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.