Transposable element mobilization during spermatogenesis in Drosophila

果蝇精子发生过程中转座元件的动员

基本信息

批准号：
10747733
负责人：
Lauren Ann Tracy
金额：
$ 4.03万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10747733
关键词：
Binding Sites Cells Circular DNA Code Complementary DNA DNA DNA Ligases DNA Repair Pathway DNA Transposable Elements DNA-Directed DNA Polymerase Development Disease Drosophila genus Drosophila melanogaster Elements Event Factor VIII Female Future Generations Genes Genetic Code Genome Germ Cells Hemophilia A Human Genome Infertility Integration Host Factors Knowledge Length Life Cycle Stages Location Modeling Monitor Mutation Nomads Oocytes Oogenesis Organism Pathway interactions Placentation Process Production RNA RNA Interference RNA-Directed DNA Polymerase Regulatory Element Reporter Repression Retrotransposon Single-Stranded DNA Small RNA Sperm Maturation Spermatocytes Spermatogenesis Sterility Study models Technology Testing Testis Time Writing XRCC1 gene Y Chromosome cell type cost egg experimental study fly gene regulatory network improved male member nanopore piRNA preference recruit sperm cell syncytin transcription factor

项目摘要

Abstract Transposons are mobile pieces of DNA that comprise significant proportions of eukaryotic genomes, including around 45% of the human genome and 30% of the fly genome. While most transposons have lost the ability to jump into new locations in the genome, several in each organism maintain the ability to mobilize. New transposon insertions are particularly consequential if they are generated in germline cells. Germline insertions are subsequently present in every cell of the organism they develop into and can be passed on to future generations. Thus, germline cells employ multilayered mechanisms to repress transposons. These repressive mechanisms are essential to largely suppress transposon for development of germline cells into mature eggs and sperm because transposon activity can contribute to sterility. Since these repressive mechanisms like the piRNA pathway render transposition events so rare, they are difficult to detect and our understanding of how transposons mobilize in the germline remains incomplete. Namely, the host factors that transposons employ to generate new insertions and the time point when they make new insertions during sperm maturation are undefined. To find potentially active transposons in the male germline, I have sequenced circular DNA from Drosophila testes with the piRNA pathway depleted. Disrupting the piRNA pathway allowed for transposon activation and revealed that the nomad transposon generates the most circular DNA in the testes. Circular DNA is a transposition intermediate of LTR retrotransposons and its presence can indicate the likelihood of a transposon being able to still make new insertions. With the knowledge that nomad is the most active transposon in the male germline, I propose to use this transposon to study how transposons mobilize in the germline. I hypothesize that transposons utilize host factors from the alt-EJ DNA repair pathway to achieve stage specific mobilization during spermatogenesis. I will utilize circular nomad DNA as a readout to find host factors required for its production and transposition reporters for nomad to precisely identify when it generates new insertions.

抽象的转座子是可移动的 DNA 片段，构成真核生物基因组的很大一部分，包括人类基因组的 45% 左右，果蝇基因组的 30% 左右。虽然大多数转座子已经失去了跳跃到基因组中的新位置，每个生物体中的几个位置都保持着动员能力。新转座子如果插入是在生殖细胞中产生的，那么插入尤其重要。种系插入是随后存在于它们发育成的有机体的每个细胞中，并可以遗传给后代。因此，生殖细胞采用多层机制来抑制转座子。这些压制机制对于很大程度上抑制转座子对生殖细胞发育成成熟卵子和精子至关重要因为转座子活性可能导致不育。由于这些抑制机制如 piRNA 途径使得转座事件如此罕见，它们很难被发现，我们对如何理解种系中动员的转座子仍然不完整。即，转座子利用的宿主因子产生新的插入，并且在精子成熟期间它们进行新插入的时间点是不明确的。为了在雄性种系中找到潜在的活性转座子，我对来自 piRNA 途径耗尽的果蝇睾丸。破坏 piRNA 通路允许转座子激活并揭示游牧转座子在睾丸中产生最环状的 DNA。环状DNA 是 LTR 逆转录转座子的转座中间体，它的存在可以表明转座子仍然能够进行新的插入。了解 nomad 是最活跃的转座子在雄性种系中，我建议使用该转座子来研究转座子如何在种系中动员。我假设转座子利用来自 alt-EJ DNA 修复途径的宿主因子来实现阶段特异性精子发生过程中的动员。我将利用循环游牧 DNA 作为读数来查找所需的宿主因子用于 nomad 的生产和转座报告器，以准确识别何时生成新的插入。