title: Chaperone-mediated assembly of centromeric chromatin in vitro.
abstract: Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, we have biochemically purified a soluble assembly complex that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription.
authors.affiliation: Fred Hutchinson Cancer Research Center and Howard Hughes Medical Institute, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
authors.forename: Takehito
authors.initials: T
authors.lastname: Furuyama
authors.forename: Yamini
authors.initials: Y
authors.lastname: Dalal
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
mesh_terms: D000818:Animals; D002503:Centromere / Q000737:chemistry / Q000378:metabolism*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D006657:Histones / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D009707:Nucleosomes / Q000378:metabolism; D056505:Retinoblastoma-Binding Protein 4
chemical_list: C110251:Caf1-55 protein, Drosophila; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C427237:Cid protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D006657:Histones; D018832:Molecular Chaperones; D009707:Nucleosomes; D056505:Retinoblastoma-Binding Protein 4