Abstract
SummaryCentromeres 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.
Highlights
The centromere is the genomic locus that directs faithful chromosome segregation
As the rare neocentromeres observed in human patients allow only observational or correlative studies, and human neocentromeres are typically observed in adults after large numbers of cell generations have passed, little is known about the molecular events that lead to neocentromere formation
We found that low but significant levels of nonkinetochore CENP-A scattered throughout chromatin flanking the 40 kb centromeric CENP-A domain may seed for neocentromere formation following removal of the original centromere
Summary
The centromere is the genomic locus that directs faithful chromosome segregation. In human cells, the ability of cells to inactivate a centromere on dicentric chromosomes (Earnshaw and Migeon, 1985) and the formation of neocentromeres at uniqueDNA sequences lacking the alpha-satellite repeats traditionally associated with centromeres (du Sart et al, 1997; Marshall et al, 2008) together reveal that the underlying DNA sequence is neither necessary nor sufficient to specify centromere formation in vertebrate cells. The ability of cells to inactivate a centromere on dicentric chromosomes (Earnshaw and Migeon, 1985) and the formation of neocentromeres at unique. In Schizosaccharomyces pombe or Candida albicans, centromere deletion has been used to drive neocentromere formation, and the yeast model systems are widely used to understand molecular mechanisms for centromere formation (Ishii et al, 2008; Ketel et al, 2009). These fungal genomes are compact and contain few noncoding regions and repetitive sequences, and it is still unclear how neocentromeres form following centromere inactivation in vertebrate cells
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