A genetic model for metazoan programmed DNA elimination

后生动物程序性 DNA 消除的遗传模型

• 批准号: 10719260
• 负责人: Jianbin Wang
• 金额: $ 29.91万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10719260
• 关键词: 3-Dimensional; Animals; Biochemistry; Bioinformatics; Biological; Biological Process; Biology; Caenorhabditis elegans; Cells; ChIP-seq; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Conserved Sequence; Cytology; DNA; DNA Transposable Elements; Data; Development; Disease; Ensure; Evolution; Excision; Family; Gene Expression Profile; Genes; Genetic; Genetic Models; Genetic Transcription; Genetic Variation; Genome; Genome Stability; Genomics; Histones; In Vitro; Life; Life Cycle Stages; Location; Longevity; Maintenance; Modeling; Molecular; Mutation; Nematoda; Organism; Phenotype; Phylogenetic Analysis; Plants; Population; Positioning Attribute; Process; Proteins; RNA Interference; Repetitive Sequence; Reproducibility; Reproduction; Role; Site; Small RNA; Somatic Cell; Untranslated RNA; Variant; Work; blastomere structure; chromosomal location; comparative genomics; embryo cell; flexibility; genetic manipulation; genome analysis; genome integrity; genome sequencing; genome-wide; genomic locus; healing; insight; member; mutant; telomere; tool; transcriptome sequencing

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.

项目摘要/摘要 基因组完整性对生命至关重要。为维持基因组的稳定而做出了巨大的努力。然而 基因组也经历恒定变化，通常是随机的，规模较小，提供了进化的机制 和适应。相反，编程的DNA消除是一种戏剧性的基因组变化形式， 在发育过程中消除的DNA量，范围为0.5％至95％。 DNA消除是 高度选择性和可重现，是不同生物的生物学不可或缺的一部分，包括单细胞 纤毛，跨动物门和某些植物的多种多细胞生物。广泛的系统发育 分布表明，消除DNA已独立发展，并且具有重要的生物学功能。一个 消除后生DNA的共同主题是消除种系表达基因和重复性 序列。这表明后生动物中消除DNA的可能功能是永久静音 某些种系序列可能对体细胞有害。尽管基因组学和细胞学进展，但 后生DNA消除的功能和机械研究受到限制，这主要是由于缺乏遗传 和功能工具。最近，我们建立并扩展了基因组观察，建立了遗传和 自由生存线虫Oscheius Tipulae中消除DNA的功能模型，br鼠的成员 家庭，包括秀丽隐杆线虫。我们表明O. tipulae中消除DNA是在8-16期间发生的 细胞胚胎。我们确定并表征了一个保守的序列（消除序列，SFE）基序 与DNA断裂位点相关，并证明了其在DNA消除中的直接作用。发生DNA断裂 在主题中，然后进行最终切除和端粒愈合。同时发生的其他休息 消除了DNA，也许是消除DNA的故障安全机制。我们透露了丰富的 和这种图案的变化在世界各地的许多O. tipulae的野生分离株中。在此提案中，我们将（1） 通过表征来自O. tipulae中DNA消除的功能，通过表征来自失败的脱位表型 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 这些突变体中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O. tipulae DNA消除的机制 使用CRISPR以及使用体外生物化学与SFE相互作用的蛋白质，生物信息学预测， 和遗传学。我们将进一步（3）通过构建端粒到凝结物来研究消除DNA的变化 O. tipulae菌株，鉴定SFE和进行比较基因组学的基因组。全面的， 该建议将在自由生命的线虫O中使用我们既定的遗传模型。 DNA消除的功能，机制和变化。这项工作将揭示有关分子细节的见解 在一代中消除DNA。