Induced Ectopic Kinetochore Assembly Bypasses the Requirement for CENP-A Nucleosomes

SummaryAccurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. Although prior work identified the centromeric histone H3-variant CENP-A as the important upstream factor necessary for centromere specification, in human cells CENP-A is not sufficient for kinetochore assembly. Here, we demonstrate that two constitutive DNA-binding kinetochore components, CENP-C and CENP-T, function to direct kinetochore formation. Replacing the DNA-binding regions of CENP-C and CENP-T with alternate chromosome-targeting domains recruits these proteins to ectopic loci, resulting in CENP-A-independent kinetochore assembly. These ectopic kinetochore-like foci are functional based on the stoichiometric assembly of multiple kinetochore components, including the microtubule-binding KMN network, the presence of microtubule attachments, the microtubule-sensitive recruitment of the spindle checkpoint protein Mad2, and the segregation behavior of foci-containing chromosomes. We additionally find that CENP-T phosphorylation regulates the mitotic assembly of both endogenous and ectopic kinetochores. Thus, CENP-C and CENP-T form a critical regulated platform for vertebrate kinetochore assembly.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (277 K)Download as PowerPoint slideHighlights► Ectopic targeting of CENP-T and CENP-C directs assembly of kinetochore-like structures ► Ectopic CENP-T/CENP-C kinetochore structures do not contain CENP-A nucleosomes ► Ectopic kinetochore structures can rescue segregation of acentric chromosomes ► CDK-dependent phosphorylation of CENP-T licenses mitotic kinetochore assembly