Dissecting the molecular factors that underlie the array size dependent centromere performance

Abstract

Centromeres are specialized regions of chromosomes essential for accurate chromosome segregation. In humans, centromeres comprise of chromosome-specific alpha-satellite (α-satellite) DNA repeats which can span kilobases to megabases in size. Telomere-to-telomere (T2T)-assembled genomes from hundreds of individuals show that human centromere sizes are highly variable; and large differences exist between centromeres of homologous chromosomes. I hypothesize that centromere array size is a genetic contributor to aneuploidy. In this study, I investigate the impact of centromeric array size on centromere function and the associated molecular factors. Variable centromere array sizes can be distinguished on homologous chromosomes using quantitative FISH and microscopy. Cell biological assessments show that out of a homologous pair, the chromosome harboring the smaller centromere is biased to mis segregate, when the spindle checkpoint is turned off. The centromere specific histone H3 variant (CENP-A), kinetochore complex proteins, and inner centromere proteins did not scale with the centromere array size differences. My findings suggest that the assembly and maturation of kinetochore subunits is independent of array size. I also inspected how centromere array size affects sister chromatid cohesion under prolonged mitosis. I found that out of a homologous pair, the smaller centromere is more prone to cohesion loss compared to its larger counterpart, a difference that can be abrogated by depleting cells of shugoshin protein, suggesting the role of the cohesin complex in mediating size-dependent cohesion differences at centromeres. I further developed tools to track the dynamics of centromere 7 in live cells, to validate findings from fixed cell FISH assays. Altogether, I present a molecular analysis of functional differences at homologous centromeres exhibiting array size variation.