰的影响。这项技术旨在实现 在预定基因组基因座处选择性引入所需修饰，以及 产生表观遗传修饰对细胞的影响的定量光学读数。 转录。这是通过表观遗传修饰剂的序列特异性募集来实现的。 将蛋白质与驱动荧光-发光融合报告基因的启动子连接。的威力 方法来自于定量光学分析和局部光学分析的结合。 招聘这将允许高通量评估的主要因果关系的影响， 个体表观遗传修饰。 第二个具体目标是开发一种用于体内可视化的成像测定， CpG转换突变。表观遗传DNA甲基化标记处的转换突变是 负责大约三分之一的人类遗传性疾病突变， 在人类结直肠癌中发现的所有p53点突变的近50%。但缺乏 这种类型的表观遗传诱导突变的成像工具阻碍了我们的研究。 了解体内该事件的时间和细胞类型特异性。所提出的系统 设计用于提供突变事件结果的直接可视化。实现这一点 通过使用突变的绿色荧光蛋白， 发色团区域的CpG转换突变。一种特定基因的转换 突变为光学信号提供了分析突变的有吸引力的机会 而不使用直接测序。诱变的原位分析将使我们能够不 不仅确定组织特异性和细胞类型特异性的体内CpG突变频率，而且 来分析突变动力学

Project Summary/Abstract Histone modification and cytosine-5 DNA methylation represent two important epigenetic marks. These two very distinct modifications work in close cooperation to control transcriptional potential, thereby influencing a wide diversity of biological processes. The past decade has seen an explosion of interest in the role of these epigenetic modifications in human heath and disease. The goal of the proposed application is to advance our understanding of these two modifications, by developing and applying two novel in vivo imaging technologies capable of visualizing the epigenetic and even genetic consequences of these modifications. The first specific aim is to develop an in vivo imaging system to assess the causal influence of individual epigenetic modifications. The technology is designed to both achieve selective introduction of a desired modification at a predetermined genomic locus, and to produce a quantitative optical readout of the effect of the epigenetic modification on transcription. This is achieved through the sequence-specific recruitment of epigenetic modifier proteins to a promoter driving a fluorescence-luminescence fusion reporter. The power of this approach comes from the combination of the quantitative optical analysis and the localized recruitment. This will allow high-throughput evaluation of the primary causal effects of individual epigenetic modifications. The second specific aim is to develop an imaging assay for the in vivo visualization of CpG transition mutations. Transition mutations at the epigenetic DNA methylation mark are responsible for approximately one-third of all human hereditary disease mutations and for nearly 50% of all p53 point mutations found in human colorectal cancer. However, the lack of imaging tools for this type of epigenetically induced mutagenesis has held back our understanding of the timing and cell-type specificity of this event in vivo. The proposed system is designed to provide direct visualization of the result of the mutation event. This is achieved through the use of a mutant green fluorescent protein that produces a fluorescent signal upon CpG transition mutation in the chromophore region. The conversion of a specific genetic mutation into an optical signal provides an attractive opportunity to analyze the mutation without employing direct sequencing. The in situ analysis of mutagenesis would allow us to not only determine tissue-specific and cell-type specific in vivo CpG mutation frequencies, but also to analyze mutation kinetics.