The cross-regulation of host DNA replication and LTR Retrotransposons

宿主DNA复制和LTR反转录转座子的交叉调节

• 批准号: 10406993
• 负责人: MIGUEL ANGEL ZARATIEGUI BIURRUN
• 金额: $ 38.85万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406993
• 关键词: Affect; Aging; Base Sequence; Biochemistry; Chromatin; Chromosomal Rearrangement; Clustered Regularly Interspaced Short Palindromic Repeats; DNA Transposable Elements; DNA biosynthesis; Defense Mechanisms; Detection; Disease; Eukaryota; Evolution; Fission Yeast; Genetic; Genetic Materials; Genome; Genomics; Goals; Investigation; Lead; Malignant Neoplasms; Meiosis; Methods; Mutagenesis; Nature; Organism; Parasites; Process; RNA; RNA Interference; RNA Interference Pathway; Regulation; Repetitive Sequence; Research; Retrotransposon; Reverse Transcription; Site; Structure; Work; genetic analysis; genome integrity; homologous recombination; innovation; next generation; novel; pressure

Project summary The proposed work is part of our long-term goal to understand host-transposon interactions that influence eukaryotic genome function and evolution. Retrotransposons are a class of transposable element capable of amplifying their copy number via reverse-transcription of RNA intermediates and insertion in a new locus. Through this transposition activity, they can multiply to the point of making up the majority of genetic material in some genomes, exerting a broad influence in genome structure and regulation. The repetitive nature of TE can also affect genome integrity through homologous recombination between their dispersed copies that can cause chromosomal rearrangements. To counteract their deleterious effects, eukaryotes have evolved multiple genome defense mechanisms that suppress Retrotransposon activity. The most important are RNA interference (RNAi) pathways that are often specifically active in the germline, protecting the genetic material that will make up the next generation. We are investigating these host-Retrotransposon interactions using the LTR Retrotransposons Tf1 and Tf2, endemic to fission yeast. In previous work, we have revealed an intense cross-regulation between the Retrotransposon and the process of host DNA replication, discovering how Retrotransposons select new insertion sites, regulate chromatin silencing, and control homologous recombination. Several aspects of these phenomena are conserved in LTR retrotransposons present in the genomes of other organisms, even while the specific factors involved are not. This suggests that common evolutionary pressures lead to convergent evolution of host-Retrotransposon interactions. As a consequence, these conserved mechanisms may be important for metazoan germline stability. In the present application, we propose a comprehensive line of research combining genetics, biochemistry, and high throughput sequence-based genomics methods to (1) ascertain universal Retrotransposon insertion site selection mechanisms, (2) resolve the determinants of LTR Retrotransposon detection by RNAi, (3) determine the mechanism by which Retrotransposons guide their own homologous recombination, and (4) investigate the effect of Retrotransposon activity in meiosis progression. In pursuit of these goals, we will make use of novel fission yeast strains in which all Retrotransposon copies have been deleted through an innovative CRISPR mutagenesis method. This advance enables us to carry out previously unfeasible genetic analyses of these highly repetitive elements. The proposed studies will provide a novel and comprehensive understanding of host- Retrotransposon interactions, and their consequences on genome regulation and stability.

项目摘要 这项工作是我们理解宿主-转座子相互作用的长期目标的一部分 影响真核生物基因组功能和进化。 反转录转座子是一类能够扩增其拷贝数的转座因子 通过RNA中间体的逆转录和插入新的基因座。通过这个 转座活动，它们可以繁殖到组成大多数遗传物质的点， 一些基因组，在基因组结构和调控中发挥广泛的影响。重复性 TE也可以通过它们分散的基因组之间的同源重组来影响基因组的完整性。 会导致染色体重排为了抵消它们的有害影响， 真核生物已经进化出多种基因组防御机制， 活动最重要的是RNA干扰（RNAi）途径，它们通常具有特异性活性 在生殖细胞中，保护构成下一代的遗传物质。 我们正在使用LTR反转录转座子研究这些宿主-反转录转座子相互作用 Tf 1和Tf 2，裂殖酵母特有的。在以前的工作中，我们已经揭示了强烈的交叉调节 逆转录转座子和宿主DNA复制过程之间的关系， 反转录转座子选择新的插入位点，调节染色质沉默，并控制同源 重组这些现象的几个方面是保守的LTR反转录转座子存在于 其他生物的基因组，即使所涉及的特定因素并不存在。这表明 共同的进化压力导致宿主-反转录转座子相互作用的趋同进化。 因此，这些保守的机制可能是重要的后生动物种系稳定性。 在本申请中，我们提出了结合遗传学， 生物化学和高通量基于序列的基因组学方法，以（1）确定通用的 反转录转座子插入位点选择机制，（2）解析LTR的决定因素 通过RNAi检测反转录转座子，（3）确定反转录转座子引导的机制 它们自身的同源重组，以及（4）研究反转录转座子活性在 减数分裂进程在追求这些目标的过程中，我们将利用新的裂变酵母菌株，其中 通过创新的CRISPR诱变方法删除了所有反转录转座子拷贝。 这一进展使我们能够对这些高度重复的基因进行以前不可行的遗传分析。 元素这些研究将为宿主提供一个新的、全面的认识， 反转录转座子相互作用及其对基因组调控和稳定性的影响。