Identifying targets of the Piwi/piRNA pathway in stem cells

识别干细胞中 Piwi/piRNA 途径的靶标

• 批准号: 8774562
• 负责人: Celina Juliano
• 金额: $ 11.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8774562
• 关键词: Address; Affect; Aging; Aging-Related Process; Animal Model; Animals; Biological Models; Caenorhabditis elegans; Caloric Restriction; Cell Aging; Cell Count; Cell Cycle; Cell Lineage; Cell Proliferation; Cell division; Cell physiology; Cells; Cnidaria; Code; Collaborations; Complex; DNA; Data; Drosophila genus; Epigenetic Process; Epithelial; Family; Gene Expression; Gene Expression Profile; Gene Family; Gene Targeting; Germ Lines; Goals; Health; Homeostasis; Hydra Polyps; Insulin; Light; Limb structure; Longevity; Maintenance; Maps; Mediating; Messenger RNA; Methods; Modeling; Molecular; Mus; Nuclear; Nutritional status; Organism; Pathway interactions; Phenotype; Phylogeny; Population; Proteins; RNA; RNA Interference; Regulation; Regulatory Pathway; Relative (related person); Reproduction; Role; Site; Small RNA; Somatic Cell; Stem cells; Testing; Time; Tissues; Transcript; Transgenic Organisms; Translating; Vertebrates; adult stem cell; base; cell age; cell type; dietary restriction; feeding; germline stem cells; human stem cells; in vivo; insight; interstitial; member; novel; piRNA; protein expression; repaired; response; senescence; stem cell population; transcription factor

DESCRIPTION (provided by applicant): Adult stem cells are required to maintain tissue homeostasis and decrease in stem cell function leads to the decline in health associated with aging. The causes of stem cell aging are poorly understood, and exploring basic mechanisms of stem cell regulation is vital to address this issue. The simple metazoan Hydra vulgaris is apparently "immortal", while other members of the genus undergo reproduction-induced senescence. The ability to compare the molecular mechanisms underlying the regulation of stem cells from two closely related animals with starkly different aging strategies is a unique and attractive feature of Hydra for aging studies; other well-established model organisms do not have seemingly "immortal" relatives. Furthermore, Hydra stem cells have attractive features for experimentation including: 1) Stem cell populations are well-characterized, 2) Due to recent advances in transgenic methods, the stem cells can be easily collected in large numbers and can be tracked and manipulated in vivo, and 3) Hydra stem cells share a gene expression profile with stem cells found in more complex animals. This includes the Piwi proteins, which are required for germline and stem cell function in animals. Our preliminary data suggest that Hydra Piwi proteins operate in a stem cell-specific pathway for maintaining tissue homeostasis and epithelial integrity in response to nutritional status. Because nutritional status is a conserved regulator of lifespan, we hypothesize that the Piwi pathway is a key player in the aging pathway of stem cells and we aim to understand how it functions in this context. Piwi proteins associate with a distinct class of small RNAs, piRNAs, and in Drosophila and mouse these Piwi/piRNA complexes modulate gene expression at the epigenetic and post-transcriptional levels. Hydra vulgaris stem cells have two strictly cytoplasmic Piwi proteins, allowing us to focus on identifyin post-transcriptional targets of the pathway. We are using a transgenic approaches to knockdown Piwi protein expression and test the function of the Piwi/piRNA pathway in each Hydra stem cell type. To identify targets of the pathway, we are: 1) Mapping piRNAs to a Hydra transcriptome to identify putative targets, 2) Testing for increased transcript levels of putative targets in piwi knockdown animals, and 3) Testing for a physical interaction between the putative transcript and Piwi/piRNA complexes. Finally, we are testing the hypothesis that the Piwi/piRNA pathway responds to nutritional status through the insulin pathway to regulate stem cell proliferation and tissue homeostasis. We aim to understand both the upstream regulators of the pathway and the downstream targets. Ultimately, we hope to gain significant insights into the regulation of aging in stem cells that will translate to more complex organisms. My long-term goal is to establish Hydra as an important new model for dissecting the regulatory control of stem cell function and aging. Once I have established my own lab I will initiate collaborations in order to test the conservation of the Piwi/piRNA pathway in stem cell regulation in vertebrates.

描述（由申请人提供）：需要成年干细胞来维持组织稳态和干细胞功能的降低会导致与衰老有关的健康下降。干细胞衰老的原因知之甚少，探索干细胞调节的基本机制对于解决此问题至关重要。简单的后代九头蛇显然是“不朽的”，而该属的其他成员经历了繁殖引起的衰老。比较从两种与截然不同的衰老策略的密切相关动物调节干细胞的分子机制的能力是一种独特的，并且 Hydra用于衰老研究的有吸引力的特征；其他良好的模型生物似乎没有“不朽的”亲戚。此外，Hydra干细胞具有吸引人的实验特征，包括：1）干细胞群体被良好特征，2）由于最近的转基因方法的最新进展，可以轻松地收集干细胞，并可以在体内跟踪和操纵3）Hydra干细胞在更复杂的动物中与干细胞共享基因表达。这包括PIWI蛋白，这些蛋白是动物种系和干细胞功能所必需的。我们的初步数据表明，Hydra Piwi蛋白在干细胞特异性的途径中运行，以维持组织稳态和上皮完整性，以响应营养状况。由于营养状况是寿命的保守调节因子，因此我们假设Piwi途径是干细胞衰老途径的关键参与者，我们旨在了解其在这种情况下的功能。 PIWI蛋白与一类不同的小RNA，PIRNA以及果蝇和小鼠相关联，这些PIWI/PIRNA复合物在表观遗传和转录后水平调节基因表达。九头蛇干细胞具有两个严格的细胞质PIWI蛋白，使我们能够专注于识别途径的转录后靶标。我们正在使用一种转基因方法来敲除PIWI蛋白表达并测试PIWI/PIRNA途径在每种Hydra干细胞类型中的功能。为了识别途径的靶标，我们是：1）将PIRNA映射到Hydra转录组中以识别推定的靶标，2）测试PIWI敲低动物中推定靶标的转录水平提高，以及3）测试假定的转录本和PIWI/PIRNA复合物之间的物理相互作用。最后，我们正在检验以下假设：PIWI/PIRNA途径通过调节干细胞增殖和组织稳态的胰岛素途径对营养状况做出反应。我们的目标是了解途径的上游调节器和下游目标。最终，我们希望对干细胞中衰老的调节有重大见解，这将转化为更复杂的生物。我的长期目标是建立Hydra作为解剖干细胞功能和衰老调节控制的重要新模​​型。一旦建立自己的实验室，我将启动协作，以测试脊椎动物干细胞调节中PIWI/PIRNA途径的保存。