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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.