title: Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres.
abstract: The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.
authors.affiliation: Institute of Cell and Molecular Biology, University of Edinburgh, UK.
authors.forename: P E
authors.initials: PE
authors.lastname: Warburton
authors.forename: C A
authors.initials: CA
authors.lastname: Cooke
authors.forename: S
authors.initials: S
authors.lastname: Bourassa
authors.forename: O
authors.initials: O
authors.lastname: Vafa
authors.forename: B A
authors.initials: BA
authors.lastname: Sullivan
authors.forename: G
authors.initials: G
authors.lastname: Stetten
authors.forename: G
authors.initials: G
authors.lastname: Gimelli
authors.forename: D
authors.initials: D
authors.lastname: Warburton
authors.forename: C
authors.initials: C
authors.lastname: Tyler-Smith
authors.forename: K F
authors.initials: KF
authors.lastname: Sullivan
authors.forename: G G
authors.initials: GG
authors.lastname: Poirier
authors.forename: W C
authors.initials: WC
authors.lastname: Earnshaw
mesh_terms: D000595:Amino Acid Sequence; D001323:Autoantibodies / Q000378:metabolism; D001324:Autoantigens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002503:Centromere / Q000737:chemistry / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D009707:Nucleosomes