Biochemical regulation and genomic targeting of TET-catalyzed cytosine oxidation

 描述(由申请者提供)：此奖学金申请寻求对某一医学博士/博士研究生的论文研究的支持，并提出一项培训计划，使她有能力成为一家主要研究机构的独立内科科学家。该研究项目旨在阐明胞嘧啶氧化的生化调节，这是一种新发现的表观遗传现象，参与了不同的过程，包括胚胎发育、多能性和恶性肿瘤。在DNA的四个碱基中，胞嘧啶为影响基因表达的化学修饰提供了主要底物。胞嘧啶甲基化形成5-甲基胞嘧啶(5mC)是细胞分化、印迹和基因组稳定性中基因沉默的重要中介。然而，最近发现了10-11种易位(Tet)酶将5mC氧化成另外三个碱基-5-羟甲基胞嘧啶(5hmC)、5-甲酰胞嘧啶(5fC)和5-羧基胞嘧啶(5caC)，这些碱基在哺乳动物基因组中保持稳定存在，表明它们可能具有不同的表观遗传学功能。到目前为止发现的最重要的功能是DNA去甲基化，因为被氧化的碱基提供了再生未修饰胞嘧啶的途径。基因组中各种胞嘧啶修饰的存在似乎是一种复杂的平衡行为，当受到干扰时，会导致许多癌症，DNA甲基化的异常基因组模式就是明证，越来越多的人将其作为癌症标志进行研究。关于Tet酶如何调节这些氧化的胞嘧啶的平衡和基因组分布的主要问题仍然存在。这项提议的目标是结合使用生化方法和创新的基因组测序技术来阐明这些前沿。目标1建议破译Tet的底物偏好如何帮助控制氧化胞嘧啶的逐步合成。Tet酶优先作用于CpG二核苷酸，有三种可能的底物(5mC、5hmC和5fC)，可以存在于一条或两条互补的DNA链上。基于高效液相色谱和质谱学的分析将被用来量化三种Tet亚型对其所有可能底物的反应性。这将标志着对Tet动力学性质的首次系统研究，将Tet分解为其每种底物和异构体的单独氧化步骤，这将阐明胞嘧啶氧化的生化调节的关键要素。目的2提出一种在单核苷酸分辨率下定位5fC和5caC的新方法。特定的碱基切除酶胸腺嘧啶DNA糖基酶(TDG)将被利用来从基因组中切割5fC和5caC，从而产生将通过下一代测序绘制出的基本位置。这将是对目前方法的重大改进，并提供第一个碱基分辨率的、全基因组的5caC图谱。该方法将应用于原始生殖细胞及其恶性对应的精原细胞瘤细胞，与Aim 1一起将为Tet酶和氧化胞嘧啶在生理和病理环境中潜在的表观遗传功能提供重要的见解。

 DESCRIPTION (provided by applicant): This fellowship application seeks support for the dissertation research of an individual MD/PhD student and proposes a training plan that will equip her to become an independent physician-scientist at a major research institution. The research project aims to elucidate the biochemical regulation of cytosine oxidation, a newly- discovered epigenetic phenomenon that factors into diverse processes, including embryonic development, pluripotency, and malignancy. Among the four bases of DNA, cytosine provides the major substrate for chemical modifications that impact gene expression. The methylation of cytosine to form 5-methylcytosine (5mC) is well established as a critical mediator of gene silencing in cellular differentiation, imprinting, and genomic stability. Recently, however, ten-eleven translocation (TET) enzymes were found to oxidize 5mC into three additional bases-5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)-that maintain a stable presence in mammalian genomes, suggesting they could have distinct epigenetic functions. The most important function discovered thus far is DNA demethylation, since the oxidized bases provide pathways to regenerate unmodified cytosine. The presence of the various cytosine modifications in the genome appears to be an intricate balancing act, which when perturbed contributes to numerous cancers, as evidenced by abnormal genomic patterns of DNA methylation that are increasingly studied as cancer signatures. Major questions remain regarding how TET enzymes regulate the balance and genomic distribution of these oxidized cytosines. The goal of this proposal is to shed light on these frontiers using a combination of biochemical approaches and innovative genome sequencing technologies. Aim 1 proposes to decipher how TET's substrate preferences help to control stepwise synthesis of the oxidized cytosines. TET enzymes preferentially act on CpG dinucleotides and have three possible substrates (5mC, 5hmC, and 5fC), which can be present on one or both complementary DNA strands. HPLC- and mass spectrometry-based assays will be used to quantify the reactivity of the three TET isoforms to all of their possible substrates. This would mark the first systematic study of TET's kinetic properties, dissected into individual oxidation steps for each of its substrates and isoforms, which would elucidate key elements of the biochemical regulation of cytosine oxidation. Aim 2 proposes a novel method of localizing 5fC and 5caC at single nucleotide resolution in the genome. The specific base excision enzyme thymine DNA glycosylase (TDG) will be exploited to cleave 5fC and 5caC from the genome, resulting in abasic sites that will be mapped by next-generation sequencing. This would represent a significant improvement over current methods and provide the first base-resolution, genome-wide map of 5caC. This method will be applied to primordial germ cells and their malignant counterpart, seminoma cells, which together with Aim 1 would provide important insights into the potential epigenetic functions of TET enzymes and oxidized cytosines in physiological and pathological settings.