title: Variable and hierarchical size distribution of L1-retroelement-enriched CENP-A clusters within a functional human neocentromere.
abstract: Human neocentromeres are fully functional centromeres that arise epigenetically from non-centromeric precursor sequences that are devoid of alpha-satellite DNA. Using chromatin immunoprecipitation (ChIP) and BAC-array analysis, we have previously described a 330 kb binding domain for CENP-A (a histone H3 variant that confers centromere-specific nucleosomal property) at the 10q25 neocentromere found on a chromosome 10-derived marker chromosome mardel(10). For the further detailed analysis of the CENP-A-associated chromatin, we have generated a high-resolution genomic array consisting of PCR fragments with an average size of 8 kb, providing an approximately 20-fold increment in analytical resolution. ChIP and PCR-array analysis reveals seven distinct CENP-A-binding clusters within the 330 kb domain, demonstrating the interspersion of CENP-A-associated nucleosomal blocks within the neocentromeric chromatin. Independent ChIP-PCR analysis verified this distribution profile and indicated that histone H3-containing nucleosomes directly intervene the CENP-A-binding clusters. The CENP-A-binding clusters are uneven in size, with the central cluster (>50 kb) being significantly larger than the flanking ones (10-30 kb), and the flanking clusters arranged in an interesting hierarchical and symmetrical configuration of alternating larger and smaller sizes around the central cluster. In silico sequence analysis indicates an approximately 2.5-fold increase in the prevalence of L1 retroelements within the CENP-A-binding clusters when compared with the non-CENP-A-binding regions. These results provide insight into the possible role of retroelements in determining the positioning of CENP-A binding at human neocentromeres, and that a hierarchical and symmetrical arrangement of CENP-A-binding clusters of varying sizes may be an important structural requirement for mammalian kinetochore assembly and/or to provide stability to withstand polar microtubule forces.
authors.affiliation: Chromosome Research Laboratory, Murdoch Childrens Research Institute, Melbourne University Department of Paediatrics, Royal Children's Hospital, Parkville, Australia.
authors.forename: Anderly C
authors.initials: AC
authors.lastname: Chueh
authors.forename: Lee H
authors.initials: LH
authors.lastname: Wong
authors.forename: Nicholas
authors.initials: N
authors.lastname: Wong
authors.forename: K H Andy
authors.initials: KH
authors.lastname: Choo
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism; D016466:CHO Cells; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics* / Q000378:metabolism; D002879:Chromosomes, Human, Pair 10 / Q000235:genetics*; D006224:Cricetinae; D004276:DNA, Satellite / Q000235:genetics / Q000502:physiology; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D005810:Multigene Family / Q000235:genetics*; D009707:Nucleosomes / Q000235:genetics / Q000502:physiology; D018626:Retroelements / Q000235:genetics* / Q000502:physiology
chemical_list: D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; D004276:DNA, Satellite; D009707:Nucleosomes; D018626:Retroelements