Novel Role for Piwi/piRNA pathway in developmental robustness

项目概要/摘要： 我们建议的长期目标是描述Piwi/皮尔纳通路的机制， 使严重环境胁迫和固有基因型变异诱导的表型变异正常化。 渠道化或发育的健壮性提供了一个框架，在这个框架中，生物体抵制展示 表型变异在面对组合的基因型变异和严重的环境压力。最近 研究表明，Hsp 90是一种分子伴侣，在管道化中起着关键作用。尽管已知 关于Hsp 90的作用，缺乏一个详细的分子机制引起了很大的争论 几十年我们的实验室最近证实了“管道化”的存在，并发现了一个主要的分子 Piwi、Hsp 90和Hop参与的信号通路。使用致敏眼发育试验，我们表明， 减少果蝇Piwi和Aubergine的母体剂量，它们与新的生殖系富集的 称为piRNA的小的非编码RNA诱导可以在群体中固定的表型变异， 在后代中稳定遗传。研究还表明，Piwi直接与啤酒花和Hsp 90相互作用， 在抑制表型变异方面与Hsp 90的作用途径相同。此外，我们证明了 首次发现Piwi以Hsp 90依赖的方式磷酸化，从而提供了关于Piwi如何磷酸化的见解。 热休克蛋白90可调节Piwi的神经通路功能。至关重要的是，这项研究还揭示了两个相关但不同的 Piwi/皮尔纳途径依赖性机制抑制表型变异：表观遗传学 固有基因型变异的沉默和转座子介导的诱变的抑制。我们的工作 揭示了抑制表型变异的途径框架。我们的假设是 Hsp 90和Hop通过磷酸化调节Piwi功能，磷酸化调节Piwi功能。 渠化通过进一步揭示这一途径每一步的内部运作，我们的目标是阐明 这是一个非常重要的机制，但人们对其了解甚少。的目标 建议是：1）发现新的组件，介导和/或调节Piwi功能的渠道，使用 遗传筛选，2）揭示Piwi介导的管道化表观遗传调控的生化机制 最后3）了解Hsp 90-Piwi/piRNA通路如何抵消由Hsp 90-Piwi/piRNA介导的环境应激。 工业污染物比如六价铬。Piwi蛋白从原生动物到人类， 在干细胞自我更新和生殖系维持中的保守功能。然而，它们在镇压 表型变异是一种新的功能，需要进一步的表征。解开机制， Piwi以果蝇为模型系统， 将提供必要的背景， 了解它在人类中的作用，并了解这一过程的功能障碍如何影响人类 发展并导致疾病。

Project Summary/Abstract: The long term goal of our proposal is to delineate the mechanisms by which Piwi/piRNA pathway normalizes phenotypic variation induced by severe environmental stress and inherent genotype variations. Canalization or developmental robustness provides a framework in which organisms resist displaying phenotypic variation in the face of combined genotype variations and severe environmental stress. Recent research has shown that Hsp90, a molecular chaperone, plays a critical role in canalization. Despite the known role for Hsp90, the lack of a detailed molecular mechanism underlying canalization has provoked great debate for decades. Our lab recently validated the existence of 'canalization' and uncovered a major molecular pathway involving Piwi, Hsp90 and Hop. Using a sensitized eye development assay, we showed that a reduction in the maternal dosage of Drosophila Piwi and Aubergine, which bind to novel germline-enriched small non-coding RNAs called piRNAs, induces phenotypic variations that can be fixed in a population and stably inherited in later generations. The study also showed that Piwi directly interacts with Hop and Hsp90 and functions in the same pathway as Hsp90 in suppressing phenotypic variations. Further, we demonstrate for the first time that Piwi becomes phosphorylated in an Hsp90 dependent manner, thus providing insights into how Hsp90 may modulate Piwi's function in canalization. Crucially, this study also revealed two related yet distinct Piwi/piRNA pathway-dependent mechanisms responsible for suppression of phenotypic variation: epigenetic silencing of inherent genotype variations and suppression of transposon-mediated mutagenesis. Our work revealed a framework of a pathway that suppresses phenotypic variation. Our working hypothesis is that Hsp90 and Hop modulate Piwi function via its phosphorylation which then modulates Piwi function in canalization. By further unraveling the inner workings of each step of this pathway, we aim to shed light on the mechanisms that mediate this very important, yet poorly understood cellular phenomenon. The aims of the proposal are to- 1) Discover new components that mediate and/or regulate Piwi function in canalization using genetic screens, 2) Unravel the biochemical mechanism of Piwi mediated epigenetic regulation in canalization and finally 3) Understand how Hsp90-Piwi/piRNA pathway counteracts environmental stress induced by industrial pollutants like hexavalent chromium. Piwi proteins are present from protozoans to humans with conserved functions in stem cell self-renewal and germ line maintenance. However their role in suppression of phenotypic variation is a novel function that requires further characterization. Unraveling the mechanism by which Piwi functions in canalization using Drosophila as a model system will provide the necessary context for understanding its roles in humans and learning how the dysfunction of this process might affect human development and cause diseases.