title: Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A.
abstract: The mechanisms that specify precisely where mammalian kinetochores form within arrays of centromeric heterochromatin remain largely unknown. Localization of CENP-A exclusively beneath kinetochore plates suggests that this distinctive histone might direct kinetochore formation by altering the structure of heterochromatin within a sub-region of the centromere. To test this hypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a subset of centromere-kinetochore components, including CENP-C, hSMC1, and HZwint-1 by a mechanism that requires the unique CENP-A N-terminal tail. The sequence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity was not detected. Experimental mistargeting of CENP-A to inactive centromeres or to acentric double-minute chromosomes was also not sufficient to assemble complete kinetochore activity. The recruitment of centromere-kinetochore proteins to chromatin appears to be a unique function of CENP-A, as the mistargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore assembly: (1) precise targeting of CENP-A, which is sufficient to assemble components of a centromere-prekinetochore scaffold; and (2) targeting of kinetochore microtubule-associated proteins by an additional mechanism present only at active centromeres.
authors.affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: A A
authors.initials: AA
authors.lastname: Van Hooser
authors.forename: I I
authors.initials: II
authors.lastname: Ouspenski
authors.forename: H C
authors.initials: HC
authors.lastname: Gregson
authors.forename: D A
authors.initials: DA
authors.lastname: Starr
authors.forename: T J
authors.initials: TJ
authors.lastname: Yen
authors.forename: M L
authors.initials: ML
authors.lastname: Goldberg
authors.forename: K
authors.initials: K
authors.lastname: Yokomori
authors.forename: W C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: K F
authors.initials: KF
authors.lastname: Sullivan
authors.forename: B R
authors.initials: BR
authors.lastname: Brinkley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D016466:CHO Cells; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004268:DNA-Binding Proteins; D015870:Gene Expression; D006367:HeLa Cells; D006657:Histones; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008938:Mitosis / Q000502:physiology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D014162:Transfection
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; C497926:CENPB protein, human; D000076247:Centromere Protein A; D051936:Centromere Protein B; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D008869:Microtubule-Associated Proteins; C060905:centromere protein C