biostudies-literatureGascoigne KEEuropean Molecular Biology OrganizationSearle Scholars ProgramMinistry of Education, Culture, Sports, Science and TechnologyMassachusetts Life Sciences CenterNational Institutes of HealthNational Institute of General Medical SciencesNIGMS NIH HHS410-22https://www.ebi.ac.uk/biostudies/studies/S-EPMC3085131biostudies-literatureUnknown145(3)Accurate 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.CellInduced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.PMC3085131GM088313R01 GM088313-03R01 GM088313Suzuki AGascoigne KETakeuchi KCheeseman IMFukagawa THori TfalseInduced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.Accurate 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.2011-01-01T00:00:00Z2011 Apr2024-11-13T14:18:42.257Z2019-03-27T00:41:06ZS-EPMC30851312152971410.1016/j.cell.2011.03.031