Functional Genomics of Stress Defense in Yeast

描述(申请人提供)：所有生物体必须保护其内部系统免受细胞压力。无论应激是由外部毒素或突变和疾病引起的，细胞都必须敏感地监测应激信号，并做出适当的反应，以维持内部平衡。尽管压力防御很重要，但关于真核生物在压力环境中生存的机制仍有许多未知之处。功能基因组学已经发现了各种基因组中许多基因的功能，主要是通过在标准条件下描述基因功能来实现的。然而，很大一部分基因仍然没有特征，其中许多很可能与压力防御有关，因此没有通过传统研究发现。这项建议将使用高通量功能基因组学、基因组表达分析、计算生物学以及遗传学和生物化学技术来识别和表征酵母中参与逆境防御的基因。目的1将利用与应激防御相关的两种新的表型来发现与真核生物应激生存相关的新基因。第一种是一种被称为“获得性抗逆性”的现象，即暴露在小剂量一种压力下的细胞对另一种致死剂量的另一种压力产生抵抗力。第二种现象是，在温和的压力处理后，即使在温和的压力被移除之后，细胞仍保持着一种抗逆性的“记忆”，这种记忆持续了许多代。我们将在高通量选择中使用这些表型来鉴定在轻度应激处理后无法获得或保持对严重应激的抗性的酵母缺失突变体。已识别的基因，以及已知的获得性抗逆性调节因子，将被表征以确定它们在这些现象中的确切作用。细胞对压力的反应是多方面的。这种反应，包括基因组表达的重组，是由一个复杂的信号网络协调的，该网络对压力做出反应。目的2将阐明酵母中复杂的应激激活信号网络，它协调基因组表达对应激的反应。通过筛选酵母缺失库中不能在胁迫处理时诱导表达的突变体，将识别胁迫依赖基因的调节因子。确定的调节器和各种已知的网络组件将被组织到一个假定的信令网络中，使用多种计算方法。这个网络随后将在基因组、遗传和生化研究的基础上进行剖析和提炼。这些实验将有助于阐明酵母中复杂的应激激活信号网络，这是人类和其他生物体中此类网络的优秀模型，同时开发可能推动这一生物学领域的计算方法。由于这些反应中的许多在人类身上是保守的，这些结果将促进人类医学中的压力最小化和疾病预防。 项目相关性：酵母使用的许多应激防御机制在人类中是保守的，因此这项提议的结果将为理解并最终调节人类健康的应激抵抗提供坚实的基础。这些结果将有广泛的应用，从最大限度地减少化疗的衰弱副作用，到减少侵入性手术、心脏病发作和中风造成的创伤，再到预防癌症。此外，了解酵母如何感知和应对压力是人类细胞如何应对类似细胞压力的一个极好的模型。

DESCRIPTION (provided by applicant): All organisms must protect their internal system from cellular stress. Whether stress arises from external toxins or mutation and disease, cells must sensitively monitor stress signals and mount the appropriate responses to maintain internal homeostasis. Despite the importance of stress defense, much remains unknown about the mechanisms eukaryotes use to survive stressful situations. Functional genomics has uncovered functions for many genes in various genomes, largely by characterizing gene function under standard conditions. However, a substantial fraction of genes remains uncharacterized, and many of these are likely to be involved in stress defense and thus have not been uncovered through traditional studies. This proposal will use high-throughput functional genomics, genomic expression analysis, computational biology, and techniques in genetics and biochemistry to identify and characterize genes involved in stress defense in yeast. Aim 1 will exploit two new phenotypes related to stress defense to uncover novel genes involved in eukaryotic stress survival. The first is a phenomenon known as `acquired stress resistance', in which cells exposed to a small dose of one stress become resistant to an otherwise lethal dose of a different stress. The second is a phenomenon in which cells retain a `memory' of stress resistance that persists for many generations after mild-stress treatment, even after the mild stress has been removed. We will use these phenotypes in high-throughput selections to identify yeast deletion mutants that cannot acquire or retain resistance to severe stress after mild-stress treatment. Identified genes, as well as known regulators of acquired stress resistance, will be characterized to define their precise roles in these phenomena. Cells respond to stress with a multi-facetted response. This response, including reorganization of genomic expression, is coordinated by a complex signaling network that responds to stress. Aim 2 will elucidate the intricate stress-activated signaling network in yeast that orchestrates genomic expression responses to stress. Regulators of stress-dependent genes will be identified by screening the yeast-deletion library for mutants unable to induce expression upon stress treatments. Identified regulators and various known network components will be organized into a putative signaling network, using numerous computational approaches. This network will be subsequently dissected and refined based on genomic, genetic, and biochemical studies. These experiments will help to elucidate the complex stress-activated signaling network in yeast, which serves as an excellent model for such networks in humans and other organisms, while developing computational approaches that are likely to advance this area of biology. As many of these responses are conserved in humans, these results will foster stress minimization and disease prevention in human medicine. Project Relevance: Many stress-defense mechanisms used by yeast are conserved in humans, and therefore the results of this proposal will provide a strong foundation for understanding, and eventually modulating, stress resistance for human health. These results will have broad application, from minimizing debilitating side effects of chemotherapy, to reducing trauma inflicted by invasive surgery, heart attacks and strokes, to preventing cancer. Furthermore, understanding how yeast sense and respond to stress is an excellent model for how human cells respond to analogous cellular stresses.