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

 描述(申请人提供)：本申请的主要目的是了解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是否可以通过一种独立于DNA复制的过程转化为其未甲基化的对应物(又名。活性胞嘧啶去甲基化)。最近发现的10-11易位(Tet)家族酶的功能为哺乳动物主动胞嘧啶去甲基化提供了重要的新见解。Tet酶能氧化DNA中的5-MDC生成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可以被胸腺嘧啶DNA糖基酶有效地从DNA上切割，随后的碱基切除修复机制的作用可以导致5-MDC最终被未甲基化的DC取代。在R21的应用中，我们建议进行实验，以探索Tet介导的氧化产物在表观遗传调控中的新机制，并根据以下两个特定目标组织拟议的研究：(1)探索从头DNA胞嘧啶甲基转移酶DNMT3a和Dnmt3b在5-HmdC、5-FodC和5-CADC直接转化为未甲基化DC过程中的细胞作用。我们将评估DNMT3a和Dnmt3b在5-HmdC、5-FodC和5-CADC转化为未甲基化DC过程中的功能，以及组蛋白表观遗传标记如何调控这一过程。(2)检测潜在的5-HmdC葡萄糖偶联衍生物在人细胞中的存在、生物合成和转录扰动。在我们新开发的锥虫DNA碱基J定量方法的基础上，我们将研究类似的糖基化5-HMDC在人类细胞中的形成，确定参与这一转换的潜在酶(S)，并评估糖基化的5-HMDC对人类细胞转录的影响。这项研究的结果将为理解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。众所周知，暴露在许多环境介质中会刺激活性氧物种的产生，这也可能导致5-MDC不经意间氧化成5-HMDC、5-FodC和5-CADC。因此，这项拟议的研究也可能为理解环境暴露如何扰乱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.