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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要 Piwi 相互作用 RNA (piRNA) 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来遗传元件（例如转座子）引起的后果在秀丽隐杆线虫中，>10,000。序列多样化的 piRNA 基因聚集在基因组中两个不同的兆碱基级区域。尽管如此，聚类在线虫物种中都是保守的，这意味着它对于 piRNA 的表达很重要。 piRNA 基因聚集在不同的基因组区域内，由 RNA 聚合酶单独转录 II (RNA pol II) 和由此产生的短 RNA 建议在 RNA pol II 处于“暂停”状态时产生该提案的目标是了解 10,000 多种 piRNA 是如何协调上调的。我们的实验室和其他实验室以种系特异性的方式鉴定了这些大的基因组结构域的转录。 SNPC-4 和 PRDE-1 因子，它们形成一个复合物，在 piRNA 基因簇中传播，特别是然而，SNPC-4/PRDE-1 的协调机制。 piRNA 基因表达尚不清楚，我认为 SNPC-4/PRDE-1 传播介导 piRNA。最近，我们的研究通过影响染色质组织和/或控制转录活性来实现生物发生。实验室开发了一种可靠的技术来大量分离胚核 (IGN)，以进行大规模基因组分析，我将用它来以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制迄今为止，我已从其中分离出胚核。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，以及通过将 IGN 技术与各种技术相结合，观察到抑制性组蛋白修饰的整体变化。在基因组方法中，我的目的是研究 SNPC-4/PRDE-1 是否通过以下方式协调 piRNA 表达：影响染色质组织（目标 1）和转录事件（目标 2）。在目标 1 中，我将进行研究。 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影响 SNPC-4/PRDE-1 结合在目标 2 中，我将研究 SNPC-4/PRDE-1 是否存在。有助于 RNA Pol II 募集以及 SNPC-4/PRDE-1 是否与控制暂停状态的因素相互作用此外，我将确定 RNA Pol II 暂停状态是否影响 SNPC-4/PRDE-1 的结合。 piRNA 基因簇的完成将增进我们对 piRNA 生物发生的理解。破译 piRNA 染色质组织和转录机制的控制机制最终，这项工作可能对种系维持和功能至关重要。与理解许多复杂基因调控位点的调控机制相关不同物种的基因组不同。