Single-cell copy number variant detection reveals the dynamics and diversity of adaptation
Single-cell copy number variant detection reveals the dynamics and diversity of adaptation
复制标题
DOI：
10.1371/journal.pbio.3000069
复制
发表时间：
2018-12-01
期刊：
影响因子：
9.8
通讯作者：
Gresham, David
中科院分区：
文献类型：
--
作者：
Lauer, Stephanie;Avecilla, Grace;Gresham, David
拷贝数变异（CNVs）是遗传变异和进化潜力的一个普遍来源，但CNVs在进化群体中的动态和多样性仍不清楚。恒化器中的长期进化实验为研究CNV形成的分子过程以及它们产生、选择和维持的时间动力学提供了理想的系统。在这里，我们开发了一种荧光CNV报告检测从头基因扩增和删除在个别细胞。我们使用酿酒酵母中的CNV报告基因来研究在不同营养限制恒化器条件下编码一般氨基酸渗透酶的GAP 1基因座上的CNV形成。我们发现，在强选择下，GAP 1 CNVs在适应性进化的早期阶段重复产生和选择，从而产生可预测的动态。含CNV谱系的分子表征表明，CNV报告基因检测不同类型的CNV，包括非整倍体、非相互易位、串联重复和复杂的CNV。尽管GAP 1与促进染色体内重组的重复序列很接近，但断点分析表明，短的反向重复序列介导了至少50%的GAP 1 CNV的形成。在DUR 3基因座的断点处也发现反向重复序列，其中在尿素限制的恒化器中选择CNV。对28个CNV断裂点的分析表明，反向重复序列的长度通常为8个核苷酸，间隔40个碱基。这些CNV的特征与来源依赖性反向重复扩增（ODIRA）一致，表明基于复制的CNV形成机制可能是基因扩增的共同来源。我们将CNV报告基因与条形码谱系追踪相结合，发现10（2）- 10（4）个独立的含CNV谱系最初在群体内竞争，导致极端的克隆干扰。然而，只有少数（18-21）的CNV谱系曾经构成超过1%的CNV亚群，随着选择的进展，CNV谱系的多样性下降。我们的研究引入了一种新的方法来研究异质细胞群体中的CNVs，并在适应性进化的背景下深入了解它们的动态，多样性和形成机制。
Copy number variants (CNVs) are a pervasive source of genetic variation and evolutionary potential, but the dynamics and diversity of CNVs within evolving populations remain unclear. Long-term evolution experiments in chemostats provide an ideal system for studying the molecular processes underlying CNV formation and the temporal dynamics with which they are generated, selected, and maintained. Here, we developed a fluorescent CNV reporter to detect de novo gene amplifications and deletions in individual cells. We used the CNV reporter in Saccharomyces cerevisiae to study CNV formation at the GAP1 locus, which encodes the general amino acid permease, in different nutrient-limited chemostat conditions. We find that under strong selection, GAP1 CNVs are repeatedly generated and selected during the early stages of adaptive evolution, resulting in predictable dynamics. Molecular characterization of CNV-containing lineages shows that the CNV reporter detects different classes of CNVs, including aneuploidies, nonreciprocal translocations, tandem duplications, and complex CNVs. Despite GAP1's proximity to repeat sequences that facilitate intrachromosomal recombination, breakpoint analysis revealed that short inverted repeat sequences mediate formation of at least 50% of GAP1 CNVs. Inverted repeat sequences are also found at breakpoints at the DUR3 locus, where CNVs are selected in urea-limited chemostats. Analysis of 28 CNV breakpoints indicates that inverted repeats are typically 8 nucleotides in length and separated by 40 bases. The features of these CNVs are consistent with origin-dependent inverted-repeat amplification (ODIRA), suggesting that replication-based mechanisms of CNV formation may be a common source of gene amplification. We combined the CNV reporter with barcode lineage tracking and found that 10(2) - 10(4) independent CNV-containing lineages initially compete within populations, resulting in extreme clonal interference. However, only a small number (18-21) of CNV lineages ever constitute more than 1% of the CNV subpopulation, and as selection progresses, the diversity of CNV lineages declines. Our study introduces a novel means of studying CNVs in heterogeneous cell populations and provides insight into their dynamics, diversity, and formation mechanisms in the context of adaptive evolution.