A genetic model for metazoan programmed DNA elimination

项目摘要/摘要 基因组的完整性对生命至关重要。为了保持基因组的稳定性，人们做出了相当大的努力。还没有 基因组也会经历不断的变化，通常是随机的，规模很小，为进化提供了机制 和适应。相比之下，程序性DNA消除是基因组变化的一种戏剧性形式， 在发育过程中消除的DNA数量，从基因组的0.5%到95%不等。DNA消除是 高度选择性和重复性，是包括单细胞在内的各种生物体生物学的组成部分 纤毛虫，动物门和一些植物中的一种多细胞生物体。广义的系统发育 分布表明DNA消除是独立进化的，具有重要的生物学功能。一个 后生动物DNA消除的共同主题是去除种系表达的基因和重复的 序列。这表明后生动物中dna消除的一个可能功能是永久沉默。 某些对体细胞有潜在危害的生殖系序列。尽管基因组学和细胞学取得了进步， 后生动物DNA消除的功能和机制研究有限，这主要是由于缺乏遗传 和实用的工具。最近，我们在基因组观察的基础上进行了扩展，并建立了一个遗传和 Rhabditidae属自由生活线虫Oscheius tipuae消除DNA的功能模型 科，包括秀丽隐杆线虫。我们发现dna的消除发生在8-16个月。 细胞胚胎。我们鉴定并鉴定了一个保守的序列(Sequence for Elevation，SFE)基序 与DNA断裂位点相关联，并展示了它在DNA消除中的直接作用。DNA断裂会发生 在主题内，接着是末端切除和端粒愈合。其他中断同时发生在 消除了DNA，可能是DNA消除的故障安全机制。我们揭示了丰富的 这一基序在世界各地的许多野生分离株中也存在变异。在这项建议中，我们将(1) 通过鉴定不能消除的表型来研究斑节对虾的DNA消除功能 CRISPR编辑了SFE突变体。我们将使用RNA-SEQ、CHIP-SEQ和小RNA测序来确定 这些突变体的RNA表达和沉默机制的变化。我们还将(2)研究分子 通过研究SFE所需的序列和基因组位置来研究泰普氏钉螺DNA消除的机制 使用CRISPR，以及使用体外生物化学、生物信息学预测与SFE相互作用的蛋白质， 和遗传学。我们将通过构建端粒到端粒来进一步研究DNA消除的变异 不同毒株的基因组，鉴定SFE，并开展比较基因组学研究。总的来说， 这项建议将使用我们在自由生活线虫O.tipuae中建立的遗传模型来研究 DNA消除的功能、机制和变异。这项工作将揭示对分子细节的洞察 在后生动物中消除DNA。

Project Summary/Abstract Genome integrity is essential to life. Considerable efforts are made to maintain the stability of genomes. Yet genomes also undergo constant changes, often random and small in scale, providing mechanisms for evolution and adaptation. In contrast, programmed DNA elimination is a dramatic form of genome change with large amounts of DNA, ranging from 0.5 to 95% of the genome, eliminated during development. DNA elimination is highly selective and reproducible and is an integral part of biology for diverse organisms, including single-cell ciliates, a variety of multicellular organisms across animal phyla and some plants. The broad phylogenetic distribution suggests DNA elimination has evolved independently and has important biological functions. A common theme for metazoan DNA elimination is the removal of both germline-expressed genes and repetitive sequences. This suggests that a possible function of DNA elimination in metazoa is to permanently silence certain germline sequences potentially harmful to somatic cells. Despite progress in genomics and cytology, functional and mechanistic studies of metazoan DNA elimination are limited, largely due to the lack of genetic and functional tools. Recently, we built upon and extended a genomic observation and established a genetic and functional model for DNA elimination in the free-living nematode Oscheius tipulae, a member of the Rhabditidae family, which includes Caenorhabditis elegans. We show that DNA elimination in O. tipulae occurs during 8-16 cell embryos. We identified and characterized a conserved sequence (Sequence For Elimination, SFE) motif associated with the DNA break sites and demonstrated its direct role in DNA elimination. DNA breaks occur within the motif, followed by end resection and telomere healing. Additional breaks occur simultaneously in the eliminated DNA, perhaps serving as a fail-safe mechanism for DNA elimination. We revealed the abundance and variations of this motif in many wild isolates of O. tipulae from around the world. In this proposal, we will (1) study the functions of DNA elimination in O. tipulae by characterizing the fail-to-eliminate phenotypes from CRISPR edited SFE mutants. We will use RNA-seq, ChIP-seq and small RNA sequencing to determine the changes of RNA expression and silencing mechanisms in these mutants. We will also (2) study the molecular mechanisms of O. tipulae DNA elimination by investigating the sequence and genomic position required for SFEs using CRISPR, as well as proteins that interact with SFEs using in vitro biochemistry, bioinformatic predictions, and genetics. We will further (3) study the variations of DNA elimination by building telomere-to-telomere genomes for divergent strains of O. tipulae, identifying SFEs, and carrying out comparative genomics. Overall, this proposal will use our established genetic model in the free-living nematode O. tipulae to examine the functions, mechanisms, and variations of DNA elimination. This work will reveal insights into the molecular details of DNA elimination in a metazoan.