Approaches for genomic mapping of 5-hydroxymethylcytosine a novel epigenetic mar

描述(由申请人提供)：在过去的十年里，表观遗传现象在细胞调控过程的研究中占据了中心地位。此外，越来越多的证据表明，表观遗传因素可能在各种人类疾病中发挥重要作用。最被理解的表观遗传机制之一是DNA甲基化。在哺乳动物基因组中，胞嘧啶(C)以两种功能状态存在：未甲基化或嘧啶环5位甲基化(5mC)。胞嘧啶和鸟嘌呤(CpG二核苷酸)是甲基化的首选靶标。最近对人脑、神经元和小鼠胚胎干细胞基因组DNA的研究发现，CG序列也含有5-羟甲基胞嘧啶(HMC)。由于它们与细胞蛋白质的相互作用是不同的，DNA中的5-羟甲基不仅模拟5-甲基，而且可能在未知的各种生物过程的表观遗传调控中发挥独立的作用，如胚胎发育、脑功能和癌症进展。然而，由于缺乏适当的分析技术，对这些耐人寻味的现象的进一步研究受到阻碍。在过去的几年里，已经开发了无数的方法来研究大的DNA区域--染色体甚至整个基因组的DNA甲基化情况。所有这些方法都是二元的--即，设计只区分胞嘧啶的两种表观遗传状态：甲基化和未修饰。该项目致力于开发能够在哺乳动物基因组中明确绘制HMC图谱的新技术。我们的策略是基于利用已知和新发现的HMC中羟甲基的酶转化来实现对含有HMC的基因组片段的选择性检测和捕获。将这些新方法与DNA微阵列分析相结合，我们将对人脑中的HMC进行表观基因组范围的图谱绘制。最后，我们将在选定的位点上进行HMC的核苷酸分辨图谱，以揭示DNA中这种新的胞嘧啶修饰的个体内和个体间的变异。 公共卫生相关性：DNA甲基化模式的变化可能使个人易患各种人类疾病，包括癌症、精神分裂症、自闭症、哮喘和糖尿病。由于缺乏适当的实验室技术，人类疾病中表观遗传失调的研究受到阻碍。我们用于分析第六碱基的新技术，即5-羟甲基胞嘧啶，将是全面基因组研究的“工具箱”中一个非常有价值的补充。

DESCRIPTION (provided by applicant): Over the last decade, epigenetic phenomena have claimed a central role in the research of cell regulatory processes. In addition, there is increasing evidence that epigenetic factors may play a significant role in various human diseases. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) are known to exist in two functional states: unmethylated or methylated at the 5-position of the pyrimidine ring (5mC). Cytosines followed by guanine (CpG dinucleotides) are the preferred targets for methylation. Recent studies of genomic DNA from the human brain, neurons and from mouse embryonic stem cells detected evidence that CG sequences also contain 5- hydroxymethylcytosine (hmC). As their interactions with cellular proteins are distinct, the 5- hydroxymethyl groups in DNA do not merely mimic 5-methyl groups, but likely play an independent role in yet unknown epigenetic regulation of various biological processes, such as embryonic development, brain function, and cancer progression. However, further studies of these intriguing phenomena are hampered by the lack of adequate analytical techniques. A myriad of methods have been developed over the past several years to investigate DNA methylation profiles across large DNA regions - chromosomes and even entire genomes. All such methods are binary - i.e., designed to distinguish only the two epigenetic states of cytosine: methylated versus unmodified. This project is dedicated to the development of new technologies that are capable of unequivocal mapping of hmC in mammalian genomes. Our strategy is based on utilization of known and newly discovered enzymatic transformations of the hydroxylmethyl group in hmC to achieve selective detection and capture of hmC-containing genomic fragments. Combining these novel approaches with DNA microarray analyses, we will perform epigenome-wide mapping of hmC in the human brain. Finally, we will perform nucleotide-resolution mapping of hmC at selected loci to unveil the intra- and inter- individual variation of this novel cytosine modification in DNA. PUBLIC HEALTH RELEVANCE: Changes in DNA methylation patterns may predispose individuals to various human diseases, including cancer, schizophrenia, autism, asthma, and diabetes. Research into the epigenetic misregulation in human disease is hampered by the lack of adequate laboratory techniques. Our new technologies for the analysis of the 6th base, namely 5-hydroxymethylcytosine, will be an incredibly valuable addition to the "tool box" of comprehensive genomic studies.