A Genetic System Able to Screen for Active DNA Demethylation

能够筛选活性 DNA 去甲基化的遗传系统

• 批准号: 7107041
• 负责人: SHUANG-YONG XU
• 金额: $ 10.4万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-25 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7107041
• 关键词: DNA methylation; animal genetic material tag; bioinformatics; enzyme mechanism; epigenetics; genetic library; genetic screening; human genetic material tag; plasmids; technology /technique development; transfection /expression vector

DESCRIPTION (provided by applicant): With the increasing interest in epigenetic modification of DNA there is a need for research reagents to help study the phenomenon. Of especial interest would be an enzyme able to demethylate DNA that might provide a direct way to measure the extent of methylation in eukaryotic DNA. Such an enzyme is expected to be present in newly fertilized oocytes as an extensive active demethylation of embryonic DNA takes place immediately following fertilization and in the absence of DNA replication. However, so far attempts to isolate and characterize such an enzyme have failed. The literature report of an enzyme able to catalyze DNA demethylation has not been successfully reproduced. Several mechanisms can be conceived for DNA demethylation such as direct removal by a demethylase or a multistep process begun by a 5-methylcytosine DNA glycosylase. Initial studies will focus on searching for an enzyme able to directly demethylate DNA. In Phase II an in vivo system, devised during Phase I, will be used to screen or select for active demethylation. The selective system will use a methylation-sensitive restriction enzyme to kill cells unable to demethylate DNA. The screen will use a Type II restriction enzyme to induce an SOS response, visible by a blue phenotype, when demethylation takes place. Additionally, an in vitro transcription-translation system will be used that could detect active demethylation. Finally, a bioinformatics approach will be used to find candidate genes for the demethylase or a component of it in the human and mouse genomes. In Phase II these selection systems will be applied to eukaryotic cDNA libraries that are candidates to contain demethylases. The proposed research will have an impact on DNA modification, epigenetic memory and cancer biology. In addition, knowledge of the mechanism of demethylation could be crucial in converting fully differentiated adult stem cells into totipotent stem cells.

描述(申请人提供)：随着人们对DNA表观遗传修饰的兴趣与日俱增，需要研究试剂来帮助研究这一现象。特别令人感兴趣的是一种能够使DNA去甲基化的酶，它可能提供一种直接测量真核DNA甲基化程度的方法。这种酶预计会出现在新受精的卵母细胞中，因为胚胎DNA的广泛主动去甲基化在受精后立即发生，并且在没有DNA复制的情况下发生。然而，到目前为止，分离和鉴定这种酶的尝试都失败了。关于一种能够催化DNA去甲基化的酶的文献报道尚未成功重现。DNA去甲基化可以设想几种机制，例如通过去甲基酶直接去除或由5-甲基胞嘧啶DNA糖基酶开始的多步骤过程。最初的研究将集中在寻找一种能够直接使DNA脱甲基化的酶。在第二阶段，在第一阶段设计的体内系统将用于筛选或选择活性去甲基化。这个选择性系统将使用甲基化敏感的限制酶来杀死无法使DNA去甲基化的细胞。当去甲基化发生时，屏幕将使用II型限制酶来诱导SOS反应，通过蓝色表型可见。此外，还将使用体外转录-翻译系统来检测活性去甲基化。最后，将使用生物信息学方法在人类和小鼠基因组中寻找去甲基酶或其组成部分的候选基因。在第二阶段，这些选择系统将被应用于包含去甲基酶的候选真核基因文库。这项拟议的研究将对DNA修饰、表观遗传记忆和癌症生物学产生影响。此外，了解去甲基化的机制对于将完全分化的成人干细胞转化为全能干细胞至关重要。