Sperm tsRNAs and their RNA modifications in diet-induced epigenetic inheritance

项目摘要 越来越多的哺乳动物证据表明，在父亲的环境中，某些后天习得的特征 暴露可能会在精子中被“记忆”并传递给后代，这意味着表观遗传 通过精子进行遗传。特别是，饮食引起的父亲代谢紊乱在哺乳动物中是可遗传的， 这表明这种类型的表观遗传在许多与代谢相关的人类疾病中具有长期影响。 到目前为止，对父亲饮食变化做出反应并将 代际表型仍然难以捉摸，但推测与DNA甲基化、染色质状态或 非编码RNA。最近，在高脂饮食(HFD)小鼠模型中，我们发现高度浓缩的tRNA来源 父本HFD后精子小RNA(TsRNAs)在表达谱和RNA修饰方面的变化 曝光。我们还表明精子tsRNAs及其RNA修饰是 父系获得性代谢紊乱的代际传递(Chen et.al.《科学》，2016)。这 这一发现提出了一个悬而未决的问题，即精子tsRNAs及其RNA修饰是如何调节 影响后代表型的胚胎发育规划(Chen et.al.NAT Rev Genet，2016)。至 为了了解其潜在的机制，我们建议：(1)通过注射精子来识别精子tsRNAs的靶点 TsRNAs(来自HFD和对照雄性)转化为受精卵，然后是比较的单细胞胚胎RNA序列2-to 4-细胞胚胎和生物信息学分析。(2)由于精子中M5C(5-甲基胞苷)的显著变化 已经发现了来自HFD男性的tsRNA，我们将确定M5C胞嘧啶RNA甲基转移酶， 通过利用HFD模型，DNMT2是精子tsRNAs将获得性代谢紊乱传递给后代所必需的 在DNMT2-/-和DNMT2/雄性小鼠中，将它们的精子tsRNA注射到正常受精卵中，然后检测 F1代的代谢表型。(3)此外，在DNMT2-/-与DNMT2/HFD模型中，我们将 用rna-seq检测精子tsRNAs中rna表达谱的变化以及rna修饰谱的变化。 通过高通量方法(基于LC-MS/MS) 量化多个RNA修饰，不仅可以检测M5C的变化，还可以发现新的RNA 修饰在存在或不存在DNMT2的情况下发生变化。拟议研究的数据将提供洞察力 精子tsRNAs介导获得性代谢紊乱代际遗传的机制 并有助于我们理解人类疾病的病因学，这些疾病源于以饮食为基础的跨代 效果。

Project Summary Increasing lines of evidence in mammals have shown that certain acquired traits during paternal environmental exposure could be “memorized” in sperm and transmitted to the future generations, implicating epigenetic inheritance via the sperm. Particularly, diet-induced metabolic disorders in the father are heritable in mammals, suggesting this type of epigenetic inheritance has a long-term impact in many metabolic-related human diseases. To date, the exact sperm “epigenetic carrier” that responds to paternal dietary changes and transmits the intergenerational phenotype has remained elusive, but presumably involves DNA methylation, chromatin status or non-coding RNAs. Recently, in a high-fat diet (HFD) mouse model, we showed that highly enriched, tRNA-derived small RNAs (tsRNAs) from sperm are altered in both expression profiles and RNA modification after paternal HFD exposure. We also showed that sperm tsRNAs, along with their RNA modifications, are required for intergenerational transmission of paternally acquired metabolic disorders (Chen et.al. Science, 2016). This discovery raises the open question of how sperm tsRNAs, along with their RNA modifications, mediate the embryonic developmental programming that affects the offspring phenotype (Chen et.al. Nat Rev Genet, 2016). To understand the underlying mechanisms, we propose to (1) identify the target of sperm tsRNAs by injecting sperm tsRNAs (from HFD and control males) into zygotes, followed by comparative single-cell embryo RNA-seq of 2- to 4-cell embryos and bioinformatics analysis. (2) Because significant alterations of m5C (5-methylcytidine) in sperm tsRNAs from HFD males have been found, we will determine whether the m5C cytosine RNA methyltransferase, DNMT2, is essential for sperm tsRNAs to transmit acquired metabolic disorder to offspring, by utilizing HFD model in Dnmt2-/- and Dnmt2+/+ male mice, and injecting their sperm tsRNAs into normal zygote followed by examining the metabolic phenotype of F1 offspring. (3) Moreover, in the Dnmt2-/- versus Dnmt2+/+ HFD model, we will examine the changes of RNA expression profiles by RNA-seq, and the RNA modification profiles in sperm tsRNAs by a high-throughput approach (based on Liquid chromatography-tandem mass spectrometry, LC-MS/MS) to quantify multiple RNA modifications, which not only will detect the changes of m5C, but also discover novel RNA modification changes in the presence or absence of DNMT2. Data from the proposed study will provide insights into the mechanism by which sperm tsRNAs mediate intergenerational inheritance of acquired metabolic disorders and facilitate our understanding of the etiology of human diseases originated from diet-based transgenerational effect.