Novel Role for Piwi/piRNA pathway in developmental robustness

描述(由申请人提供)： 我们建议的长期目标是描述Piwi/piRNA途径通过何种机制使由严重的环境胁迫和固有的基因变异引起的表型变异正常化。通道化或发育稳健性提供了一个框架，在这种框架中，生物体在面对组合的基因变异和严重的环境压力时，拒绝表现出表型变异。最近的研究表明，Hsp90是一种分子伴侣，在管道形成过程中起着关键作用。尽管Hsp90的作用已为人所知，但几十年来，缺乏一个详细的分子机制来支持导管形成，引发了巨大的争论。该实验室最近证实了“管化”的存在，并发现了一条涉及Piwi、Hsp90和Hop的主要分子途径。利用致敏的眼睛发育试验，证明了Piwi果蝇和Aubergine果蝇的母体剂量减少，它们与称为piRNAs的新的生殖系丰富的非编码小RNA结合，诱导表型变异，这些表型变异可以固定在群体中，并稳定地在后代中遗传。研究还表明，Piwi与Hop和Hsp90直接相互作用，并以与Hsp90相同的途径抑制表型变异。此外，第一次证明了Piwi以Hsp90依赖的方式被磷酸化，从而为Hsp90如何调节Piwi在管化中的功能提供了洞察力。重要的是，这项研究还揭示了两个相互关联但又不同的Piwi/piRNA途径依赖的机制，负责抑制表型变异：表观遗传沉默固有的基因型变异和抑制转座子介导的突变。这项工作揭示了抑制表型变异的途径的框架。工作假说是Hsp90和Hop通过其磷酸化来调节Piwi的功能，然后在管化过程中调节Piwi的功能。通过进一步解开这一途径每一步的内部工作机制，我们的目标是阐明调节这一非常重要但知之甚少的细胞现象的机制。该提案的目的是-1)利用遗传筛选发现介导和/或调节Piwi功能的新成分，2)揭开Piwi介导的表观遗传调控的生化机制，最终了解Hsp90-Piwi/piRNA途径如何抵消六价铬(CrVI)等工业污染物诱导的环境压力。Piwi蛋白从原生动物到人类都存在，在干细胞自我更新和生殖系维持方面具有保守的功能。然而，它们在抑制表型变异中的作用是一个新的功能，需要进一步鉴定。以果蝇为模型系统，了解其在人类中的作用，并了解这一过程的功能障碍如何影响人类，解开Piwi在导管形成中的作用机制，将为发育和引发疾病提供必要的背景。 公共卫生相关性：环境中有毒污染物引起的压力对人类发展具有有害影响。这项提议采用跨学科的方法来发现新的成分和基本的细胞机制，这些新的成分和基本的细胞机制可以在未来用作抵消这种环境压力的工具。

DESCRIPTION (provided by applicant): 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. This lab recently validated the existence of 'canalization' and uncovered a major molecular pathway involving Piwi, Hsp90 and Hop. Using a sensitized eye development assay, a reduction in the maternal dosage of Drosophila Piwi and Aubergine was demonstrated, 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, it was demonstrated 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. This work revealed a framework of a pathway that suppresses phenotypic variation. The 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 (CrVI). 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 understanding its roles in humans and learning how the dysfunction of this process might affect human will provide the necessary context for development and cause diseases. PUBLIC HEALTH RELEVANCE: Stress induced by toxic pollutants in the environment has deleterious effect on human development. This proposal takes an inter-disciplinary approach to discover new components and fundamental cellular mechanisms that can be used as tools in the future to counteract such environmental stress.