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

基本信息

批准号：
10456922
负责人：
Sevinc Ercan
金额：
$ 19.94万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10456922
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 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

项目摘要

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.

项目概要染色体分离缺陷会损害基因组完整性，并且是癌症 A 的一个标志。染色体分离问题的常见表型是之间存在 DAPI 染色的 DNA 这些“后期桥”的特征部分尚不清楚。由于假设它们的内容是不变的或随机的，因为这种假设可能是错误的。先前的研究已经确定了导致着丝粒、端粒或端粒特异富集的桥的突变体这些是使用荧光原位杂交 (FISH) 或分析的候选序列。迄今为止，有一种公正的方法来确定与它们结合的蛋白质。后期桥中包含的人类基因组序列的补体和频率尚不可用。了解染色体分离缺陷在序列水平上的主要影响将有助于连接他们参与了表征癌症和衰老中基因组完整性问题的大型测序项目。例如，如果某些与癌症相关的突变偏向于特定的非整倍体，其后果可能是根据受影响的基因，细胞类型具有特异性。确定桥 DNA 含量的关键障碍是技术限制。在这里，我们提出了一个限制。创新结合两种技术开发 Bridge-seq：激光捕获显微切割 (LCM) 我们将在后期桥上分离 DNA 并进行高通量测序来鉴定基因组序列。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在目标 1 中，我们将使用 Bridge-seq 建立条件。在目标 2 中，可以强烈诱导富含 rDNA 的 DAPI 染色桥。评估使用 Bridge-seq 研究一系列条件下的后期桥含量的有效性，以及不同的突变，我们将分析显示影响染色体其他区域的突变体的桥梁，包括端粒和着丝粒在目标 3 中我们将应用 Bridge-seq 来分析和比较后期。我们的公正分析将为人类本质提供新的见解。基因组序列容易受到分离缺陷的影响，并最终导致基因组不稳定。