Chromatin clusters containing CENP-A, a histone H3 variant, are found in centromeres of multicellular eukaryotes. This study examines the ability of alpha-satellite (alphoid) DNA arrays in different lengths to nucleate CENP-A chromatin and form functional kinetochores de novo. Kinetochore assembly was followed by measuring human artificial chromosome formation in cultured human cells and by chromatin immunoprecipitation analysis. The results showed that both the length of alphoid DNA arrays and the density of CENP-B boxes had a strong impact on nucleation, spreading and/or maintenance of CENP-A chromatin, and formation of functional kinetochores. These effects are attributed to a change in the dynamic balance between assembly of chromatin containing trimethyl histone H3-K9 and chromatin containing CENP-A/C. The data presented here suggest that a functional minimum core stably maintained on 30-70 kb alphoid DNA arrays represents an epigenetic memory of centromeric chromatin.

To investigate the mechanism of chromatin assembly at human centromeres, we isolated cultured human cell lines in which a transfected alpha-satellite (alphoid) YAC was integrated ectopically into the terminal region of host chromosome 16, where it was stably maintained. Centromere activity of the alphoid YAC was suppressed at ectopic locations on the host chromosome, as indicated by the absent or reduced assembly of CENP-A and -C. However, long-term culture in selective medium, or shortterm treatment with the histone deacetylase inhibitor Trichostatin A (TSA), promoted the re-assembly of CENP-A, -B and -C at the YAC site and the release of minichromosomes containing the YAC integration site. Chromatin immunoprecipitation analyses of the re-formed minichromosome and the alphoid YAC-based stable human artificial chromosome both indicated that CENP-A and CENP-B assembled only on the inserted alphoid array but not on the YAC arms. On the YAC arms at the alphoid YAC integration sites, TSA treatment increased both the acetylation level of histone H3 and the transcriptional level of a marker gene. An increase in the level of transcription was also observed after long-term culture in selective medium. These activities, which are associated with changes in chromatin structure, might reverse the suppressed chromatin state of the YAC at ectopic loci, and thus might be involved in the epigenetic change of silent centromeres on ectopic alphoid loci.

Human artificial chromosomes (HACs) provide a unique opportunity to study kinetochore formation and to develop a new generation of vectors with potential in gene therapy. An investigation into the structural and the functional relationship in centromeric tandem repeats in HACs requires the ability to manipulate repeat substructure efficiently. We describe here a new method to rapidly amplify human alphoid tandem repeats of a few hundred base pairs into long DNA arrays up to 120 kb. The method includes rolling-circle amplification (RCA) of repeats in vitro and assembly of the RCA products by in vivo recombination in yeast. The synthetic arrays are competent in HAC formation when transformed into human cells. As short multimers can be easily modified before amplification, this new technique can identify repeat monomer regions critical for kinetochore seeding. The method may have more general application in elucidating the role of other tandem repeats in chromosome organization and dynamics.

The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

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