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异的功能解释 在人类基因组中发现。这一挑战由于这些变异导致复杂的性状， 疾病通过相互作用和重复DNA元件的遗传变异。评估表型 所有基因相互作用的结果都是一个不可能的数字游戏。为了优先考虑某些变体 组合，我将使用具有强大遗传学的模式生物，即酵母S。cerevisiae和C. elegans，识别和表征对复杂表型有很大影响的遗传相互作用。我提议三个 利用我们以前的研究成果的项目。这些项目是统一的，他们的重点是遗传相互作用（即。 上位性），尽管它们涉及不同类型的变体组合和不同的机制。第一个项目聚焦 rDNA是一种高度可变的重复DNA元件。rDNA拷贝数的变化影响基因表达， 复制、基因组稳定性和线粒体丰度。像其他重复基因座一样，rDNA倾向于相互作用， 由于其突变率高，与其他变体发生上位性关系。利用新开发的C.线虫作图种群 和机器人使能的表型，我们的初步数据表明，rDNA拷贝数的变化影响寿命， 上位性的适应性目前正在对抗应激能力和生育能力等健康寿命特征进行高通量分析。 我们将继续对与rDNA上位性有关的最重要的基因组位点进行精细定位， 同一性，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， 询问rDNA拷贝数变异影响复制，基因组稳定性和线粒体的途径 丰饶。为了能够精确地高通量测量模式生物和人类中的rDNA拷贝数， 我们将优化一种有前途的FISH技术。第二个项目依赖于详细的基因型-表型图， 酵母交配途径中的基因。选择具有小和中等效应的单核苷酸变体， 我们将联合收割机的变异在两个基因和测试的交配效率的组合，同时也干扰强 遗传修饰和施加共同的压力。为此，我们开发了一种测序策略， 同时表型化成对基因之间的数万个单核苷酸变体组合。第三 项目将应用我们最近开发的显性负性多肽技术，在基因组规模上进行鉴定 蛋白质相互作用表面及其动力学。在酵母中，我们将探索遗传相互作用在多大程度上反映了直接的 蛋白质相互作用我们将询问蛋白质相互作用表面如何容易（或不容易）被突变扰动， 进化的分歧，或药物或压力，扰乱蛋白质折叠。这三个项目的成果将共同 对上位性、人类遗传学的可检验假设和新的检验技术进行广泛而深入的评估 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.