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

de novo DNMT 在毒物诱导的 DNA 甲基化改变中的作用

基本信息

批准号：
8813984
负责人：
NEELAKANTESWAR Aluru
金额：
$ 47.9万
依托单位：
WOODS HOLE OCEANOGRAPHIC INSTITUTION
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2019-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8813984
关键词：
Address Adult Affect Alleles Applications Grants Biological Assay Biological Models Cardiovascular Diseases Chemicals Child Chromatin Cytosine Cytosine Nucleotides DNA DNA Methylation DNA Modification Methylases DNA Modification Process DNA Sequence Development Developmental Disabilities Diabetes Mellitus Embryo Embryonic Development Enzymes Epidemiology Epigenetic Process Exposure to Gametogenesis Gene Expression Gene Silencing Generations Genes Genome Genomic Imprinting Human Hypertension In Vitro Knock-out Light Long-Term Effects Maintenance Measures Mediating Methylation Methyltransferase Modeling Onset of illness Orthologous Gene Pattern Play Process Protein Isoforms Public Health RNA Sequences Recombinants Regulation Research Research Personnel Risk Role Specificity System Testing Toxic Environmental Substances United States Vertebrates Zebrafish base bisulfite sequencing chromatin immunoprecipitation critical period early life exposure environmental chemical genome-wide improved in vivo methyl group methylation pattern methylome mutant paralogous gene preference public health relevance response teleost fish toxicant transcriptome sequencing

项目摘要

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.

描述（适用提供）：对环境化学物质的发展暴露是一个重大的公共卫生问题。仅在美国，六分之一的孩子就有某种形式的发育障碍。其中许多案例归因于发展对毒物的暴露。也有很强的迹象表明，其中一些暴露会增加成人发作疾病的风险，例如糖尿病，高血压和心血管疾病。最近的研究已经实施了表观遗传机制，例如DNA甲基化在发育暴露于毒物的持续作用中。 DNA甲基化在发育中起着至关重要的作用，它涉及通过通过DNA甲基转移酶（DNMT）酶向胞嘧啶核丁基添加甲基的DNA化学修饰。 DNMT3酶是为了建立从头甲基化模式的原因，但尚不清楚它们如何靶向特定的DNA序列进行甲基化以及如何改变毒性改变甲基化模式。在此R01赠款的应用中，研究人员提出的是研究DNMT3在正常发育中的作用以及发育暴露对毒物的长期影响。研究人员使用斑马鱼（斑马鱼（Zebrafish），是一个公认的发展和毒理学模型系统，提出了表征DNMT3功能并确定其在对环境毒物反应中的作用。在AIM 1中，研究人员将测试DNMT3S显示DNA靶特异性的假设，每个DNMT3旁系同源物均针对一组独特的基因，从而建立了DNA甲基化模式。他们将通过使用偶发甲基化测定法确定体外的DNMT3靶特异性，并表征重组DNMT3同工型的序列特异性，并使用DNMT3-敲除斑马鱼在体内。在AIM 2中，研究者将检验以下假设：有毒物质改变了DNA甲基氮于模式，并且这些改变是DNMT3依赖性的。他们将在敏感的开发窗口中将野生型和DNMT3-KNOCKOUT斑马鱼胚胎暴露于与环境相关的毒物中，并使用降低的表示Bisulfite测序（RRBS）确定对DNA甲基化模式的影响，Bisulfite测序（RRB），一种基因组全基因组方法，用于测量CPG富含基因组的DNA甲基化。在AIM 3中，研究人员将检验以下假设：毒性暴露的胚胎中不同甲基化的DNA区域显示DNMT3S的占用率增加。他们将将斑马鱼胚胎暴露于诱导DNA甲基化变化并使用染色质免疫沉淀（CHIP）的毒素占用症的毒物中，然后是硫酸盐测序（CHIP-BS-seq）。总体而言，拟议的研究将证明DNMT3在正常发育过程中建立DNA甲基化模式和介导毒物诱导的作用方面的功能。