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

 描述（由申请人提供）：本申请的主要目的是获得对Tet-induced 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作用的机制见解。CpG二核苷酸位点胞嘧啶C5位甲基化是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是否可以通过不依赖于DNA复制的过程转化为其未甲基化的对应物（a.k.a.活性胞嘧啶去甲基化）。最近发现的10 - 11易位（泰特）酶家族的功能提供了重要的新的见解活性胞嘧啶去甲基化在哺乳动物。泰特酶可氧化DNA中的5-mdC以产生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，5-FodC和5-CadC可以通过胸腺嘧啶DNA糖基化酶从DNA有效地切割，并且随后通过碱基切除修复机制的作用可以导致5-mdC被未甲基化的dC最终取代。在R21申请中，我们提出了实验来探索Tet-mediated氧化产物在表观遗传调控中的新机制，并且所提出的研究根据以下两个具体目标组织：（1）探索从头DNA胞嘧啶甲基转移酶DNMT 3a和DNMT 3b在5-HmdC、5-FodC和5-CadC向未甲基化dC的直接转化中的细胞作用。我们将评估DNMT 3a和DNMT 3b在人类细胞中5-HmdC，5-FodC和5-CadC向未甲基化dC转化中的功能，以及这一过程如何受到组蛋白表观遗传标记的调节。(2)研究5-HmdC潜在葡萄糖缀合衍生物在人细胞中的发生、生物合成和转录干扰。基于我们新开发的用于定量锥虫DNA中碱基J的方法，我们将检查人类细胞中类似糖基化5-HmdC的形成，鉴定参与这种转化的潜在酶，并评估糖基化5-HmdC对人类细胞中转录的影响。拟议的研究结果将产生重要的新知识，了解Tet-mediated氧化产物的5-mdC在哺乳动物的表观遗传调控的作用。已知暴露于许多环境因子会刺激活性氧物质的产生，这也可能导致5-mdC意外氧化为5-HmdC、5-FodC和5-CadC。因此，拟议的研究也可能为理解环境暴露如何干扰基因调控的表观遗传机制提供新的知识。

 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.