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

项目摘要 大多数哺乳动物的反转录转座子在发育和生理上是严格沉默的，但诱导 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，有一部分 在重新激活的反转录转座子中，特别是LTR反转录转座子，赋予基因调控作用，在 至少部分是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。这种反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。 哺乳动物着床前胚胎是探索胚胎功能的一个很好的实验系统。 反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种哺乳动物中都观察到了，并且全球反转录转座子的表达谱 所有哺乳动物物种都是相似的。使用已发表的来自小鼠、人 灵长类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， 它们驱动着植入前特异的近端基因亚型。有趣的是，大多数反转录转座子 序列和整合是物种特有的，但许多反转录转座子启动子产生基因 编码进化保守蛋白质的异构体。因此，这些数据表明反转录转座子 启动子可以调节保守的蛋白质序列，并赋予它们物种特有的基因 监管。这种进化上保守的逆转座子驱动的基因亚型之一，CDK2ap1N(MT2B2)， 编码N端截短的CDK2ap1异构体，CDK2ap1是细胞增殖的负调控因子，通过 抑制CDK2。CDK2ap1N(MT2B2)是由MT2B2启动子产生的，其缺失在小鼠体内产生 细胞增殖减少，着床受损，胚胎致死率降低。这是首批研究之一 展示了反转录转座子元件在发育过程中的重要作用。在这里，我们假设 反转录转座子介导的基因调控在哺乳动物着床前发挥重要作用 发展。利用生物信息学预测和实验验证相结合，我们建议 全面准确地对小鼠、灵长类和家畜中的逆转座子启动子进行分类 植入前胚胎，并阐明逆转座子介导的不同分子机制 基因调控。此外，我们将采用高效的CRISPR技术CRISPR-EZ，以 为选定的反转录转座子启动子或相应的 典型的基因亚型。我们将比较反转录转座子依赖的基因异构体和 规范基因亚型，阐明其作用的分子机制，并探索其进化 这种规定的意义。综上所述，这些拟议的研究将产生一个全面的 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 通过计算和实验研究反转录转座子函数的新范式。

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.