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

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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.