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.

项目概要/摘要 基因组完整性对于生命至关重要。为了维持基因组的稳定性付出了相当大的努力。然而 基因组也会不断发生变化，通常是随机的且规模较小，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，具有大量的 发育过程中消除的 DNA 量（占基因组的 0.5% 至 95%）。 DNA 消除是 高度选择性和可重复性，是包括单细胞在内的多种生物体生物学的一个组成部分 纤毛虫，跨动物门和一些植物的多种多细胞生物。广泛的系统发育 分布表明DNA消除是独立进化的并且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基因和重复基因 序列。这表明后生动物 DNA 消除的一个可能功能是永久沉默 某些种系序列可能对体细胞有害。尽管基因组学和细胞学取得了进展， 后生动物 DNA 消除的功能和机制研究有限，很大程度上是由于缺乏遗传 和功能工具。最近，我们建立并扩展了基因组观察，并建立了遗传和 自由生活的线虫 Oscheius Tipulae（杆状线虫科的成员）中 DNA 消除的功能模型 家族，其中包括秀丽隐杆线虫。我们发现 O. Tipulae 中的 DNA 消除发生在 8-16 年间。 细胞胚胎。我们鉴定并表征了一个保守序列（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。 DNA断裂发生 在主题内，然后进行末端切除和端粒愈合。额外的中断同时发生在 消除了 DNA，也许可以作为消除 DNA 的自动防故障机制。我们揭示了丰富性 以及来自世界各地的许多 O. Tipulae 野生分离株中该基序的变异。在本提案中，我们将 (1) 通过表征 O.tipulae 中无法消除的表型来研究 O.tipulae 中 DNA 消除的功能 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来了解 O. Tipulae DNA 消除机制 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。我们将进一步（3）通过构建端粒到端粒来研究 DNA 消除的变化 O. Tipulae 不同菌株的基因组，鉴定 SFE，并进行比较基因组学。全面的， 该提案将使用我们在自由生活的线虫 O. Tipulae 中建立的遗传模型来检查 DNA 消除的功能、机制和变化。这项工作将揭示对分子细节的见解 后生动物中 DNA 的消除。