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

项目摘要 大多数哺乳动物反转录转座子在发育和生理学上是严格沉默的，但在哺乳动物中， 在特定的发育过程中可以观察到一些反转录转座子。有趣的是， 的重新激活的反转录转座子，特别是LTR反转录转座子，赋予基因调控作用，在 至少部分地，通过充当替代启动子来驱动具有近端蛋白编码的嵌合转录物， 基因.这种逆转录转座子启动子经常改变基因结构和/或基因表达，但它们的表达是不稳定的。 功能的重要性在很大程度上仍不清楚。 哺乳动物植入前胚胎是一个很好的实验系统，以探讨功能 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种哺乳动物中观察到， 所有哺乳动物物种都是相似的。使用已发表的来自小鼠、人类、 灵长类动物和家畜的植入前胚胎，我们发现了数百个反转录转座子启动子， 其驱动前体特异性的近端基因同种型。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，然而许多反转录转座子启动子产生基因， 编码进化上保守的蛋白质。因此，这些数据表明，逆转录转座子 启动子可以调控保守的蛋白质序列，赋予其物种特异性基因 调控这种进化上保守的反转录转座子驱动的基因亚型之一，Cdk 2ap 1 α N（MT 2B 2）， 编码Cdk 2ap 1的N末端截短同种型，Cdk 2ap 1是细胞增殖的负调节因子， 抑制Cdk 2。Cdk 2ap 1启动子N（MT 2B 2）由MT 2B 2启动子产生，其在小鼠中的缺失产生 细胞增殖减少、植入受损和胚胎致死。这是最早的研究之一， 证明了反转录转座子元件在发育中的基本功能。在这里，我们假设 反转录转座子介导基因调控在哺乳动物着床前发育中起重要作用 发展使用生物信息学预测结合实验验证，我们建议， 对小鼠、灵长类动物和家畜中的反转录转座子启动子进行了全面准确的分类 植入前胚胎，并阐明逆转录转座子介导的不同的分子机制， 基因调控此外，我们将采用高效的CRISPR技术CRISPR-EZ， 产生所选逆转录转座子启动子或相应的 典型基因亚型我们将比较反转录转座子依赖的基因亚型和 典型的基因亚型，阐明其作用的分子机制，并探讨其进化 这种规范的意义。这些拟议的研究将产生一个全面的 胚胎植入前发育过程中反转录转座子依赖的基因调控图谱，并提供了一个 新的范例，研究反转录转座子功能的计算和实验。

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.