title: Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.
abstract: 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.
authors.affiliation: Whitehead Institute for Biomedical Research, Department of Biology, Nine Cambridge Center, MA 02142, USA.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Gascoigne
authors.forename: Kozo
authors.initials: K
authors.lastname: Takeuchi
authors.forename: Aussie
authors.initials: A
authors.lastname: Suzuki
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hori
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
authors.forename: Iain M
authors.initials: IM
authors.lastname: Cheeseman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D000076247:Centromere Protein A; D002645:Chickens; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D020090:Chromosome Segregation; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism*; D010766:Phosphorylation; D014714:Vertebrates / Q000378:metabolism*
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D009707:Nucleosomes; C060905:centromere protein C