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

Bridge-seq，分析人类基因组分离缺陷的新工具

• 批准号: 10303448
• 负责人: Sevinc Ercan
• 金额: $ 24.06万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303448
• 关键词: Address; Affect; Aging; Anaphase; Aneuploidy; Benchmarking; Binding Proteins; Cell Aging; Cells; Centromere; Chromosome Segregation; Chromosome Structures; Chromosomes; Complement; DNA; DNA Sequence; Data; Defect; Development; Disease; Dominant-Negative Mutation; Fluorescent in Situ Hybridization; Frequencies; Genes; Genome; Genomic Instability; Genomic Segment; Genomics; High-Throughput Nucleotide Sequencing; Human; Human Characteristics; Human Genome; Immunofluorescence Immunologic; Lead; Malignant Neoplasms; Methods; Mitosis; Mitotic Chromosome; Molecular; Mutation; Normal Cell; PTPRJ gene; Phenotype; Ploidies; Poly(ADP-ribose) Polymerases; Process; Protein Analysis; Proteins; Recombinant DNA; Ribosomal DNA; Sampling; Sequence Analysis; Sister Chromatid; Site; Specificity; Stains; System; Tankyrase; Techniques; Technology; Testing; age related; base; cancer cell; cell type; cohesin; condensin; genome integrity; innovation; insight; knock-down; laser capture microdissection; mutant; normal aging; response; segregation; success; telomere; tool; whole genome

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.

项目摘要 染色体分离的缺陷对基因组完整性有害，并且是癌症的标志。一个 染色体分离问题的常见表型是在 后期隔离染色体质量。这些“后期桥”的特征很差，部分是 由于假设其内容是不变的或随机的。这个假设可能是错误的，因为 先前的研究已经确定了导致桥的突变体，该突变体特异性地富含着丝粒，远程远程远程或 核糖体DNA。这些是使用荧光原位杂交（FISH）或 与它们结合的蛋白质的免疫荧光分析。迄今为止，一种公正的方法来确定 没有后期桥中包含的人类基因组序列的补体和频率。 了解序列级别染色体隔离缺陷的主要效果将使连接 他们参加了大型测序项目，这些项目表征了癌症和衰老中的基因组完整性问题。为了 实例，如果某些癌症相关的突变偏向特定的非整倍性，它们的后果可能 基于受影响的基因为细胞类型。 确定桥梁的DNA含量的关键障碍是技术限制。在这里，我们建议 两种开发桥梁式的技术的创新组合：激光捕获显微解剖（LCM） 在后期桥和高通量测序上分离DNA，以鉴定基因组序列。我们将 开发桥梁式并测试其在影响基于基因组不同区域的多个系统中的可行性 关于正常，癌症和衰老细胞中的突变。在AIM 1中，我们将建立使用桥梁塞克的条件 允许对富含rDNA的DAPI染色桥的稳健诱导的条件。在AIM 2中 评估使用桥梁在各种条件下研究后期桥含量的有效性 不同的突变，我们将分析来自显示的突变体的桥梁，以影响其他染色体区域， 包括端粒和中心粒。在AIM 3中，我们将应用Bridge-Seq分析和比较动画 衰老和癌细胞中的桥梁。我们的公正分析将为人类本质提供新的见解 容易受到隔离缺陷并最终驱动基因组不稳定性的基因组序列。