Causes and consequences of gene copy number change in adapting yeast populations

描述(申请人提供)：基因和染色体拷贝数的变化在从癌症到耐药的基因组进化的系统中被广泛观察到。尽管非整倍体对许多与人类健康相关的重要现象很重要，但在确定细胞如何适应这种极端的基因剂量变化方面所做的工作很少。我的实验室之前使用真核细胞酵母模型所做的工作发现，非整倍体可能对高生长速度的细胞有害，但可能有利于细胞在具有挑战性的环境中生长。在恒化器培养的营养限制生长中，基因组的特定片段被重复地发现扩增和缺失。在某些情况下，近端原因是显而易见的，例如营养转运蛋白基因的放大，但在其他情况下，例如影响大基因组片段的情况下，驱动力仍然不清楚。恒化器允许对选择和生长条件进行精确控制，重要的是，每个种群的完整冰冻历史，使该系统成为研究非整倍体在适应强而窄的选择中的作用的理想方法。我建议利用这个系统来实现以下具体目标：目标1：确定在实验进化的培养中存在的一套拷贝数变化。利用之前进行的一系列进化实验，我们将使用阵列比较基因组杂交和下一代测序来调查种群的拷贝数变化。目标2：确定基因组重排的适合性后果。在AIM 1中发现的高频重排将被重建，并在与匹配的祖先菌株的直接竞争分析中进行适合性测试。重排将在多个选择性条件下进行交叉测试，以质疑特异性。结果将与携带多个突变的进化菌株进行比较，以确定它们的健康益处在多大程度上归功于基因组重排。目的3.剖析各基因在非整倍体染色体上的适合度贡献。为了测试每个基因对非整倍体片段的贡献，我们将利用由每个酵母基因组成的菌株集合，这些基因的剂量范围从单个拷贝到扩增到多个拷贝。通过相互竞争这些菌株，并通过条形码测序测量它们的丰度，我们可以同时确定与每个基因相关的适合度效应。这些数据将被整合，并与来自AIM 2的菌株的适合度效果进行比较。我们还将与每个基因都返回到wt拷贝数的非整倍体菌株竞争，以确定哪些基因是改善适合度所必需的。这种方法的结合将是全基因组范围内首次尝试剖析与非整倍体相关的适合性变化的确切分子原因。

DESCRIPTION (provided by applicant): Changes in gene and chromosome copy number are widely observed in systems from cancer to drug resistance to genome evolution. Despite the importance of aneuploidy to many important phenomena related to human health, little work has been done to determine how cells adapt to such extreme changes in gene dosage. Prior work from my lab using the model eukaryote yeast has found that aneuploidy can be detrimental to cells at high growth rates, but can be beneficial to cells growing in challenging environments. In nutrient- limited growth in chemostat culture, specific segments of the genome are reproducibly found amplified and deleted. In some cases, the proximal cause is obvious, such as amplification of nutrient transporter genes, but in others, such as those affecting large genome segments, the driving force remains opaque. The chemostat allows precise control over selection and growth conditions, and, importantly, a complete frozen history of each population, making this system ideal to study the role of aneuploidy in adaptation to strong, narrow selection. I propose to leverage this system to accomplish the follow specific aims: Aim 1: Determine the suite of copy number changes present in experimentally evolved cultures. Using a series of previously performed evolution experiments, we will survey populations for copy number changes using array comparative genomic hybridization and next generation sequencing. Aim 2: Determine the fitness consequences of genome rearrangements. Rearrangements found at high frequency in Aim 1 will be reconstructed and tested for fitness in direct competition assays versus matched ancestral strains. Rearrangements will be cross-tested in multiple selective conditions to query specificity. Results will be compared to evolved strains carrying multiple mutations to determine how much of their fitness benefit is due to genome rearrangements. Aim 3. Dissect the fitness contributions of each gene on the aneuploid chromosomes. To test the contribution of each gene on an aneuploid segment, we will take advantage of strain collections consisting of every yeast gene present at dosages ranging from deletion of a single copy to amplification to many copies. By competing these strains against each other and measuring their abundance via barcode sequencing, we can determine the fitness effect associated with every gene simultaneously. These data will be integrated and compared to the fitness effects of strains from Aim 2. We will also compete aneuploid strains in which each gene is returned to wt copy number to determine which genes are necessary for fitness improvements. This combination of approaches will be the first genome-wide attempt to dissect the precise molecular causes of the fitness changes associated with aneuploidy.