Novel Role for Piwi/piRNA pathway in developmental robustness

描述（由申请人提供）： 我们提案的长期目标是描述Piwi/皮尔纳途径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的机制。渠化或发育稳健性提供了一个框架，其中生物体在面对组合的基因型变异和严重的环境胁迫时抵抗表现出表型变异。最近的研究表明，热休克蛋白90，分子伴侣，发挥关键作用的渠道。尽管热休克蛋白90的作用是已知的，但缺乏一个详细的分子机制，管道化引起了几十年的争论。该实验室最近证实了“渠道化”的存在，并发现了一个涉及Piwi，Hsp 90和Hop的主要分子途径。使用致敏眼发育测定，证明了果蝇Piwi和Aubergine的母体剂量的减少，其结合到称为piRNA的新的种系富集的小的非编码RNA，诱导可以在群体中固定并在后代中稳定遗传的表型变异。该研究还表明，Piwi直接与Hop和Hsp 90相互作用，并且在抑制表型变异方面与Hsp 90的作用途径相同。此外，首次证明Piwi以Hsp 90依赖性方式磷酸化，从而提供了Hsp 90如何调节Piwi在管道化中的功能的见解。至关重要的是，这项研究还揭示了两种相关但不同的Piwi/皮尔纳途径依赖性机制，负责抑制表型变异：固有基因型变异的表观遗传沉默和转座子介导的诱变抑制。这项工作揭示了抑制表型变异的途径的框架。工作假设是Hsp 90和Hop通过其磷酸化调节Piwi功能，然后调节Piwi在通道化中的功能。通过进一步揭示这一途径每一步的内部工作，我们的目标是阐明介导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）使用遗传筛选发现介导和/或调节Piwi功能的新组分，2）揭示Piwi介导的管道化表观遗传调节的生化机制，最后3）了解Hsp 90-Piwi/皮尔纳途径如何抵消工业污染物如六价铬（CrVI）诱导的环境应激。Piwi蛋白存在于原生动物和人类中，在干细胞自我更新和生殖系维持中具有保守功能。然而，它们在抑制表型变异中的作用是一种新的功能，需要进一步表征。以果蝇为模型系统，揭示Piwi在管道化中的作用机制，了解Piwi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，将为发育和导致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有有害影响。该提案采取跨学科的方法来发现新的成分和基本的细胞机制，这些成分和机制可以在未来用作对抗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.