The Role of Retrotransposon Activity in Mammalian Pre-Implantation Development

逆转录转座子活性在哺乳动物植入前发育中的作用

• 批准号: 10594575
• 负责人: Andrew Joseph Modzelewski
• 金额: $ 24.89万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-18 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594575
• 关键词: 5' Untranslated Regions; Acceleration; Area; Automobile Driving; Bioinformatics; Biological; Biology; Biosensor; CRISPR/Cas technology; Cell Cycle; Cell Cycle Progression; Cell division; Cells; Chimerism; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Community Developments; Complex; Consumption; Crowding; Data; Data Set; Defect; Development; Disease; Electroporation; Elements; Embryo; Embryo Transfer; Embryonic Development; Event; Exons; Family; Fertility; Gene Structure; Genes; Genetic; Genetic Transcription; Genome; Glucose; Goals; Health; Homeostasis; Human; Human Genome; Immunofluorescence Immunologic; In Vitro; Individual; Infertility; Knock-out; Knockout Mice; Metabolic; Methods; Monitor; Morula; Mus; Mutation; N-terminal; Nature; Open Reading Frames; Phase; Phenotype; Placenta; Poly A; Polyadenylation; Positioning Attribute; Pre-Eclampsia; Pre-implantation Embryo Development; Pregnancy; Pregnancy Complications; Pregnancy loss; Process; Protein Biosynthesis; Protein Isoforms; Proteins; Proteome; Proteomics; Pyruvate; RNA; RNA Splicing; Recurrence; Regulation; Reporting; Research; Retrotransposon; Role; Signal Transduction; Site; Specific qualifier value; Stress; Surveys; Techniques; Technology; Testing; Time; Training; Transcript; Translations; Uterus; Western Blotting; Work; blastocyst; career; confocal imaging; embryo cell; gene regulatory network; genetic manipulation; genome editing; implantation; in vivo; insight; mouse genome; natural Blastocyst Implantation; novel; overexpression; placenta previa; preimplantation; promoter; ribosome profiling; single cell analysis; transcriptome; transcriptome sequencing; uterine receptivity

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.

摘要 人类和小鼠近一半的基因组都来自古老的逆转录病毒整合。虽然沉默， 在几乎所有细胞中，反转录转座子再激活是发生在植入前胚胎中的公认现象。 在少数报告中，逆转录转座子家族表达的破坏导致胚胎死亡，这表明 基本功能，但其原因完全未知。大多数逆转录转座子 通过突变失活。尽管如此，许多保留了完整元件的调节和结构特征， 逆转录转座子影响附近基因的报道。逆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，但是重新分析单细胞植入前小鼠胚胎 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有在 在限定的时间窗口内，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“嵌合转录本”，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。作为一种原理证明， 使用实验室开发的基于电穿孔的CRISPR胚胎编辑方法来生成 两个嵌合启动子的反转录转座子缺失。驱动Rpl 41的MT 2B 1启动子的缺失导致 在延迟的全球翻译，造成压力诱导的逮捕。第二，缺失MT2C_Mm启动子， 细胞周期调节因子Cdk 2ap 1，导致小窝，身体异常，胚胎间隔，拥挤， 植入不合适的子宫部位，使人联想到人类妊娠并发症前置胎盘， 植入和潜在的先兆子痫。因此，申请人假设逆转录转座子再激活和 植入前的转录本嵌合体对于早期胚胎发育和植入是必不可少的。期间 在K99阶段，申请人将接受三项尖端技术的培训： 共聚焦成像，单细胞/胚胎蛋白质印迹与艾米赫尔博士和超低输入核糖体分析 尼古拉斯·因戈利亚博士的胚胎这些合作和额外的课程将帮助申请人掌握 实现长期学术和职业目标所需的技术。为此，Aim 1将有助于理解 M2TC_Mm：CKD 2ap 1缺失的植入缺陷的细胞原因，提供了对新的 解释人类怀孕的相关问题。Aim 2将致力于阐明M2 TB 1：Rp 141在全球范围内的作用。 翻译以及深入了解发育中的胚胎的氧化和代谢需求。目标1 其中2个将在K99阶段完成。在R 00阶段，目标3将致力于确定 反转录转座子对植入前胚胎的影响。与大卫·里索博士一起， 胚胎核糖体分析和匹配的RNA-SEQ数据将有助于解开和进一步测试复杂的基因重组。 通过反转录转座子再激活来连接植入前胚胎。由于这些重新激活并不是唯一的 这项工作将建立一种方法来研究这种被忽视但重要的新型调控网络 并为迄今为止没有明确遗传原因的人类发育表型提供了额外的解释。