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

描述（由申请人提供）：基因和染色体拷贝数的变化在从癌症到耐药性到基因组进化的系统中广泛观察到。尽管非整倍体对许多与人类健康相关的重要现象很重要，但几乎没有工作来确定细胞如何适应基因剂量的这种极端变化。我的实验室先前使用真核酵母模型的工作发现，非整倍性对高生长率的细胞是有害的，但对在具有挑战性的环境中生长的细胞是有益的。在恒化器培养物中的营养限制生长中，可再现地发现基因组的特定区段扩增和缺失。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扩增，但在其他情况下，例如影响大基因组片段的那些，驱动力仍然不透明。恒化器允许精确控制选择和生长条件，重要的是，每个种群的完整冷冻历史，使该系统成为研究非整倍性在适应强，窄选择中的作用的理想选择。我建议利用这个系统来实现以下具体目标：目标1：确定实验进化文化中存在的拷贝数变化套件。使用一系列先前进行的进化实验，我们将调查人口的拷贝数变化，使用阵列比较基因组杂交和下一代测序。目的2：确定基因组重排的适应性后果。在目标1中以高频率发现的重排将被重建，并在直接竞争测定中测试与匹配的祖先菌株的适应性。将在多个选择性条件下交叉测试重排，以查询特异性。结果将与携带多个突变的进化菌株进行比较，以确定它们的适应性益处有多少是由于基因组重排。目标3。剖析非整倍体染色体上每个基因的适应度贡献。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，所述酵母基因以从单个拷贝的缺失到扩增到多个拷贝的剂量存在。通过这些菌株相互竞争，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应性效应。这些数据将被整合并与目标2菌株的适应性效应进行比较。我们还将竞争非整倍体菌株，其中每个基因恢复到野生型拷贝数，以确定哪些基因是必要的健身改善。这种方法的结合将是第一次全基因组尝试解剖与非整倍体相关的适应性变化的精确分子原因。

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.