Toward a mechanistic understanding of genetic interactions

后基因组时代的一个关键挑战是对大量单核苷酸变体的功能解释 发现于人类基因组中。这一挑战因以下事实而变得更加复杂：这些变异导致了复杂的性状和 疾病之间的相互作用和重复DNA元素的遗传变异。评估表型 所有基因相互作用的结果相当于一场不可能的数字游戏。为了确定某些变量的优先顺序 我将使用具有强大遗传学的模式生物，即酵母S.cerevisiae和蠕虫C. 识别和表征对复杂表型有较大影响的遗传交互作用。我建议有三个 利用我们之前研究成果的项目。这些项目因其对遗传相互作用的关注而团结在一起(即 上位性)，尽管它们涉及不同类型的变异组合和不同的机制。第一个项目的重点是 在rDNA上，一种高度可变的重复DNA元件。RDNA拷贝数的变化会影响基因的表达， 复制、基因组稳定性和线粒体丰度。像其他重复的基因座一样，rdna易于相互作用。 由于其高突变率，与其他变异体具有上位性。利用新开发的线虫作图种群 和机器人使能的表型，我们的初步数据显示，rDNA拷贝数变化影响寿命和 上位性健身。对抗应激和生育能力等健康跨度特征的高通量分析正在进行中。 我们将用rDNA对与上位性有关的最重要的基因组座位进行精细定位，因为他们的 身份，可能是dna复制或修复基因，可能指向rdna变异的分子机制。 影响表型。在酵母和蠕虫中，我们将使用遗传学和基因组学的整个工具箱直接 询问rDNA拷贝数变化影响复制、基因组稳定性和线粒体的途径 富足。为了能够对模型生物和人类中的rDNA拷贝数进行准确的高通量测量， 我们将优化一项前景看好的鱼类技术。第二个项目依赖于我们的详细的基因-表型图谱 为酵母交配途径中的基因建立的。选择小效应和中效应的单核苷酸变体， 我们将结合两个基因的变异，并测试组合的交配效率，同时也扰乱强 遗传修饰剂和施加共同压力。为此，我们开发了一种排序策略，使我们能够 基因对之间同时表现出数以万计的单核苷酸变异组合。第三 该项目将应用我们最近开发的一项显性负性多肽技术，以在基因组规模上进行鉴定 蛋白质相互作用面及其动力学。在酵母中，我们将探索基因相互作用在多大程度上直接反映了 蛋白质的相互作用。我们将询问蛋白质相互作用表面是否容易受到突变、 进化分化，或者是药物或压力扰乱了蛋白质的折叠。这三个项目的成果加在一起将 广泛而深入地评估上位性，人类遗传学的可测试假说和测试的新技术 他们。

A key challenge of the post-genomic era is the functional interpretation of the vast numbers of single nucleotide variants found in human genomes. This challenge is compounded by the fact that these variants contribute to complex traits and diseases by interacting with one another and with genetic variation in repetitive DNA elements. Assessing the phenotypic consequences of all genetic interactions amounts to an impossible numbers game. In order to prioritize certain variant combinations, I will use model organisms with powerful genetics, namely the yeast S. cerevisiae and the worm C. elegans, to identify and characterize genetic interactions with large impact on complex phenotypes. I propose three projects that capitalize on our previous studies. These projects are united by their focus on genetic interactions (i.e. epistasis), albeit they address different types of variant combinations and different mechanisms. The first project focuses on rDNA, a highly variable repetitive DNA element. Variation in rDNA copy number impacts gene expression, replication, genome stability, and mitochondrial abundance. Like other repetitive loci, rDNA is predisposed to interact epistatically with other variants because of its high mutation rate. Using newly developed C. elegans mapping populations and robotics-enabled phenotyping, our preliminary data show that rDNA copy number variation affects lifespan and fitness through epistasis. High-throughput analyses of healthspan traits such as stress resistance and fertility are ongoing. We will pursue fine-mapping of the most significant genomic loci implicated in epistasis with rDNA because their identity, possibly DNA replication or repair genes, may point to the molecular mechanism by which rDNA variation affects phenotype. In both yeast and worms, we will use the entire tool box of genetics and genomics to directly interrogate the pathways by which rDNA copy number variation affects replication, genome stability, and mitochondrial abundance. To enable accurate high-throughput measurements of rDNA copy number in model organisms and humans, we will optimize a promising FISH technology. The second project relies on the detailed genotype–phenotype maps we established for genes in the yeast mating pathway. Selecting single nucleotide variants of small and intermediate effects, we will combine variants in two genes and test the combinations for mating efficiency while also perturbing strong genetic modifiers and applying common stresses. To do so, we developed a sequencing strategy that allows us to simultaneously phenotype tens of thousands of single nucleotide variant combinations between pairs of genes. The third project will apply a technology of dominant negative polypeptides that we recently developed to identify at genome scale protein interaction surfaces and their dynamics. In yeast, we will explore to what extent genetic interactions reflect direct protein interactions. We will ask how easily (or not) protein interaction surfaces are perturbed by mutation, by evolutionary divergence, or by drugs or stress that perturb protein folding. Together, the results of these three projects will yield a broad and deep assessment of epistasis, testable hypotheses for human genetics and novel technologies for testing them.