Functional Genomics of Stress Defense in Yeast

描述（由申请人提供）：所有生物必须保护其内部系统免受细胞压力。无论压力是来自外部毒素还是突变和疾病，细胞都必须敏感地监测压力信号，并采取适当的反应来维持内部稳态。尽管应激防御很重要，但真核生物在应激环境中生存的机制仍不为人所知。功能基因组学揭示了不同基因组中许多基因的功能，主要是通过在标准条件下表征基因功能。然而，仍有相当一部分基因未被表征，其中许多可能与压力防御有关，因此尚未通过传统研究发现。本提案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鉴定和表征酵母中参与应激防御的基因。目的1将利用两种与应激防御相关的新表型来发现参与真核生物应激生存的新基因。第一种是一种被称为“获得性应激抵抗”的现象，在这种现象中，细胞暴露于小剂量的一种压力下，就会对另一种致命剂量的压力产生抵抗力。第二种现象是，细胞在轻度应激处理后，甚至在轻度应激被消除后，仍然保留了抗应激的“记忆”，这种“记忆”会持续许多代。我们将在高通量选择中使用这些表型来鉴定在轻度胁迫处理后不能获得或保持对严重胁迫的抗性的酵母菌缺失突变体。已确定的基因，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来协调的。目的2将阐明酵母中复杂的应激激活信号网络，该网络协调基因组表达对应激的反应。胁迫依赖性基因的调节因子将通过筛选酵母缺失文库来确定在胁迫处理下无法诱导表达的突变体。确定的调节器和各种已知的网络组件将被组织成一个假定的信号网络，使用多种计算方法。该网络随后将根据基因组、遗传和生化研究进行剖析和完善。这些实验将有助于阐明酵母中复杂的应激激活信号网络，为人类和其他生物的这种网络提供了一个很好的模型，同时开发出可能推进这一生物学领域的计算方法。由于许多这些反应在人类中是保守的，这些结果将促进人类医学中的压力最小化和疾病预防。

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.