Transposable element mobilization during spermatogenesis in Drosophila

果蝇精子发生过程中转座元件的动员

• 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
• 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

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.

摘要 转座子是移动的DNA片段，其构成真核生物基因组的重要部分，包括 大约45%的人类基因组和30%的苍蝇基因组。虽然大多数转座子已经失去了 在基因组中跳跃到新的位置时，每个生物体中有几个保持动员的能力。新转座子 如果插入在生殖系细胞中产生，则它们特别重要。生殖系插入是 随后存在于它们发育成的生物体的每一个细胞中，并且可以传递给后代。 因此，生殖系细胞采用多层机制来抑制转座子。这些压抑机制 对于在很大程度上抑制转座子以使生殖系细胞发育成成熟的卵子和精子至关重要 因为转座子的活性会导致不育。由于这些抑制机制，如皮尔纳， 途径使转座事件如此罕见，它们很难检测，我们对转座事件的理解是如何进行的。 转座子在生殖系中的移动仍然不完全。也就是说，转座子用来 产生新的插入，以及在精子成熟过程中它们产生新插入的时间点是 未定义。为了在雄性生殖细胞系中找到潜在的活跃转座子，我对来自 具有皮尔纳途径耗尽的果蝇睾丸。破坏皮尔纳通路允许转座子 激活，并揭示了游牧转座子在睾丸中产生最多的环状DNA。环状DNA 是LTR反转录转座子的转座中间体，其存在可以指示LTR反转录转座子的转座可能性。 转座子仍然能够进行新的插入。我们知道nomad是最活跃的转座子 在雄性生殖系中，我建议使用这种转座子来研究转座子如何在生殖系中移动。我 假设转座子利用来自alt-EJ DNA修复途径宿主因子来实现阶段特异性 精子发生过程中的动员。我将利用环状游牧DNA作为读出器， 用于其生产和转座报告基因，以便Nomad精确识别何时产生新的插入。