Epigenetic assembly of centromeric chromatin at ectopic α-satellite sites on human chromosomes

Abstract

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.

Epigenetic engineering: histone H3K9 acetylation is compatible with kinetochore structure and function

“…Centromere identity is maintained epigenetically through the local deposition of CENP-A and at least in part through the associated chromatin context (Allshire and Karpen, 2008;Black and Bassett, 2008;Nakano et al, 2003;Nakashima et al, 2005;Okada et al, 2007). Classically, centromeres were assumed to be composed of heterochromatin.…”

“…This special chromatin, which has been termedx 'centrochromatin' (Sullivan and Karpen, 2004), suggests a functional link between the local chromatin environment and kinetochore function. Indeed, previous work from our laboratories indicates that the local chromatin state is crucial for the maintenance of a functional kinetochore structure (Bergmann et al, 2011;Cardinale et al, 2009;Nakano et al, 2008;Nakano et al, 2003;Nakashima et al, 2005).…”

Human centromere repositioning “in progress”

“…The histone deacetylase inhibitor TSA was shown previously to induce centromere activity in ␣-satellite DNA that has been transfected into human cells and integrated at a noncentromeric site, resulting in functionally dicentric chromosomes and breakage of these chromosomes to form minichromosomes (21). We therefore treated a PD-NC4-containing lymphocyte cell line with TSA to determine whether the inactive alphoid centromere can be induced to reassemble a kinetochore.…”

Gene Therapy Progress and Prospects: Episomally maintained self-replicating systems

“…26,28 The latter event is particularly unfavorable since it may cause activation of a new centromere at ectopic position. 29 The molecules obtained from the de novo and top-down approach share some important similarities. They are mitotically stable and maintained at low copy numbers and are 1-10 Mb in size and mainly composed of alphoid DNA confirming the primary role of the centromeric DNA in chromosome maintenance.…”