Understanding how aneuploidy disrupts quiescence in the model eukaryote Saccharomyces cerevisiae

摘要 非整倍体，即细胞携带错误数目的染色体的状态，是人类面临的一个主要问题 健康。非整倍体在哺乳动物发育过程中是有毒的，也是导致妊娠丢失的主要原因。降下来 由21三体引起的综合征(DS)是为数不多的存活的非整倍体综合征之一，但受影响的个体 包括过早衰老在内的终生问题。尽管进行了密集的研究，非整倍体毒性的原因是 仍然不完全被理解，这给理解DS带来了挑战。相比之下，非整倍体是非常 在人类癌症中很常见，大多数肿瘤都能耐受，甚至可能受益于额外的染色体。它是 不清楚癌细胞是如何克服非整倍体的压力的，因为我们还不完全了解 非整倍体首先影响细胞。这项提议将利用一个极其强大和独特的系统来 研究非整倍体在一个重要的模式系统--发芽酵母野生株中的后果 酿酒酵母。酵母是解剖细胞生物学的强大模型，因为许多 机制和防御策略在人类身上是保守的。我们最近有了一个令人兴奋的发现 健康酵母菌株的染色体复制扰乱了营养反应和静止，a 保守的细胞程序对生长控制和细胞维持和更新很重要。多个菌株 遗传背景和携带不同的染色体扩增显示出共同的表型，包括 营养耗竭、代谢异常、静息诱导的缺陷引起的不完全细胞周期停滞 沉默，并最终缩短了按时间顺序计算的寿命。这是值得注意的，因为类似的缺陷 在DS和许多癌症中都可以看到静止的标志--如果静止的中断是保守的 对于非整倍体的反应，它可能会对未来的研究产生变革性的影响。这笔赠款将阐明如何 在一个重要的真核模型系统中，非整倍体打破了静止状态。目标1将使用动态 转录学和单细胞显微镜来表征缺陷的时间顺序，测试几个初始 假设，并牵连到上游监管机构。它还将区分共同的和特定于染色体的 效果。AIM 2将使用条形码质粒过度表达文库来识别互补的基因 非整倍体缺陷在进入静止状态的过程中。集成目标1和目标2的结果将定义时间 非整倍体酵母菌株中存在缺陷并参与静止期的基因和过程图谱。它还将指出 对于直接由染色体复制引起的上游缺陷(S)，其进一步的研究将扩大我们的 对非整倍体目标3的理解将使用基因组、蛋白质组、单细胞和单分子分析来 定义和表征“SSD1Q颗粒”，一种含有RNA结合蛋白的相分离颗粒 SSD1，它是健康酵母中非整倍体耐受的基础。因为有很多人 酵母中的机制在包括人类在内的高等生物中是保守的，这个项目将扩展我们的基本 了解非整倍体，并对多种人类疾病产生深远影响。

ABSTRACT Aneuploidy, the state in which cells carry an incorrect number of chromosomes, is a major problem for human health. Aneuploidy is toxic during mammalian development and a leading cause of pregnancy loss. Down syndrome (DS) due to trisomy 21 is one of the few viable aneuploid syndromes, but affected individuals have life-long problems including premature aging. Despite intense study, the reasons for aneuploidy toxicity are still incompletely understood, presenting challenges for understanding DS. In contrast, aneuploidy is very common in human cancers, where most tumors tolerate and may even benefit from extra chromosomes. It is unclear how cancer cells overcome the stress of aneuploidy, because we don’t fully understand how aneuploidy affects cells in the first place. This proposal will utilize an extremely powerful and unique system to study the consequence of aneuploidy in an important model system, wild strains of budding yeast Saccharomyces cerevisiae. Yeast is a powerful model for dissecting cellular biology, because many of the mechanisms and defense strategies are conserved in humans. We recently made an exciting discovery that chromosome duplication in healthy yeast strains disrupts nutrient responses and quiescence, a conserved cellular program important for growth control and cell maintenance and renewal. Strains of multiple genetic background and carrying different chromosome amplifications display shared phenotypes, including incomplete cell-cycle arrest upon nutrient depletion, metabolic aberrations, defects in quiescence-induced silencing, and ultimately reduced chronological life span. This is remarkable, because defects in similar markers of quiescence are seen in both DS and many cancers – if disruption of quiescence is a conserved response to aneuploidy, it could have transformative impacts for future studies. This grant will elucidate how aneuploidy disrupts quiescence in an important eukaryotic model system. Aim 1 will use dynamic transcriptomics and single-cell microscopy to characterize the temporal order of defects, test several initial hypotheses, and implicate upstream regulators. It will also distinguish common versus chromosome-specific effects. Aim 2 will use a barcoded plasmid over-expression library to identify genes that complement aneuploid defects along the progression to quiescence. Integrating Aim 1 and 2 results will define a temporal map of genes and processes defective in aneuploid yeast strains and involved in quiescence. It will also point to the upstream defect(s) directly caused by chromosome duplication, whose further study will expand our understanding of aneuplodiy Aim 3 will use genomic, proteomic, single-cell and single-molecule analysis to define and characterize the “Ssd1 Q granule”, a phase separated granule containing the RNA-binding protein Ssd1, which we previously showed is fundamental for aneuploidy tolerance in healthy yeast. Since many mechanisms in yeast are conserved in higher organisms including humans, this project will expand our basic understand of aneuploidy and have far-reaching impact relevant for multiple human diseases.