Role of de novo DNMTs in Toxicant Induced Alterations in DNA Methylation

 描述（由申请人提供）：环境化学品的发育暴露是一个重大的公共健康问题。仅在美国，六分之一的儿童有某种形式的发育障碍。其中许多病例是由于发育过程中暴露于有毒物质所致。也有强有力的迹象表明，其中一些接触会增加患糖尿病、高血压和心血管疾病等成人发病疾病的风险。最近的研究表明，表观遗传机制，如DNA甲基化在发育暴露于有毒物质的持续影响。DNA甲基化在发育中起着至关重要的作用，它涉及通过DNA甲基转移酶（DNMT）酶将甲基基团添加到胞嘧啶核苷酸来对DNA进行化学修饰。DNMT 3组酶负责建立从头甲基化模式，但尚不清楚它们如何靶向特定的DNA序列进行甲基化，以及毒物如何改变甲基化模式。在这项R 01资助申请中，研究人员建议调查DNMT 3在正常发育中的作用，以及发育暴露于有毒物质的长期影响。使用斑马鱼，一个完善的发育和毒理学模型系统，研究人员建议表征DNMT 3功能，并确定它们在对环境毒物的反应中的作用。在目标1中，研究人员将测试DNMT 3显示DNA靶特异性的假设，每个DNMT 3靶向一组独特的基因，从而建立DNA甲基化模式。他们将通过使用附加型甲基化测定和表征重组DNMT 3同种型的序列特异性在体外确定DNMT 3靶特异性，并使用DNMT 3敲除斑马鱼在体内确定DNMT 3靶特异性。在目标2中，研究人员将测试毒物改变DNA甲基化模式的假设，并且这些改变是DNMT 3依赖的。他们将野生型和DNMT 3敲除斑马鱼胚胎在发育的敏感窗口期间暴露于环境相关的有毒物质，并使用还原亚硫酸氢盐测序（RRBS）确定对DNA甲基化模式的影响，RRBS是一种全基因组方法，用于测量基因组中富含CpG的区域的DNA甲基化。在目标3中，研究人员将检验这一假设，即暴露于有毒物质的胚胎中差异甲基化DNA区域显示DNMT 3的占用增加。他们将斑马鱼胚胎暴露于诱导DNA甲基化变化的有毒物质中，并使用染色质免疫沉淀（ChIP）然后进行亚硫酸氢盐测序（ChIP-BS-Seq）来确定染色质占有率。总体而言，拟议的研究将证明DNMT 3在正常发育过程中建立DNA甲基化模式和介导毒物诱导效应方面的功能。

 DESCRIPTION (provided by applicant): Developmental exposure to environmental chemicals is a significant public health problem. In the United States alone, one in six children has some form of developmental disability. Many of these cases are attributed to developmental exposure to toxicants. There are also strong indications that some of these exposures increase the risk of adult-onset diseases such as diabetes, hypertension and cardiovascular diseases. Recent studies have implicated epigenetic mechanisms such as DNA methylation in the persistent effects of developmental exposure to toxicants. DNA methylation plays an essential role in development and it involves the chemical modification of DNA by the addition of a methyl group to the cytosine nucleotide by DNA methyltransferase (DNMT) enzymes. DNMT3 group of enzymes are responsible for the establishment of de novo methylation patterns, but it is not known how they target specific DNA sequences for methylation, and how toxicants alter the methylation patterns. In this R01 grant application, the investigators propose to investigate the role of DNMT3s in normal development, as well as in the long-term effects of developmental exposure to toxicants. Using zebrafish, a well-established developmental and toxicological model system, the investigators propose to characterize DNMT3 functions and to determine their role in the response to environmental toxicants. In aim 1, the investigators will test the hypothesis that DNMT3s show DNA target specificity, with each DNMT3 paralog targeting a unique set of genes, thereby establishing DNA methylation patterns. They will determine DNMT3 target specificity in vitro by using an episomal methylation assay and characterizing the sequence specificity of recombinant DNMT3 isoforms, and in vivo using DNMT3- knockout zebrafish. In aim 2, the investigators will test the hypothesis that toxicants alter DNA methylatio patterns, and that these alterations are DNMT3-dependent. They will expose wild-type and DNMT3-knockout zebrafish embryos to environmentally relevant toxicants during sensitive windows of development, and determine the effects on DNA methylation patterns using Reduced Representation Bisulfite Sequencing (RRBS), a genome-wide approach for measuring DNA methylation in CpG rich regions of the genome. In Aim 3, the investigators will test the hypothesis that differentially methylated DNA regions in toxicant-exposed embryos show increased occupancy by DNMT3s. They will expose the zebrafish embryos to a toxicant that induces DNA methylation changes and determine chromatin occupancy using Chromatin immunoprecipitation (ChIP) followed by bisulfite sequencing (ChIP-BS-Seq). Overall, the proposed studies will demonstrate the functions of DNMT3s in establishing DNA methylation patterns during normal development and in mediating toxicant-induced effects.