title: CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly.
abstract: Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoid(tetO)). We also obtained cell lines bearing the alphoid(tetO) array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly.
authors.affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Shono
authors.affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Jun-ichirou
authors.initials: J
authors.lastname: Ohzeki
authors.affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Otake
authors.affiliation: Wellcome Trust Centre for Cell Biology University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: Nuno M C
authors.initials: NM
authors.lastname: Martins
authors.affiliation: Public Relations Team, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nagase
authors.affiliation: Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
authors.affiliation: Developmental Therapeutic Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.affiliation: Wellcome Trust Centre for Cell Biology University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK.
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.affiliation: Laboratory of Cell Engineering, Department of Frontier Research, Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan masumoto@kazusa.or.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
mesh_terms: D001324:Autoantigens / Q000235:genetics / Q000378:metabolism*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism*
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; C484297:CENPI protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C060905:centromere protein C
keywords: CENP-A; CENP-C; CENP-I; Centromere; HAC; Human artificial chromosome; Kinetochore