Roles of Nuclear Receptors in Generation of Heritable Epimutations in Germ Cells

 描述（由申请人提供）：越来越明显的是，哺乳动物胎儿在子宫内暴露于环境内分泌干扰物可能会通过破坏调节基因表达的表观遗传机制而导致跨代疾病。F2代和后代的疾病表型可能彼此相似，因为这些表型可能来自一组常见的生殖系传播的表位突变。相比之下，F1表型不是来自种系表位突变;因此，它不需要与F2+代的表型相似。然而，许多动物研究报告了F1代和F2+代的表型非常相似。为了解释这个有点矛盾的现象，我们假设，核受体表达的体细胞和生殖细胞可能是负责的表型通常观察到的F1和F2+代，但通过不同的机制。具体而言，虽然在体细胞中表达的核受体可能直接影响激素靶基因的转录以激发F1表型，但在生殖细胞中，相同的核受体可能导致相同基因组的表型突变，从而导致F2+表型。为了获得支持这一假设的实验证据，我们的特定目标1旨在确定小鼠胎儿暴露于已知引起跨代肥胖的内分泌干扰物是否会在生殖细胞基因组中受影响的核受体的结合序列处引入特定的表观遗传变化。从12.5 dpc至18.5 dpc，将工程化用于生殖系特异性GFP表达的妊娠雌性转基因小鼠暴露于三丁基锡、罗格列酮或双酚A，并通过FACS从其胎儿中收集GFP+生殖系细胞。将通过深度测序（RNA-seq、亚硫酸氢盐-seq和ChIP-seq）确定F1种系细胞基因组中mRNA表达、DNA甲基化和核激素受体结合的全基因组谱。为了鉴定可转代传播的生殖系表观突变，我们还将确定在18.5 dpc时F2和F3胎仔的GFP+生殖系细胞中的转录组和表观遗传标记分布。为了评价跨代肥胖表型和生殖细胞传递的表型突变，我们将检查8周龄F1、F2和F3幼仔棕色和白色脂肪组织的脂肪贮库重量、脂肪细胞大小和脂肪细胞数量。还将对选定的脂肪组织标本进行转录组和表观基因组分析。我们的特定目标2旨在确定小鼠和人类原始生殖细胞样细胞（PGC-LC）暴露于致肥胖内分泌干扰物是否会导致核受体结合序列的特定表观遗传变化，从而影响细胞对体外相关激素的敏感性。我们将使用深度测序确定激素应答基因的转录组、表观基因组标记分布和全基因组配体敏感性谱。RNAi敲除和CRISPR/Cas9基因敲除实验将确定在形成由RNAi诱导的表突变中每种核受体和泰特家族DNA羟甲基转移酶的需要。该项目的成功完成将为未来研究胎儿暴露于内分泌干扰物引起的跨代疾病的分子机制奠定新的理论和方法学基础，包括体内和体外模型系统。

 DESCRIPTION (provided by applicant): It has been becoming increasingly evident that in utero exposure of mammalian fetuses to environmental endocrine disruptors may cause transgenerational diseases through disruption of the epigenetic mechanisms regulating gene expression. The disease phenotypes of the F2 and later generations may resemble each other because such phenotypes might be derived from a common set of germline-transmitted epimutations. In contrast, the F1 phenotype is not derived from the germline epimutations; therefore, it does not need to be similar to phenotypes of the F2+ generations. However, many animal studies have reported remarkably similar phenotypes of the F1 and the F2+ generations. To explain this somewhat paradoxical phenomenon, we hypothesize that the nuclear receptors expressed in both the somatic cells and the germline cells may be responsible for the phenotypes commonly observed in the F1 and the F2+ generations but through distinct mechanisms. Specifically, while nuclear receptors expressed in the somatic cells may directly affect transcription of the hormone target genes to provoke the F1 phenotypes, in the germline cells the same nuclear receptors might cause epimutations at the same set of genes to cause the F2+ phenotypes. To obtain experimental evidence supporting this hypothesis, our Specific Aim 1 aims to determine whether fetal exposure of mice to endocrine disruptors known to cause transgenerational obesity introduces specific epigenetic changes at the binding sequences of the affected nuclear receptors in the genome of the germline cells. Pregnant female transgenic mice engineered for germline-specific GFP expression will be exposed to tributyltin, rosiglitazone, or Bisphenol A from 12.5 dpc until 18.5 dpc, and the GFP+ germline cells will be collected from their fetuses by FACS. Genome-wide profiling of mRNA expression, DNA methylation, and nuclear hormone receptor binding in the genome of the F1 germline cells will be determined by deep sequencing (RNA- seq, Bisulfite-seq, and ChIP-seq). To identify the transgenerationally transmittable germline epimutations, we will also determine transcriptomes and epigenetic mark distributions in the GFP+ germline cells of F2 and F3 fetuses at 18.5 dpc. To evaluate the transgenerational obesity phenotype and the germline-conveyed epimutations, we will examine fat depot weight, adipocyte size, and adipocyte number of brown and white adipose tissues in 8-weeks-old F1, F2, and F3 pups. Selected adipose tissue specimens will also be subjected to transcriptomal and epigenomic analyses. Our Specific Aim 2 aims to determine whether exposure of mouse and human primordial germ cell-like cells (PGC-LCs) to the obesogenic endocrine disruptors causes specific epigenetic changes at the nuclear receptor binding sequences to affect cellular sensitivity to the relevant hormonal agents in vitro. We will determine transcriptomes, epigenomic mark distributions, and genome-wide ligand sensitivity profiles of the hormone responsive genes using deep sequencing. RNAi knockdown and CRISPR/Cas9 gene knockout experiments will establish the requirement of each nuclear receptor and the Tet family DNA hydroxymethyltransferases in formation of the exposure-induced epimutations. Successful completion of this project will establish a novel theoretical and methodological foundation, including in vivo and in vitro model systems, for future research on the molecular mechanisms of transgenerational diseases caused by fetal exposure to endocrine disruptors.