Retrotransposon derived promoters drive alternative host gene isoforms with important developmental functions

项目概要 大多数哺乳动物逆转录转座子在发育和生理学上都被严格沉默，但诱导 在特定的发育过程中可以观察到一些逆转录转座子。有趣的是，有一部分 重新激活的逆转录转座子，特别是 LTR 逆转录转座子，赋予基因调节作用， 至少部分地，通过充当替代启动子来驱动具有近端蛋白质编码的嵌合转录本 基因。此类逆转录转座子启动子经常改变基因结构和/或基因表达，但它们的 功能重要性在很大程度上仍不清楚。 哺乳动物植入前胚胎是探索功能的优秀实验系统 逆转录转座子的重要性。植入前胚胎中反转录转座子的动态诱导 在所有 8 个检查的哺乳动物物种中均观察到，并且总体逆转录转座子表达谱 哺乳动物物种之间的情况相似。使用已发表的来自小鼠、人类、 在灵长类动物和家畜植入前胚胎中，我们发现了数百个逆转录转座子启动子， 驱动着床前特异性的近端基因亚型。有趣的是，大多数逆转录转座子 序列和整合是物种特异性的，但许多逆转录转座子启动子产生基因 编码进化上保守的蛋白质的亚型。因此，这些数据表明逆转录转座子 启动子可以调节保守的蛋白质序列并赋予它们物种特异性基因 监管。这种进化上保守的逆转录转座子驱动的基因亚型之一，Cdk2ap1N(MT2B2)， 编码 Cdk2ap1 的 N 端截短亚型，Cdk2ap1 是细胞增殖的负调节因子 抑制 Cdk2。 Cdk2ap1N(MT2B2) 由 MT2B2 启动子产生，在小鼠中缺失该启动子会产生 细胞增殖减少、植入受损和胚胎致死。这是第一项研究 证明逆转录转座子元件在发育中的重要功能。在这里，我们假设 逆转录转座子介导的基因调控在哺乳动物植入前发挥重要作用 发展。利用生物信息学预测与实验验证相结合，我们建议 全面准确地对小鼠、灵长类动物和家畜中的逆转录转座子启动子进行分类 植入前胚胎，并阐明逆转录转座子介导的多种分子机制 基因调控。此外，我们将采用高效的 CRISPR 技术 CRISPR-EZ， 产生选定的逆转录转座子启动子或相应的小鼠缺失突变体 典型的基因亚型。我们将比较逆转录转座子依赖性基因亚型和 典型的基因亚型，阐明其作用的分子机制并探索进化 这种监管的意义。总而言之，这些拟议的研究将产生全面的 植入前发育过程中逆转录转座子依赖性基因调控图谱，并提供 通过计算和实验研究反转录转座子功能的新范式。

Project Summary Most mammalian retrotransposons are strictly silenced in development and physiology, yet induction of some retrotransposons can be observed during specific developmental processes. Interestingly, a portion of the reactivated retrotransposons, particularly LTR retrotransposons, confer a gene regulatory role, at least in part, by acting as alternative promoters to drive chimeric transcripts with proximal protein-coding genes. Such retrotransposon promoters frequently alter gene structure and/or gene expression, yet their functional importance remains largely unclear. Mammalian preimplantation embryos are an excellent experimental system to probe the functional importance of retrotransposons. Dynamic induction of retrotransposons in preimplantation embryos have been observed in all 8 mammalian species examined, and the global retrotransposon expression profiles across mammalian species are similar. Using published single-cell RNA-seq data from mouse, human, primate and livestock preimplantation embryos, we discovered hundreds of retrotransposon promoters, which drive preimplantation-specific, proximal gene isoforms. Interestingly, most retrotransposon sequences and integrations are species specific, yet many retrotransposon promoters yield gene isoforms that encode evolutionarily conserved proteins. Hence, these data suggest that retrotransposon promoters can regulate conserved protein sequences and bestow them with species-specific gene regulation. One of such evolutionarily conserved retrotransposon driven gene isoform, Cdk2ap1N(MT2B2), encodes an N-terminally truncated isoform for Cdk2ap1, a negative regulator for cell proliferation by repressing Cdk2. Cdk2ap1N(MT2B2) is generated by an MT2B2 promoter, whose deletion in mice yield reduced cell proliferation, impaired implantation and embryonic lethality. This is among the first study demonstrating an essential function of a retrotransposon element in development. Here, we hypothesize that retrotransposon-mediate gene regulation play an essential role in mammalian preimplantation development. Using bioinformatics prediction combined with experimental validation, we propose to comprehensively and accurately categorize retrotransposon-promoters in mouse, primate and livestock preimplantation embryos, and elucidate the diverse molecular mechanisms for retrotransposon-mediated gene regulation. Additionally, we will employ a highly efficient CRISPR technology, CRISPR-EZ, to generate mouse deletion mutants for selected retrotransposon promoters or for the corresponding canonical gene isoforms. We will compare the roles of retrotransposon-dependent gene isoform and the canonical gene isoform, elucidate the molecular mechanisms of their action and explore the evolutionary significance of such regulation. Taken together, these proposed studies will generate a comprehensive atlas of retrotransposon-dependent gene regulation during preimplantation development, and provide a new paradigm to investigate retrotransposon functions both computationally and experimentally.