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

逆转录转座子衍生的启动子驱动具有重要发育功能的替代宿主基因亚型

基本信息

批准号：
10651867
负责人：
Lin He
金额：
$ 55.58万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10651867
关键词：
5&apos Untranslated Regions Amino Acid Sequence Atlases Automobile Driving Bioinformatics Biological Process CRISPR/Cas technology Callithrix Catalogs Categories Cattle Cell Proliferation Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Code Data Development Developmental Process Didelphidae Economics Elements Embryo Event Evolution Exons Family Family suidae Gene Expression Gene Expression Regulation Gene Structure Genes Genome Genome engineering Goat Human Impairment Livestock Macaca mulatta Mammals Mediating Molecular Molecular Biology Mus N-terminal Open Reading Frames Phenotype Physiology Play Poly A Polyadenylation Pre-implantation Embryo Development Primates Protein Isoforms Proteins Publishing RNA Regulation Regulator Genes Repression Retrotransposon Role Signal Transduction System Technology Time Transcript Transcriptional Regulation Validation blastocyst cohort comparative genomics gene conservation genome editing implantation in vivo mammalian genome mouse genetics mouse genome mutant next generation sequencing preimplantation promoter single-cell RNA sequencing transcriptome sequencing

项目摘要

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.

项目摘要大多数哺乳动物的逆转录座子在发育和生理学上都严格沉默，但诱导在特定的发育过程中，可以观察到一些逆转座子。有趣的是，一部分在重新激活的逆转录子，尤其是LTR逆转录座子中，赋予了基因调节作用，至少部分地，通过充当替代启动子来驱动嵌合转录本用代理蛋白质编码基因。这种逆转录座子启动子经常改变基因结构和/或基因表达，但是它们功能重要性在很大程度上不清楚。哺乳动物植入前胚胎是探测功能的极好的实验系统逆转座子的重要性。植入前胚胎中逆转座子的动态诱导在检查的所有8种哺乳动物物种中都观察到了我们，并且全局逆转录了表达曲线整个哺乳动物都相似。使用来自小鼠人类的已发表的单细胞RNA-seq数据，灵长类动物和牲畜前植入胚胎，我们发现了数百个逆转座子启动子，驱动植入前特异性的，近端基因同工型。有趣的是，大多数逆转座子序列和集成是特定规范的，但许多逆转座子启动子产生基因编码进化构成蛋白质的同工型。因此，这些数据表明逆转录座子启动子可以调节组成的蛋白质序列，并以规格特异性基因授予它们规定。这种进化构成的返回跨座子驱动的基因同工型，CDK2AP1N（MT2B2），编码CDK2AP1的N末端截断的同工型，这是一种负调节剂，用于细胞增殖的负调节剂抑制CDK2。 CDK2AP1N（MT2B2）由MT2B2启动子产生，其在小鼠中的缺失产生细胞增殖减少，植入和胚胎致死性。这是第一个研究之一展示了逆转录座子元素在开发中的基本功能。在这里，我们假设该逆转录跨座子基因调节在哺乳动物植入术中起着至关重要的作用发展。使用生物信息学预测与实验验证相结合，我们建议全面，准确地对鼠标，灵长类动物和牲畜中的逆转录子促进剂进行分类植入前胚胎，并阐明了逆转录盆地介导的潜水员分子机制基因调节。此外，我们将采用高效的CRISPR技术CRISPR-EZ来为选定的逆转座子启动子或相应的小鼠缺失突变体生成小鼠缺失突变体规范基因同工型。我们将比较逆转座子依赖性基因同工型的作用和规范基因同工型，阐明其作用的分子机制并探索进化这种调节的意义。综上所述，这些拟议的研究将产生全面的在植入前发育过程中，逆转座子依赖性基因调节的地图集，并提供新的范式以计算和实验性地研究后盆地的功能。