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: 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
• 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

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.

项目总结 染色体分离的缺陷不利于基因组的完整性，也是癌症的一个标志。一个 染色体分离问题的常见表型是存在DAPI染色的DNA之间 在后期分离染色体组。在某种程度上，这些“后期桥梁”的特征并不理想。 因为假设它们的内容是不变的或随机的。这一假设可能是错误的，因为 以前的研究已经确定了导致特定富含着丝粒、端粒或 核糖体DNA。这些候选序列是使用荧光原位杂交(FISH)或 结合它们的蛋白质的免疫荧光分析。到目前为止，一个无偏见的方法来确定 后期桥中包含的人类基因组序列的互补性和频率是不可用的。 在序列水平上了解染色体分离缺陷的主要影响将使 它们被用于描述癌症和衰老中的基因组完整性问题的大型测序项目。为 例如，如果某些癌症相关突变偏向特定的非整倍体，其后果可能 根据受影响的基因确定特定的细胞类型。 确定桥梁DNA含量的关键障碍是技术限制。在这里，我们提出一种 两种技术的创新结合开发Bridge-seq：激光捕获显微切割(LCM) 在后期桥分离DNA，并进行高通量测序以鉴定基因组序列。我们会 开发Bridge-Seq并在影响基因组不同区域的多个系统中测试其可行性 正常细胞、癌症细胞和衰老细胞的突变。在目标1中，我们将为Bridge-seq的使用创造条件 允许强健地诱导富含rDNA的DAPI染色的桥的条件。在目标2中， 评估使用Bridge-Seq在一系列条件和条件下研究后期Bridge内容的有效性 不同的突变，我们将分析突变对染色体其他区域产生影响的桥， 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 在衰老和癌细胞之间架起桥梁。我们不偏不倚的分析将为人类的本性提供新的见解 易受分离缺陷影响并最终导致基因组不稳定的基因组序列。