Bridge-seq, a new tool to analyze human genome segregation defects

项目摘要 染色体分离缺陷对基因组完整性有害，是癌症的标志。一 染色体分离问题的常见表型是染色体之间存在DAPI染色的DNA， 分裂后期分离染色体团。这些“后期桥”的特征很差，部分原因是 这是因为假设它们的内容是不变的或随机的。这个假设可能是错误的，因为 先前的研究已经鉴定了导致在着丝粒、端粒或端粒中特异性富集的桥的突变体。 核糖体DNA这些是使用荧光原位杂交（FISH）或荧光原位杂交（FISH）分析的候选序列。 免疫荧光分析与它们结合的蛋白质。到目前为止，一个公正的方法来确定 后期桥中所含的人类基因组序列的互补性和频率不可用。 在序列水平上了解染色体分离缺陷的主要影响将使连接 他们的大型测序项目表征基因组完整性问题的癌症和老化。为 例如，如果某些癌症相关突变偏向特定的非整倍性，其后果可能 根据受影响的基因进行细胞类型特异性。 确定桥的DNA含量的关键障碍是技术限制。在这里，我们提出一个 两种技术的创新组合开发Bridge-seq：激光捕获显微切割（LCM）， 分离后期桥DNA和高通量测序以鉴定基因组序列。我们将 开发Bridge-seq并测试其在影响基因组不同区域的多个系统中的可行性， 正常细胞、癌症细胞和衰老细胞的突变。在目标1中，我们将使用 允许稳健诱导富集rDNA的DAPI染色桥的条件。在目标2中， 评估使用Bridge-seq在一系列条件下研究后期桥内容的有效性， 不同的突变，我们将分析桥梁从突变显示影响其他地区的染色体， 包括端粒和着丝粒。在目标3中，我们将使用Bridge-seq对后期进行分析和比较 衰老和癌细胞之间的桥梁。我们公正的分析将为人类的本质提供新的见解 基因组序列易受分离缺陷的影响，并最终导致基因组不稳定。

PROJECT SUMMARY Defects in chromosome segregation are detrimental to genome integrity and is a hallmark of cancer. A common phenotype of chromosome segregation problems is the presence of DAPI stained DNA between segregating chromosome masses in anaphase. These “anaphase bridges” are poorly characterized, in part due to the assumption that their content is invariant or random. This assumption may be false because previous studies have identified mutants that lead to bridges specifically enriched in centromeric, telomeric or ribosomal DNA. These were candidate sequences analyzed using fluorescence in situ hybridization (FISH) or immunofluorescence analyses of proteins that bind to them. To date, an unbiased method to determine the complement and frequency of human genomic sequences contained in the anaphase bridges is not available. Knowing the primary effect of chromosome segregation defects at the sequence level will enable connecting them to the large sequencing projects characterizing genome integrity problems in cancers and aging. For instance, if certain cancer-associated mutations skew towards specific aneuploidies, their consequences may be cell-type specific based on the genes that are affected. The critical barrier to determining the DNA content of the bridges is a technical limitation. Here, we propose an innovative combination of two technologies to develop Bridge-seq: laser capture microdissection (LCM) to isolate DNA at anaphase bridges and high-throughput sequencing to identify genomic sequences. We will develop Bridge-seq and test its feasibility in multiple systems affecting different regions of the genome based on mutations in normal, cancer, and aging cells. In aim 1, We will establish the conditions for Bridge-seq using conditions that allow for a robust induction of DAPI-staining bridges that are enriched for rDNA. In aim 2, to assess the validity of using Bridge-seq to study anaphase bridge content across a range of conditions and different mutations, we will analyze bridges from mutants shown to effect other regions of chromosomes, including telomeres and centromeres. In Aim 3 we will apply Bridge-seq to analyze and compare anaphase bridges in aging and cancer cells. Our unbiased analysis will provide new insights into the nature of human genomic sequences that are vulnerable to segregation defects and that ultimately drive genomic instability.