Deciphering the germline-specific mechanisms regulating piRNA gene expression from large genomic domains

破译大基因组区域调节 piRNA 基因表达的种系特异性机制

• 批准号: 10551195
• 负责人: Nancy Sanchez
• 金额: $ 3.26万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10551195
• 关键词: Abbreviations; Affect; Binding; Biochemical; Biogenesis; Bioinformatics; Biological Assay; Caenorhabditis elegans; Cell Nucleus; ChIP-seq; Chemicals; Chromatin; Chromosomes; Complex; DNA Binding; DNA Polymerase II; Data; Environment; Event; Exhibits; Fertility; Gene Cluster; Gene Expression; Genes; Genetic Transcription; Genome; Genomic Segment; Genomic approach; Genomics; Germ; Germ Cells; Germ Lines; Global Change; Goals; Health; Human; Individual; Infertility; Maintenance; Mediating; Nematoda; Nucleosomes; Pathway interactions; Pattern; Phenotype; Positioning Attribute; Production; Proteins; Prothrombin; RNA; RNA Interference; RNA Polymerase II; Regulation; Regulator Genes; Research; Resolution; SANT Domain; Sampling; Small Nuclear RNA; Small RNA; Specificity; TATA Box; Techniques; Testing; Tissues; Transcriptional Regulation; Transposase; Work; chromatin immunoprecipitation; genetic element; genome integrity; histone methyltransferase; histone modification; inhibitor; insight; mutant; novel; piRNA; preservation; protective pathway; protein complex; recruit; tool; transcription factor; transcriptome sequencing

Project Summary/Abstract The Piwi-interacting RNA (piRNA) pathway is a conserved small RNA pathway that protects germ cells from consequences arising from active foreign genetic elements such as transposons. In C. elegans, >10,000 sequence-diverse piRNA genes cluster in two distinct megabase-scale regions in the genome. piRNA clustering is conserved across nematode species, implying that it is important for piRNA expression. Despite being clustered within distinct genomic regions, piRNA genes are individually transcribed by RNA Polymerase II (RNA pol II) and the resulting short RNAs are suggested to be produced when RNA pol II is in its “paused” state. The goal of this proposal is to understand how over 10,000 piRNAs are coordinately upregulated from these large genomic domains in a germline-specific manner. Our lab and others identified the transcription factors SNPC-4 and PRDE-1, which form a complex that spreads across piRNA gene clusters specifically in the germ line to promote piRNA production. However, the mechanism by which SNPC-4/PRDE-1 coordinates piRNA gene expression is unknown. I hypothesize that SNPC-4/PRDE-1 spreading mediates piRNA biogenesis by affecting chromatin organization and/or controlling transcriptional activity. Recently, our lab developed a reliable technique to isolate germ nuclei (IGN) at quantities for large scale genomic assays, which I will use to define at high resolution and specificity the germline-specific patterns of chromatin organization and transcriptional machinery of piRNA gene clusters. To date, I have isolated germ nuclei from wildtype and prde-1 mutants and investigated three candidate histone modifications using ChIP-seq, and observed a global change in repressive histone modifications. By combining the IGN technique with a variety of genomic approaches, I aim to investigate whether SNPC-4/PRDE-1 coordinate piRNA expression by influencing chromatin organization (Aim 1), and transcriptional events (Aim 2). In Aim 1, I will investigate whether SNPC-4/PRDE-1 affect chromatin accessibility across the piRNA gene clusters and whether the local chromatin environment affects SNPC-4/PRDE-1 binding. In Aim 2, I will investigate whether SNPC-4/PRDE-1 aids in RNA Pol II recruitment and whether SNPC-4/PRDE-1 interact with factors that control the paused state of RNA Pol II. In addition, I will determine if the RNA Pol II paused state affects SNPC-4/PRDE-1 binding at piRNA gene clusters. Completion of these aims will advance our understanding of piRNA biogenesis by deciphering the mechanisms that control chromatin organization and transcriptional machinery of the piRNA gene clusters, which is essential for germline maintenance and function. Ultimately, this work is likely to be relevant to understanding the mechanisms that underlie regulation of complex gene regulatory loci in many different genomes across species.

项目总结/摘要 Piwi相互作用RNA（皮尔纳）途径是一种保守的小RNA途径，可保护生殖细胞免受 由活跃的外源遗传因子如转座子引起的后果。In C. elegans，> 10，000 序列多样性皮尔纳基因在基因组中的两个不同的兆碱基规模区域中聚集。皮尔纳 聚类在线虫物种中是保守的，这意味着它对皮尔纳表达是重要的。尽管 皮尔纳基因在不同的基因组区域内聚集，由RNA聚合酶单独转录， RNA聚合酶II（RNA聚合酶II）和由此产生的短RNA被认为是当RNA聚合酶II处于其“暂停”时产生的。 状态该提案的目标是了解超过10，000种piRNA如何从基因组中协同上调。 这些大的基因组结构域以种系特异性的方式。我们的实验室和其他人鉴定了 因子SNPC-4和PRDE-1，它们形成一种复合物，在皮尔纳基因簇中特异性传播， 生殖系以促进皮尔纳产生。然而，SNPC-4/PRDE-1协调的机制 皮尔纳基因表达是未知的。我假设SNPC-4/PRDE-1扩散介导皮尔纳 通过影响染色质组织和/或控制转录活性来实现生物发生。最近我们 实验室开发了一种可靠的技术来大量分离生殖核（IGN）用于大规模基因组测定， 我将用它来定义高分辨率和特异性的生殖细胞特异性染色质模式 皮尔纳基因簇的组织和转录机制。到目前为止，我已经分离出生殖细胞核， 野生型和prde-1突变体，并使用ChIP-seq研究了三种候选组蛋白修饰， 观察到抑制性组蛋白修饰的全球变化。通过将IGN技术与各种 在基因组方法中，我的目的是研究SNPC-4/PRDE-1是否协调皮尔纳表达， 影响染色质组织（Aim 1）和转录事件（Aim 2）。在目标1中，我将研究 SNPC-4/PRDE-1是否影响皮尔纳基因簇的染色质可及性，以及 染色质环境影响SNPC-4/PRDE-1结合。在目标2中，我将研究SNPC-4/PRDE-1是否 帮助RNA Pol II募集以及SNPC-4/PRDE-1是否与控制暂停状态的因子相互作用 RNA Pol II。此外，我将确定RNA Pol II暂停状态是否影响SNPC-4/PRDE-1结合， 皮尔纳基因簇。这些目标的完成将通过以下方式推进我们对皮尔纳生物发生的理解： 破译控制染色质组织和皮尔纳转录机制的机制 基因簇，这是至关重要的生殖细胞的维护和功能。最终，这项工作很可能是 相关的理解机制，在许多复杂的基因调控位点的调节， 不同物种的基因组。