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

项目摘要/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 由活跃的外来遗传因素引起的后果，如转座子。在线虫中，&gt；10,000 序列多样化的piRNA基因聚集在基因组中两个不同的百万碱基规模的区域。PIRNA 聚集在线虫物种中是保守的，这意味着它对piRNA的表达是重要的。尽管 PiRNA基因聚集在不同的基因组区域，由RNA聚合酶单独转录 Ii(Rna PolII)和由此产生的短rna被认为是在rna polII处于暂停状态时产生的。 州政府。该提案的目标是了解10,000多个piRNA是如何从 这些大的基因组区域以种系特有的方式存在。我们的实验室和其他人确认了转录 SNPC-4和Prde-1因子，它们形成了一个复合体，它跨piRNA基因簇传播，特别是在 该胚系可促进piRNA的生产。然而，SNPC-4/PRDE-1协调的机制 PiRNA基因的表达尚不清楚。我假设SNPC-4/Prde-1传播介导了piRNA 通过影响染色质组织和/或控制转录活性的生物发生。最近，我们的 Lab开发了一种可靠的技术来大量分离生殖核(IGN)，用于大规模的基因组分析， 我将用它来以高分辨率和特异度定义生殖系特有的染色质模式 PiRNA基因簇的组织和转录机制。到目前为止，我已经从 野生型和prde-1突变体，并使用芯片序列研究了三个候选组蛋白修饰，以及 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 在基因组学方法中，我的目标是研究SNPC-4/Prde-1是否通过 影响染色质组织(目标1)和转录事件(目标2)。在目标1中，我将调查 SNPC-4/Prde-1是否影响染色质跨piRNA基因簇的可及性以及局部 染色质环境影响SNPC-4/Prde-1结合。在目标2中，我将调查SNPC-4/PRDE-1 RNA Pol II募集中的辅助作用以及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暂停状态是否会影响SNPC-4/Prde-1在 PiRNA基因簇。完成这些目标将通过以下方式促进我们对piRNA生物发生的理解 破译控制染色质组织的机制和piRNA的转录机制 基因簇，这对生殖系的维持和功能是必不可少的。最终，这项工作很可能是 与了解许多复杂基因调控基因座的调控机制有关 不同物种的基因组不同。

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.