Investigating how the conserved ZNFX-1 protein regulates epigenetic inheritance and germline immortality in Caenorhabditis elegans

项目摘要 生殖细胞永生对于生育和物种生存是必不可少的。为了无限增殖，生殖细胞依赖于 关于维持基因组完整性和表观遗传程序的机制。在动物中，相互作用的小RNA 与PIWI家族的ArgAerte蛋白-称为piRNAs-一起，充当转录组和 胚系中表观基因组的完整性，通过识别和沉默转座子元件以及通过调节 生殖系基因表达。在许多动物中，piRNAs对生殖系健康是必不可少的。缺陷型piRNA 生物发生或功能激活转座子的表达和移动，增加DNA损伤，破坏生殖细胞 发展，并降低生育率。 在秀丽线虫中，具有相反活动的小RNA途径合作维持生殖细胞 生存和生育。最近的研究发现，ZNFX-1是蠕虫表观遗传的调节因子。ZNFX- 1是一个高度保守的UPF1类解旋酶，具有C-末端的NF-X1型锌指结构域。ZNFX-1突变体 激活表观遗传沉默的记者，在某些情况下，他们让正常活跃的记者保持沉默，这表明 ZNFX-1平衡了相反的表观遗传程序。小RNA的靶向模式在ZNFX-1中重新分布。 1个突变体，表明ZNFX-1决定了沉默和反沉默所需的小RNA的来源 小路。初步数据还显示，znfx-1零突变导致致死性生殖系表型升高。 温度，这表明ZNFX-1保持平衡的表观遗传信号对生殖系永生至关重要。 这项建议试图使用遗传、计算和生物化学方法来检验假设 ZNFX-1被招募来作为小RNA生物发生的靶点和识别位点。目标1中的研究将决定如何 ZNFX-1通过识别它如何以及在哪里与靶转录本结合来调节小RNA的生物发生，以及如果它 沿着RNA展开或移动。AIM 2的研究将确定ZNFX-1如何通过以下方式调节生殖系不朽 鉴定ZNFX-1的功能结构域、具有同源结构域的潜在冗余蛋白和小分子 生殖系不朽的RNA和转录组特征。这些研究将揭示动物如何传播可遗传的 表观遗传信息以及表观遗传途径如何维持生殖细胞永生。此外，建议的 研究将提供遗传学和表观遗传学、定量生物化学和计算方面的培训 方法，并准备研究员在计算和系统生物学博士后和未来的职业生涯为 一名独立调查员。

Project Summary Germ cell immortality is essential for fertility and for species survival. To proliferate indefinitely, germ cells depend on mechanisms that maintain genome integrity and epigenetic programs. In animals, small RNAs that interact with Argonaute proteins of the PIWI family—called piRNAs—serve as a vanguard of transcriptome and epigenome integrity in the germline, by identifying and silencing transposable elements and by regulating germline gene expression. In many animals, piRNAs are essential for germline health. Defective piRNA biogenesis or function activates transposon expression and mobility, increases DNA damage, disrupts germ cell development, and reduces fertility. In Caenorhabditis elegans, small RNA pathways with opposing activities collaborate to maintain germ cell survival and fertility. Recent studies identified ZNFX-1 as a regulator of epigenetic inheritance in worms. ZNFX- 1 is a highly conserved UPF1-like helicase with C-terminal NF-X1-type zinc finger domains. znfx-1 mutants activate epigenetically silenced reporters, and in some cases they silence normally active reporters, indicating that ZNFX-1 balances opposing epigenetic programs. The targeting pattern of small RNAs redistributes in znfx- 1 mutants, suggesting that ZNFX-1 determines the origin of small RNAs required for silencing and anti-silencing pathways. Preliminary data also show that znfx-1 null mutations cause a mortal germline phenotype at elevated temperatures, suggesting that ZNFX-1 maintains balanced epigenetic signals essential for germline immortality. This proposal seeks to use genetic, computational, and biochemical approaches to test the hypothesis that ZNFX-1 is recruited to targets and identifies sites of small RNA biogenesis. Studies in Aim 1 will determine how ZNFX-1 regulates small RNA biogenesis by identifying how and where it binds to target transcripts, and if it unwinds or moves along RNA. Studies in Aim 2 will determine how ZNFX-1 regulates germline immortality by identifying functional domains of ZNFX-1, potentially redundant proteins with homologous domains, and small RNA and transcriptome features of germline immortality. These studies will reveal how animals transmit heritable epigenetic information and how epigenetic pathways maintain germ cell immortality. In addition, the proposed research will provide training in genetics and epigenetics, quantitative biochemistry, and computational approaches, and prepare the fellow for a postdoc in computational and systems biology and a future career as an independent investigator.