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.

项目摘要/摘要 PIWI相互作用RNA（PIRNA）途径是一种配置的小RNA途径，可保护生殖细胞免受 由活跃的外国遗传因素（例如转座子）产生的后果。在秀丽隐杆线虫中，> 10,000 基因组中两个不同的巨型尺度区域中的序列多样性piRNA基因聚类。 pirna 聚集在线虫物种之间是保守的，这意味着它对piRNA表达很重要。尽管 被聚集在不同的基因组区域中，piRNA基因被RNA聚合酶单独转录 II（RNA POL II）和所得的短RNA被建议在RNA POL II处于“暂停”中时产生 状态。该提案的目的是了解如何协调10,000多名PIRNA 这些大型基因组领域以种系特异性方式。我们的实验室和其他人确定了转录 因子SNPC-4和PRDE-1，它们形成了一种复合物，该复合物在PIRNA基因簇上散布专门在 促进皮纳生产的种系。但是，SNPC-4/PRDE-1坐标的机制 piRNA基因表达未知。我假设SNPC-4/PRDE-1扩散培养基Pirna 生物发生通过影响染色质组织和/或控制转录活性。最近，我们的 实验室开发了一种可靠的技术，以大规模基因组测定的量分离出种系核（IGN）， 我将用它来高分辨率和特异性定义染色质的种系特异性模式 PIRNA基因簇的组织和转录机械。迄今为止，我已经将菌毛与 WildType和Prde-1突变体，并使用芯片序列研究了三个候选组蛋白修饰，并研究了 观察到反射性组蛋白修饰的全球变化。通过将IGN技术与多样 在基因组方法中，我旨在研究SNPC-4/PRDE-1是否通过 影响染色质组织（AIM 1）和转录事件（AIM 2）。在AIM 1中，我将调查 SNPC-4/PRDE-1是否影响piRNA基因簇的染色质可及性以及局部 染色质环境会影响SNPC-4/PRDE-1结合。在AIM 2中，我将调查SNPC-4/PRDE-1是否 RNA POL II招聘的辅助以及SNPC-4/PRDE-1是否与控制暂停状态的因素相互作用 RNA pol II。此外，我将确定RNA POL II是否暂停状态会影响SNPC-4/PRDE-1结合 pirna基因簇。这些目标的完成将使我们通过 解密控制PIRNA的染色质组织和转录机械的机制 基因簇，这对于生殖线维持和功能至关重要。最终，这项工作可能是 与理解许多在许多人中调节复杂基因调节局部的机制有关 跨物种的不同基因组。