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

 描述(由申请人提供)：发育过程中接触环境化学品是一个重大的公共卫生问题。仅在美国，就有六分之一的儿童患有某种形式的发育障碍。其中许多病例是由于发育过程中接触毒物造成的。也有强有力的迹象表明，其中一些接触会增加成人发病疾病的风险，如糖尿病、高血压和心血管疾病。最近的研究表明，表观遗传机制，如DNA甲基化，与发育过程中毒物暴露的持续影响有关。DNA甲基化在发育过程中起着至关重要的作用，它涉及到通过DNA甲基转移酶(DNMT)在胞嘧啶核苷酸上加一个甲基来对DNA进行化学修饰。DNMT3组酶负责建立从头开始的甲基化模式，但尚不清楚它们如何针对特定的DNA序列进行甲基化，以及毒物如何改变甲基化模式。在这份R01拨款申请中，研究人员建议调查DNMT3在正常发育中的作用，以及在发育暴露于毒物的长期影响中的作用。利用斑马鱼，一个成熟的发育和毒理学模型系统，研究人员建议表征DNMT3的功能，并确定它们在环境毒物反应中的作用。在目标1中，研究人员将检验这一假设，即DNMT3显示出DNA靶标特异性，每个DNMT3 Paralog针对一组唯一的基因，从而建立DNA甲基化模式。他们将在体外通过使用异构体甲基化分析和鉴定重组DNMT3亚型的序列特异性来确定DNMT3靶标特异性，并在体内使用DNMT3敲除斑马鱼。在目标2中，研究人员将检验毒物改变DNA甲基化模式的假设，以及这些改变是DNMT3依赖的。他们将在发育的敏感时期将野生型和DNMT3基因敲除的斑马鱼胚胎暴露于与环境相关的毒物，并使用简化表示亚硫酸盐测序(RRBS)来确定对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CpG丰富区域的DNA甲基化。在目标3中，研究人员将检验这一假设，即暴露于毒物的胚胎中差异甲基化的DNA区域显示DNMT3的占有率增加。他们将斑马鱼胚胎暴露在一种毒物下，这种毒剂会导致DNA甲基化变化，并使用染色质免疫沉淀(CHIP)和亚硫酸氢盐测序(CHIP-BS-SEQ)来确定染色质的占有率。总体而言，拟议的研究将证明DNMT3在正常发育过程中建立DNA甲基化模式和在调节毒物诱导的效应中的功能。

 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.