The Role of Retrotransposon Activity in Mammalian Pre-Implantation Development

摘要 近一半的人类和小鼠基因组都来自古老的逆转录病毒整合。在静音状态下 在几乎所有的细胞中，反转录转座子重新激活是一种公认的现象，发生在植入前的胚胎中。 在一些报告中，逆转座子家族表达的中断导致胚胎死亡，这表明 基本功能，但其原因完全未知。大多数反转录转座子已经被 通过突变使其失活。尽管如此，许多公司仍保留了完整要素的监管和结构特征，其中 反转录转座子对邻近基因影响的报道。反转录转座子的高度重复性使得 研究它们的个体功能很困难，然而对单细胞植入前小鼠胚胎的重新分析 揭示了动态表达的反转录转座子家族的惊人水平。大多数反转录转座子只是活跃的 在定义的时间窗口期间，有时跨越单个细胞分裂。有趣的是，这些基因座的一个子集 与附近的蛋白质编码基因拼接，产生“嵌合转录”，形成数百种新的 胚胎特异性启动子、外显子和多聚腺苷酸化位点。作为一个原则证明，一个高效的 使用实验室开发的基于电穿孔的CRISPR胚胎编辑方法生成 两个嵌合启动子的反转录转座子缺失。驱动Rpl41结果的MT2B1启动子的缺失 在延迟的全局翻译中，导致应激诱导的停顿。第二，缺失Mt2c_mm启动子 细胞周期调节因子CDK2ap1导致产仔数少、身体异常、胚胎间隔、拥挤和 植入不合适的子宫部位，让人想起人类妊娠并发症前置胎盘， 植入物和潜在的先兆子痫。因此，申请人假设反转录转座子重新激活和 着床前的转录嵌合体是早期胚胎发育和着床所必需的。在.期间 在K99阶段，申请者将接受三项尖端技术的培训：自动化活细胞旋转磁盘 共聚焦成像、艾米·赫尔博士的单细胞/胚胎Western Blot和超低输入核糖体图谱 尼古拉斯·英戈利亚博士的胚胎。这些合作和额外的课程作业将帮助申请者掌握 实现长期学术和职业目标所需的技能。为此，Aim1将帮助理解 M2TC_mm：CKD2ap1缺失植入缺陷的细胞学原因 有关人类怀孕问题的解释。AIM2将致力于阐明M2TB1：Rpl41在全球 翻译以及洞察发育中胚胎的氧化和代谢需求。目标1 和2个将在K99阶段完成。在R00阶段，AIM 3将致力于确定 反转录转座子对植入前胚胎的影响。与Davide Risso博士一起，对 胚胎核糖体图谱和匹配的RNA-SEQ数据将有助于解开和进一步测试复杂的Re。 通过反转录转座子再激活连接着床前胚胎。因为这些重新激活并不是 老鼠，这项工作将建立一种方法来调查这个被忽视但重要的新调控网络 并对到目前为止还没有明确的遗传原因的人类发育表型提供了额外的解释。

Abstract Nearly half of both human and mouse genomes originate from ancient retroviral integrations. While silenced in nearly all cells, retrotransposon reactivation is a recognized phenomenon occurring in preimplantation embryos. In a handful of reports, disruption of retrotransposon family expression resulted in embryonic lethality, suggesting essential functions, but the cause of this is completely unknown. The majority of retrotransposons have been inactivated through mutation. Still, many retain regulatory and structural features of intact elements, with rare reports of retrotransposon influence of nearby genes. The highly repetitive nature of retrotransposons has made studying their individual functions difficult, however re-analysis of single cell pre-implantation mouse embryos revealed striking levels of dynamically expressed retrotransposon families. Most retrotransposons are only active during defined windows of time, sometimes spanning a single cell division. Interestingly, a subset of these loci are spliced with nearby protein coding genes, generating “chimeric transcripts” that form hundreds of novel embryonic specific promoters, exons and polyadenylation sites. As a proof-of-principle, a highly efficient electroporation based CRISPR embryo editing method developed in the lab was used to generate retrotransposon deletions of two chimeric promoters. The deletion of the MT2B1 promoter driving Rpl41 results in delayed global translation, causing stress induced arrest. The second, deletion of the MT2C_Mm promoter of the cell cycle regulator Cdk2ap1, results in small litters, physical abnormalities, embryo spacing, crowding and implantation into unsuitable uterine sites, reminiscent of the human pregnancy complications placenta previa, accreta and potentially pre-eclampsia. Thus, the applicant hypothesizes that retrotransposon reactivation and transcript chimerism in preimplantation is essential for early embryonic development and implantation. During the K99 phase, the applicant will train in three cutting edge technologies: automated live cell spinning disk confocal imaging, single cell/embryo Western Blot with Dr. Amy Herr and ultra-low input Ribosome Profiling of Embryos with Dr. Nicholas Ingolia. These collaborations and additional coursework will help the applicant master the techniques needed for long term academic and career goals. To this end, Aim1 will serve to understand the cellular cause of the implantation defects of the M2TC_Mm:CKD2ap1 deletion, offering insight into novel explanations for related human pregnancy issues. Aim2 will work to elucidate the role of M2TB1:Rpl41 in global translation as well as to gain insight into the oxidative and metabolic needs of the developing embryo. Aims 1 and 2 will be completed during the K99 phase. During the R00 phase, Aim 3 will work to determine the extent of retrotransposon influence on the preimplantation embryo. Together with Dr. Davide Risso, parallel analysis of the embryo ribosome profiling and matched RNA-SEQ data will help to unravel and further test the complex re- wiring of the preimplantation embryo by retrotransposon reactivation. As these reactivations are not unique to the mouse, this work will establish a method to investigate this overlooked but important novel regulatory network and offer additional explanations to human developmental phenotypes that thus far have no clear genetic cause.