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

 描述(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控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共同的生殖系传递的表型突变。相反，F1表型不是来自生殖系表型；因此，它不需要与F2代的表型相似。然而，许多动物研究报告说，F1和F2代的表型非常相似。为了解释这一有点矛盾的现象，我们假设在体细胞和生殖系细胞中表达的核受体可能与F1和F2代中常见的表型有关，但机制不同。具体地说，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，从而引发F1表型，但在生殖细胞中，相同的核受体可能会导致同一组基因的表型突变，从而导致F2表型。为了获得支持这一假说的实验证据，我们的具体目标1旨在确定小鼠胎儿暴露于已知的导致跨代肥胖的内分泌干扰物是否会在生殖系细胞基因组中受影响的核受体的结合序列上引入特定的表观遗传学变化。从12.5dpc到18.5dpc，转基因孕鼠将暴露在三丁基锡、罗格列酮或双酚A中，并通过流式细胞仪从胎儿中收集GFP生殖系细胞。对F1胚系细胞基因组中的mRNA表达、DNA甲基化和核激素受体结合的全基因组图谱将通过深度测序(RNA-seq、亚硫酸盐-seq和芯片-seq)来确定。为了确定可跨代传递的生殖系表型突变，我们还将测定18.5DPC时F2和F3胎儿的GFP生殖系细胞中的转录本和表观遗传标记分布。为了评估跨代肥胖表型和生殖系传递的表型，我们将检测8周龄F1、F2和F3幼崽的脂肪库重、脂肪细胞大小和棕色和白色脂肪组织中的脂肪细胞数量。选定的脂肪组织样本也将接受转录和表观基因组分析。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-LCS)暴露于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学变化，从而影响细胞对相关激素药物的敏感性。我们将使用深度测序来确定激素反应基因的转录本、表观基因组标记分布和全基因组配体敏感性图谱。RNAi敲除和CRISPR/Cas9基因敲除实验将确定每个核受体和Tet家族DNA羟甲基转移酶在形成暴露诱导的表观突变中的要求。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.