Project Summary/Abstract In order to fully grasp the molecular origins of genome instability, the field must understand at a molecular level how centromeres work to promote the stable transmission of chromosomes. Genome instability underlies a variety of human pathologies, including cancer and reproductive aging. Our long-term goal is to determine how the evolutionarily conserved cohesin complex maintains genome integrity through its roles in chromosome segregation, chromosome organization, and double-strand break repair. Loss of sister chromatid cohesion is speculated to be a major contributor to chromosome instability. The objective of this application is to produce a molecular model for how cohesin operates at individual human centromeres to achieve centromeric cohesion and accurate chromosome segregation. The central hypothesis is that cohesin and DNA catenation together create centromere-unique landscapes of sister chromatid cohesion to prevent chromosome instability. The variation in human centromeres and centromeric cohesion may therefore impact the transmission of each chromosome. We will test the idea that centromere-specific cohesion must be considered as a genetic determinant of sister chromatid cohesion and segregation in order to have a complete model for how chromosomal instability occurs through two specific aims: 1) discover the landscape of centromeric cohesion at individual human centromeres and 2) examine how chromosome centromeric cohesion maintains euploidy. Under the first aim, calibrated paired-end ChIP seq will be used to map cohesin binding relative to kinetochore proteins and human centromeric arrays in human tissue culture cells. This approach will be complemented by superresolution imaging of the same three components (centromeres, kinetochores, and cohesin) in cells, and will include imaging-based determination of centromere-specific cohesion. Together these approaches will produce a linear and 3D map of cohesion within and around individual human centromeres. In the second aim we will examine how centromere-specific patterns of centromeric cohesion prevent chromosome missegregation events in cultured cells and in xenograft tumor tissue. The outcome will be fundamental principles of centromeric array-based cohesion fatigue and resulting patterns of chromosome instability. The research is innovative because it incorporates the latest information on human centromeric DNA arrays, a new working model for the organization of centromeric DNA by cohesion, and new quantitative molecular, genomic, and imaging tools to probe how centromeric cohesion enforces accurate sister chromatid segregation. The proposed research is significant because centromeric arrays may be unrecognized genetic determinants of chromosome instability. Many types of cancer are associated with seemingly random patterns of instability that may have molecular origins in unique centromeric cohesion profiles. Furthermore, many cancers are associated with mutations that impact chromosome segregation machinery, such as cohesin. The outcome of this project will be a more complete picture of the mechanisms underlying chromosomal instability.

Project Narrative The proposed research is relevant to public health because it addresses the molecular origins of chromosome instability, which is prevalent in cancer and reproductive aging. We will test the hypothesis that each person’s unique complement of centromeres contains molecular and genetic information that biases how accurately chromosomes are transmitted from cell to cell. The project is relevant to the NCI mission to discover molecular and genetic origins of human genome stability.