Assessing transgenerational effects of Phthalates on primordial germ cells

描述（由申请人提供）：最近的研究表明，在子宫内暴露于环境毒素可以在几个连续的代中产生表型变化。尽管最初的研究表明，突变可以传播这些跨代表型，但必须将这些信息传递给下一代的细胞尚未得到研究。原始生殖细胞（PGCs）是卵子或精子的胚胎缔造者，在妊娠中期经历快速的表观遗传重编程，其时间框架与暴露于某些毒素后观察到的跨代效应敏感性重叠。我们研究的长期目标是阐明PGC发育的遗传和表观遗传机制。本申请的目的是评估一类环境化学物质邻苯二甲酸盐对暴露个体（F1）及其后代（F3）小鼠PGC发育和表观遗传重编程的影响。我们的中心假设是，生物学上相关剂量的邻苯二甲酸酯破坏了发育过程中涉及PGCs重编程的DNA甲基化变化，导致基因表达改变；因此，与PGCs重编程调控相关区域的错甲基化破坏了种系中关键重编程机制的表达。这一假设是基于先前关于增塑剂双酚a和邻苯二甲酸盐混合物产生的跨代表型的报告，以及邻苯二甲酸盐改变人类和小鼠印迹位点DNA甲基化的证明。我们的假设将通过以下两个特定目的进行验证：(1)建立邻苯二甲酸盐暴露对F1和F3代小鼠PGC的表型影响；(2)确定邻苯二甲酸盐对小鼠PGC表观基因组和转录组的直接和跨代影响。在第一个目标中，我们将在子宫内暴露于一系列环境相关剂量的邻苯二甲酸二（2-乙基己基）酯（DEHP）和邻苯二甲酸二正丁酯（DBP）后，询问F1和F3代的PGC表型。剂量产生的表型可能为第二个目标提供信息，在这个目标中，我们将通过检测组蛋白修饰和半定量的全球甲基化和全基因组甲基化分析来调查F1和F3 PGCs的表观遗传重编程。由此产生的F1和F3 PGC转录组的变化，连同表观遗传数据，将揭示在DNA甲基化水平上观察到的失调的后果，使预测其他组织的表型成为可能，并可以确定负责传播观察到的表观遗传突变的候选表观遗传调节因子。这项提议的工作具有创新性，因为它使用了先进的全基因组方法，并且与以前的工作不同，它将直接检查负责代际传播的细胞中的表观遗传变化和潜在后果。这项工作具有重要意义，因为它将确定邻苯二甲酸盐暴露在多大程度上改变了PGCs的重编程，并可能确定跨代表观遗传的候选机制。

DESCRIPTION (provided by applicant): Recent studies suggest that in utero exposure to environmental toxins can produce phenotypic changes over several successive generations. Although initial work suggests that epimutations propagate these transgenerational phenotypes, the cells that must carry this information to the next generation have not been examined. Primordial germ cells (PGCs), the embryonic founders of the egg or sperm, undergo rapid epigenetic reprogramming during mid-gestation in a timeframe that overlaps with the observed sensitivity to transgenerational effects following exposure to certain toxins. The long-term goal of our research is to elucidate genetic and epigenetic mechanisms underlying PGC development. The objective of this application is to assess the impact of one class of environmental chemical, phthalates, on mouse PGC development and epigenetic reprogramming in the exposed individual (F1) and subsequent generations (F3). Our central hypothesis is that biologically relevant doses of phthalates disrupt DNA methylation changes involved in reprogramming of PGCs during development, leading to gene expression changes; in order to be heritable through generations, it follows that mismethylation of regions associated with the regulation of reprogramming in PGCs disrupts expression of critical reprogramming machinery in the germline. This hypothesis is based upon prior reports of transgenerational phenotypes produced by a mixture of plasticizers BPA and phthalates as well as the demonstration that phthalates alter DNA methylation at the imprinted loci in humans and mice. Our hypothesis will be tested with the following two specific aims: (1) to establish phenotypic consequences of phthalate exposure on F1 and F3 generation mouse PGCs and (2) to determine direct and transgenerational effects of phthalates on the mouse PGC epigenome and transcriptome. In the first aim, we will interrogate PGC phenotypes in the F1 and F3 generation following in utero exposure to a range of environmentally relevant doses of di-(2-ethylhexyl) phthalate (DEHP) and di-n-butyl phthalate (DBP). Doses producing phenotypes may be informative for the second aim, in which we will survey epigenetic reprogramming in F1 and F3 PGCs by examining histone modifications and global methylation semi-quantitatively and by genome-wide methylation analysis. Resulting changes in the F1 and F3 PGC transcriptome, together with the epigenetic data, will reveal the consequences of observed dysregulation at the level of DNA methylation, enable prediction of phenotypes in other tissues, and could identify candidate epigenetic regulators responsible for propagating observed epimutations. The proposed work is innovative as it uses advanced, genome-wide approaches and, apart from previous work, will directly examine epigenetic changes and potential consequences in the very cell responsible for trangenerational transmission. The proposed work is significant as it will determine the extent to which phthalate exposure alters reprogramming in PGCs and potentially identify candidate mechanisms of transgenerational epigenetic inheritance.