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

项目摘要 越来越多的证据表明，哺乳动物在父系环境中获得的某些特征， 暴露可以在精子中“记忆”并传递给后代，这意味着表观遗传 通过精子遗传特别是，父亲的饮食诱导的代谢紊乱在哺乳动物中是可遗传的， 这表明这种表观遗传对许多代谢相关的人类疾病具有长期影响。 到目前为止，确切的精子“表观遗传载体”，响应父亲的饮食变化，并传递 代际表型仍然难以捉摸，但可能涉及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，高浓度的，tRNA衍生的 来自精子的小RNA（tsRNA）在父本HFD后在表达谱和RNA修饰方面都发生了改变 exposure.我们还发现，精子的tsRNA，沿着它们的RNA修饰， 父系获得性代谢紊乱的代际传播（Chen et.al. Science，2016）。这 这一发现提出了一个悬而未决的问题，即精子的tsRNA沿着它们的RNA修饰，是如何介导精子的增殖的。 影响后代表型的胚胎发育编程（Chen et.al. Nat Rev Genet，2016）。到 了解潜在的机制，我们建议（1）通过注射精子来确定精子tsRNA的靶点 将tsRNA（来自HFD和对照雄性）导入受精卵中，随后进行2-至3-的比较性单细胞胚胎RNA-seq。 4-细胞胚胎和生物信息学分析。(2)因为精子中m5 C（5-甲基胞苷）的显著改变 已经发现来自HFD男性的tsRNA，我们将确定m5 C胞嘧啶RNA甲基转移酶， 利用HFD模型，DNMT 2是精子tsRNA将获得性代谢紊乱传递给后代所必需的， 在Dnmt 2-/-和Dnmt 2 +/+雄性小鼠中，并将其精子tsRNA注射到正常受精卵中，随后检查 F1代的代谢表型。(3)此外，在Dnmt 2-/-与Dnmt 2 +/+ HFD模型中，我们将 通过RNA-seq检测RNA表达谱的变化，以及精子中的RNA修饰谱 通过高通量方法（基于液相色谱-串联质谱法，LC-MS/MS）， 定量多种RNA修饰，不仅可以检测m5 C的变化，还可以发现新的RNA 在DNMT 2存在或不存在下的修饰变化。拟议研究的数据将提供见解 精子tsRNA介导获得性代谢紊乱代际遗传的机制 并促进我们了解人类疾病的病因起源于饮食为基础的跨代 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.