Visualizing the effect of epigenetic modifications on transcription and mutation

描述（由申请人提供）： 组蛋白修饰和胞嘧啶-5 DNA甲基化是两个重要的表观遗传标记。这两种非常不同的修饰密切合作来控制转录潜力，从而影响多种生物过程。在过去的十年中，人们对这些表观遗传修饰在人类健康和疾病中的作用产生了极大的兴趣。拟议的应用程序的目标是通过开发和应用两种新的体内成像技术，能够可视化这些修改的表观遗传，甚至遗传后果，以促进我们对这两种修改的理解。 第一个具体目标是开发一种体内成像系统，以评估个体表观遗传修饰的因果影响。该技术被设计成既实现在预定基因组基因座处选择性引入所需修饰，又产生表观遗传修饰对转录的影响的定量光学读出。这是通过表观遗传修饰蛋白的序列特异性募集到驱动荧光-发光融合报告基因的启动子来实现的。这种方法的力量来自于定量光学分析和局部募集的结合。这将允许对个体表观遗传修饰的主要因果效应进行高通量评估。 第二个具体目标是开发一种用于CpG转换突变的体内可视化的成像测定。表观遗传DNA甲基化标记处的转换突变导致约三分之一的人类遗传性疾病突变，并导致近50%的人类结直肠癌中发现的所有p53点突变。然而，这种类型的表观遗传学诱导的诱变成像工具的缺乏阻碍了我们的时间和细胞类型的特异性，这种事件在体内的理解。所提出的系统被设计为提供突变事件的结果的直接可视化。这是通过使用突变的绿色荧光蛋白来实现的，所述突变的绿色荧光蛋白在发色团区域中的CpG转换突变时产生荧光信号。将特定的基因突变转化为光学信号提供了一个有吸引力的机会来分析突变，而不采用直接测序。诱变的原位分析将使我们不仅能够确定组织特异性和细胞类型特异性的体内CpG突变频率，而且还可以分析突变动力学。 公共卫生相关性： 在过去的十年中，人们越来越多地认识到表观遗传对发病机制的贡献。本研究旨在开发成像技术，以促进我们对所选表观遗传标记对转录潜力和体内诱变的因果作用的理解，长期目标是利用这些知识来改善我们针对表观遗传修饰的治疗或诊断策略。

DESCRIPTION (provided by applicant): 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 health 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. PUBLIC HEALTH RELEVANCE: Project Narrative During the past decade, there has been an increasing recognition of epigenetic contribution to pathogenesis. This study aims to develop imaging technologies to advance our understanding of the causal role of selected epigenetic marks on transcriptional potential and on mutagenesis in vivo, with the long term goal of using this knowledge to improve our therapeutic or diagnostic strategies targeting epigenetic modifications.