Environmental and genetic regulation of copy number variation (CNV)

描述（由申请人提供）： 本申请涉及广泛的挑战领域（08）“基因组学”和特定的挑战主题（08-ES-106）“环境暴露在拷贝数变异（CNV）中的作用”。CNV事件是太小而不能被核型分析识别的缺失或重复。自发性CNV最近被认为是人群中散发性疾病的主要来源，可能最值得注意的是自闭症谱系障碍（ASD）。CNVs形成的机制尚不清楚，尽管环境因素和内在细胞因素都可能影响CNVs的发生率。在这个提议中，我们描述了使用酵母酿酒酵母作为模型，以找出环境污染物和各种突变如何控制CNVs的速率。我们将检查大的（> 1 kb缺失或重复）和小的（1- 1000 bp）CNV事件。为了研究环境因素和污染物对大CNV事件的影响（具体目标1），我们将使用一种报告基因，其中可以选择重复，并通过筛选检测缺失。我们将使用包含两个酵母基因SFA 1和CUP 1的盒，这两个基因分别赋予对甲醛和铜的基因剂量依赖性耐受性。使用将该盒整合到不同位置的酵母菌株，我们将测量在标准实验室条件下生长的野生型菌株中的盒扩增速率。然后，我们将确定响应于各种DNA损伤剂和常见农用化学品的扩增速率。我们还将优化甲酰胺-铜抗性测定的高通量版本，用于不同化学库的初步筛选，以鉴定与CNV刺激相关的未知环境污染物。为了确定扩增中涉及哪些机制（分散重复序列之间的同源重组、微同源介导的重组、非同源末端连接或非整倍性），我们将使用DNA微阵列和分离完整染色体DNA的凝胶系统（CHEF凝胶）表征具有CNV事件的菌株。在具体目标2中，我们将对与CNV形成相关的细胞机制进行研究，重点关注自发事件和低剂量电离辐射刺激的事件。我们将确定CNV的发生率以及在细胞周期的不同阶段暴露于辐射的细胞中的改变类型。此外，我们将检查减数分裂细胞中的自发CNV。还将检查一组DNA修复和复制受损的突变株，以表征CNV形成的遗传控制。在具体目标3中，我们将研究不同类型的CNV事件，即复制或删除小于1000 bp的事件。对于这种分析，我们将使用高通量DNA测序的亚培养未经处理的酵母菌株和菌株处理的低水平的辐射。分析应提供急需的洞察这类CNV的患病率，以及其形成的机制。在这个建议中，我们研究了环境压力和遗传背景的改变对酵母中从头拷贝数变异（DNA序列的缺失和重复）频率的影响。在过去的几年中，越来越清楚的是，拷贝数变异（CNV）与许多人类疾病有关。因此，增加CNV发生率的因素可能会增加这些疾病的发生率。

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (08), "Genomics", and the specific Challenge Topic (08-ES-106), "The role of environmental exposure in copy number variation (CNV)". CNV events are deletions or duplications that are too small to be recognized by karyotypic analysis. Spontaneous CNVs have been recently recognized as a major source for sporadic disease in human populations, perhaps most notably in Autism Spectrum Disorders (ASD). The mechanisms underlying the formation of CNVs are not known, although it is likely that both environmental and intrinsic cellular factors affect the rate of CNVs. In this proposal, we describe the use of the yeast Saccharomyces cerevisiae as a model to find out how environmental contaminants and various mutations control the rate of CNVs. We will examine both large (>1kb deletions or duplications) and small (1- 1000 bp) CNV events. To investigate the effect of environmental factors and contaminants on large CNV events (Specific Aim 1), we will use a reporter in which duplications can be selected and deletions can be detected by screening. We will use a cassette that contains two yeast genes SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. Using yeast strains in which this cassette is integrated into various positions, we will measure the rate of cassette amplifications in a wild-type strain growing under standard lab conditions. We will then determine the rate of amplifications in response to various DNA damaging agents, and common agrochemicals. We will also optimize a high-throughput version of the formaldehyde-copper resistance assay for use in preliminary screens of diverse chemical libraries to identify unknown environmental contaminants associated with CNV stimulation. To determine which mechanisms are involved in the amplifications (homologous recombination between dispersed repeats, microhomology- mediated recombination, non-homologous end joining or aneuploidy), we will characterize the strains with CNV events using DNA microarrays and a gel system that separates intact chromosomal DNAs (CHEF gels). In Specific Aim 2, we will conduct an investigation of cellular mechanisms linked to CNV formation, focusing on spontaneous events and those stimulated by low-dose ionizing radiation. We will determine the rates of CNV and the types of alterations in cells exposed to radiation at different stages of the cell cycle. In addition, we will examine spontaneous CNVs in meiotic cells. A set of mutant strains with compromised DNA repair and replication will also be examined to characterize the genetic control of CNV formation. In Specific Aim 3, we will investigate a different class of CNV events, those that duplicate or delete less than 1000 bp. For this analysis, we will use high-throughput DNA sequencing of sub-cultured untreated yeast strains and of strains treated with low levels of radiation. The analysis should provide much needed insight into the prevalence of this class of CNV, as well as into the mechanism of its formation. In this proposal, we examine the effects of environmental stresses and alterations of the genetic background on the frequency of de novo copy number variation (deletions and duplications of DNA sequences) in yeast. During the past few years, it has become increasingly clear that copy number variation (CNV) is associated with many human diseases. Thus, factors that elevate the rate of CNV are likely to elevate the frequency of those diseases.