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.

项目总结/摘要 基因组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。然而 基因组也经历着不断的变化，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，程序化DNA消除是基因组变化的一种戏剧性形式， 大量的DNA，范围从0.5%到95%的基因组，在发育过程中消除。DNA消除是 具有高度选择性和可重复性，是多种生物体（包括单细胞生物体）生物学的组成部分 纤毛虫是动物门和一些植物中的多种多细胞生物。广泛的系统发育 分布表明DNA消除是独立进化的，具有重要的生物学功能。一 后生动物DNA消除的一个共同主题是去除生殖系表达的基因和重复序列。 序列的这表明后生动物DNA消除的一个可能功能是永久沉默 某些对体细胞潜在有害的生殖系序列。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是有限的，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，并建立了一个遗传和 小杆线虫Oscheius tipulae的DNA消除功能模型 家族，包括秀丽隐杆线虫。结果表明，O. tipulae发生在8-16 细胞胚胎我们鉴定并表征了一个保守序列（Sequence For Elimination，SFE）基序 与DNA断裂位点相关，并证明其在DNA消除中的直接作用。DNA断裂 在基序内，随后是末端切除和端粒愈合。额外的中断同时发生在 消除了DNA，可能是DNA消除的自动防故障机制。我们揭示了 在O. tipulae来自世界各地。在本提案中，我们将（1） 研究O.通过表征未能消除的表型， CRISPR编辑的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 机制O.通过研究SFE所需的序列和基因组位置来消除tipulae DNA 使用CRISPR，以及使用体外生物化学，生物信息学预测， 和遗传学。我们将进一步（3）通过构建端粒-端粒模型来研究DNA消除的变化 基因组的分歧株O. tipulae，识别SFE，并进行比较基因组学。总的来说， 该建议将使用我们在自由生活的线虫O中建立的遗传模型。tipulae检查 功能，机制和DNA消除的变化。这项工作将揭示分子细节的见解 DNA消除的过程。