title: A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance.
abstract: Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes.
authors.affiliation: Department of Biochemistry, Stanford University Medical School, Stanford, CA 94305.
authors.forename: Frederick G
authors.initials: FG
authors.lastname: Westhorpe
authors.affiliation: Department of Biochemistry, Stanford University Medical School, Stanford, CA 94305.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Fuller
authors.affiliation: Department of Biochemistry, Stanford University Medical School, Stanford, CA 94305 astraigh@stanford.edu.
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Straight
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division; D002474:Cell-Free System; D002503:Centromere / Q000378:metabolism*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000378:metabolism*; D020090:Chromosome Segregation; D006657:Histones / Q000378:metabolism; D006801:Humans; D009707:Nucleosomes / Q000235:genetics; D017434:Protein Structure, Tertiary; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D002352:Carrier Proteins; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; C562324:M18BP1 protein, Xenopus; D009707:Nucleosomes; D029867:Xenopus Proteins; C060905:centromere protein C