title: Chromosome engineering allows the efficient isolation of vertebrate neocentromeres.
abstract: Centromeres are specified by sequence-independent epigenetic mechanisms in most organisms. Rarely, centromere repositioning results in neocentromere formation at ectopic sites. However, the mechanisms governing how and where neocentromeres form are unknown. Here, we established a chromosome-engineering system in chicken DT40 cells that allowed us to efficiently isolate neocentromere-containing chromosomes. Neocentromeres appear to be structurally and functionally equivalent to native centromeres. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis with 18 neocentromeres revealed that the centromere-specific histone H3 variant CENP-A occupies an ∼40 kb region at each neocentromere, which has no preference for specific DNA sequence motifs. Furthermore, we found that neocentromeres were not associated with histone modifications H3K9me3, H3K4me2, and H3K36me3 or with early replication timing. Importantly, low but significant levels of CENP-A are detected around endogenous centromeres, which are capable of seeding neocentromere assembly if the centromere core is removed. In summary, our experimental system provides valuable insights for understanding how neocentromeres form.
authors.affiliation: Department of Molecular Genetics, National Institute of Genetics and The Graduate University for Advanced Studies SOKENDAI, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Wei-Hao
authors.initials: WH
authors.lastname: Shang
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hori
authors.forename: Nuno M C
authors.initials: NM
authors.lastname: Martins
authors.forename: Atsushi
authors.initials: A
authors.lastname: Toyoda
authors.forename: Sadahiko
authors.initials: S
authors.lastname: Misu
authors.forename: Norikazu
authors.initials: N
authors.lastname: Monma
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hiratani
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Maeshima
authors.forename: Kazuho
authors.initials: K
authors.lastname: Ikeo
authors.forename: Asao
authors.initials: A
authors.lastname: Fujiyama
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kimura
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Fukagawa
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002503:Centromere / Q000235:genetics* / Q000378:metabolism; D000076247:Centromere Protein A; D002645:Chickens / Q000235:genetics*; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D002875:Chromosomes / Q000235:genetics*; D019175:DNA Methylation; D004261:DNA Replication; D044127:Epigenesis, Genetic; D005818:Genetic Engineering; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D001324:Autoantigens; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone