Enzymatic Conversions of Tet-mediated Oxidation Products of 5-Methylcytosine

Tet 介导的 5-甲基胞嘧啶氧化产物的酶促转化

• 批准号: 8987741
• 负责人: Yinsheng Wang
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8987741
• 关键词: Affect; Anabolism; Base Excision Repairs; Binding Proteins; Biochemical; Bioinformatics; Cell physiology; Cells; Chromatin; Cleaved cell; CpG dinucleotide; Cytosine; DNA; DNA Modification Methylases; DNA biosynthesis; DNMT3B gene; DNMT3a; Deoxycytidine; Environmental Exposure; Enzymes; Epigenetic Process; Excision; Exposure to; Family; Gene Expression; Gene Expression Regulation; Generations; Genetic Transcription; Genomic DNA; Glucose; Glucosyltransferase; Histones; Human; Knowledge; Laboratories; Leishmania; Light; Mammalian Cell; Mammals; Mastigophora; Mediating; Methods; Methylation; Methyltransferase; Organism; Orthologous Gene; Outcome; PWWP Domain; Positioning Attribute; Process; Protein translocation; Proteins; Published Comment; Reactive Oxygen Species; Recruitment Activity; Research; Role; Site; Testing; Thymine; Thymine DNA Glycosylase; Toxic Environmental Substances; Trypanosoma; Universities; Uracil; Veins; Work; analog; base; carboxyl group; demethylation; environmental agent; epigenetic regulation; genetic manipulation; insight; mammalian genome; novel; nucleobase; oxidation; public health relevance; research study; restoration

 DESCRIPTION (provided by applicant): The major objective of this application is to gain mechanistic insights into the roles of Tet-induced oxidation products of 5-methyl-2'-deoxycytidine (5-mdC) in epigenetic regulation in mammals. Methylation at the C5 position of cytosine at CpG dinucleotide sites constitutes a major mechanism of epigenetic regulation in mammals. It was not clear whether 5-mdC in the mammalian genome can be converted to its unmethylated counterpart through a process that is independent of DNA replication (a.k.a. active cytosine demethylation). Recent discovery of the functions of ten-eleven translocation (Tet) family of enzymes offered important new insights into active cytosine demethylation in mammals. Tet enzymes can oxidize 5-mdC in DNA to yield 5- hydroxymethyl-2'-deoxycytidine (5-HmdC), 5-formyl-2'-deoxycytidine (5-FodC) and 5-carboxyl-2'-deoxycytidine (5-CadC). In addition, 5-FodC and 5-CadC can be efficiently cleaved from DNA by thymine DNA glycosylase, and subsequent action by the base excision repair machinery can result in the ultimate replacement of 5-mdC with an unmethylated dC. In this R21 application, we propose experiments to explore the novel mechanisms of Tet-mediated oxidation products in epigenetic regulation and the proposed research is organized according to the following two specific aims: (1) To exploit the cellular roles of de novo DNA cytosine methyltransferases, DNMT3a and DNMT3b, in the direct conversions of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC. We will assess the functions of DNMT3a and DNMT3b in the transformations of 5-HmdC, 5-FodC and 5-CadC to unmethylated dC in human cells and how this process is modulated by histone epigenetic marks. (2) To examine the occurrence, biosynthesis and transcriptional perturbation of potential glucose-conjugated derivatives of 5-HmdC in human cells. Based on our newly developed method for the quantification of base J in trypanosome DNA, we will examine the formation of the analogous glycosylated 5-HmdC in human cells, identify the potential enzyme(s) involved in this conversion, and assess the impact of the glycosylated 5-HmdC on transcription in human cells. The outcome of the proposed research will yield important new knowledge for understanding the role of Tet-mediated oxidation products of 5-mdC in epigenetic regulation in mammals. Exposure to many environmental agents is known to stimulate the generation of reactive oxygen species, which could also result in the inadvertent oxidation of 5-mdC to 5-HmdC, 5-FodC and 5-CadC. Thus, the proposed research may also provide new knowledge for understanding how environmental exposure perturbs epigenetic mechanisms of gene regulation.

 描述（由申请人提供）：本申请的主要目的是深入了解 Tet 诱导的 5-甲基-2'-脱氧胞苷 (5-mdC) 氧化产物在哺乳动物表观遗传调控中的作用。 CpG二核苷酸位点的胞嘧啶C5位置构成哺乳动物表观遗传调控的主要机制目前尚不清楚哺乳动物基因组中的5-mdC是否可以被调节。通过独立于 DNA 复制的过程（又称活性胞嘧啶去甲基化）转化为未甲基化的对应物。最近发现的 10-11 易位 (Tet) 家族酶的功能为哺乳动物中活性胞嘧啶 Tet 酶的功能提供了重要的新见解。可以氧化DNA中的5-mdC产生5-羟甲基-2'-脱氧胞苷（5-HmdC）， 5-甲酰基-2'-脱氧胞苷 (5-FodC) 和 5-羧基-2'-脱氧胞苷 (5-CadC) 此外，胸腺嘧啶 DNA 糖基化酶可以有效地将 5-FodC 和 5-CadC 从 DNA 上切割下来。在该 R21 中，碱基切除修复机制的后续作用可导致最终用未甲基化的 dC 取代 5-mdC。应用中，我们提出实验来探索 Tet 介导的氧化产物在表观遗传调控中的新机制，并且拟议的研究是根据以下两个具体目标组织的：（1）利用 de novo DNA 胞嘧啶甲基转移酶、DNMT3a 和 DNMT3a 的细胞作用。 DNMT3b，在 5-HmdC、5-FodC 和 5-CadC 直接转化为未甲基化 dC 的过程中，我们将评估 DNMT3b 的功能。 DNMT3a 和 DNMT3b 在人类细胞中 5-HmdC、5-FodC 和 5-CadC 向非甲基化 dC 的转化中以及组蛋白表观遗传标记如何调节该过程 (2) 检查潜在葡萄糖的发生、生物合成和转录扰动。 -人体细胞中 5-HmdC 的共轭衍生物基于我们新开发的碱基 J 定量方法。锥虫DNA，我们将检查人类细胞中类似糖基化5-HmdC的形成，识别参与这种转化的潜在酶，并评估糖基化5-HmdC对人类细胞转录的影响。这项研究将为理解 Tet 介导的 5-mdC 氧化产物在哺乳动物表观遗传调控中的作用提供重要的新知识。已知暴露于许多环境因子会刺激繁殖。活性氧物种，这也可能导致 5-mdC 无意中氧化为 5-HmdC、5-FodC 和 5-CadC 因此，拟议的研究也可能为理解环境如何扰乱基因的表观遗传暴露机制提供新的知识。规定。