title: In vitro centromere and kinetochore assembly on defined chromatin templates.
abstract: During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin that recapitulates essential steps of centromere and kinetochore assembly in vitro. We show that reconstituted CENP-A chromatin when added to cell-free extracts is sufficient for the assembly of centromere and kinetochore proteins, microtubule binding and stabilization, and mitotic checkpoint function. Using chromatin assembled from histone H3/CENP-A chimaeras, we demonstrate that the conserved carboxy terminus of CENP-A is necessary and sufficient for centromere and kinetochore protein recruitment and function but that the CENP-A targeting domain--required for new CENP-A histone assembly--is not. These data show that two of the primary requirements for accurate chromosome segregation, the assembly of the kinetochore and the propagation of CENP-A chromatin, are specified by different elements in the CENP-A histone. Our unique cell-free system enables complete control and manipulation of the chromatin substrate and thus presents a powerful tool to study centromere and kinetochore assembly.
authors.affiliation: Department of Biochemistry, Stanford Medical School, Beckman 409A, Stanford, California 94305-5307, USA.
authors.forename: Annika
authors.initials: A
authors.lastname: Guse
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Carroll
authors.forename: Ben
authors.initials: B
authors.lastname: Moree
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Fuller
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Straight
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism; D002457:Cell Extracts; D002474:Cell-Free System; D002503:Centromere / Q000378:metabolism*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020090:Chromosome Segregation; D017124:Conserved Sequence; D006657:Histones / Q000378:metabolism; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008938:Mitosis; D009865:Oocytes; D017434:Protein Structure, Tertiary; D013698:Templates, Genetic; D014982:Xenopus laevis
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D002457:Cell Extracts; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones