Chromosome Engineering Allows the Efficient Isolation of Vertebrate Neocentromeres

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.

Stepwise unfolding supports a subunit model for vertebrate kinetochores

“…According to the lengths measured for CENP-A fibers, these values ranged from 8-45 Kbp. The smaller number likely corresponds to fibers that were not completely unfolded, whereas the larger number (from unfolded CENP-C OFF and CENP-S KO chromosomes) was remarkably close to the estimated 50-60 kb of DNA in chicken kinetochores determined by quantitative fluorescence microscopy (46) and the ∼40 kb of DNA occupied by CENP-A in chicken nonrepetitive centromeres and neocentromeres determined by ChIP (47).…”

Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin

“…S5). The absence of methylated H3K4 has also been reported for some neocentromeres (46), plant centromeres (48), and the filamentous fungus Neurospora crassa (49). Thus, there may be significant flexibility in the pattern of histone modifications that can occur within a centromeric core.…”

“…Interestingly, some neocentromeres in human and chicken cells also lack flanking heterochromatin (45,46). Unlike most metazoan centromeres, these neocentromeres are not embedded in repetitive DNA sequences, suggesting that heterochromatin is correlated with the presence of repetitive sequences rather than the centromere per se (46).…”

“…Unlike most metazoan centromeres, these neocentromeres are not embedded in repetitive DNA sequences, suggesting that heterochromatin is correlated with the presence of repetitive sequences rather than the centromere per se (46). Thus, it may be that in the pericentromeric regions the presence of topology adjusters, such as cohesin and condensin, is more critical than a particular pattern of histone modifications and chromatin proteins (47).…”

Maize centromeres expand and adopt a uniform size in the genetic background of oat

“…Insertion of a marker gene in the centromeres of Schizosaccharomyces pombe chromosomes results in complete silencing of the gene (Allshire et al 1995). Similarly, neocentromeres in multiple species generally form in gene-poor regions (Lomiento et al 2008;Alonso et al 2010;Shang et al 2013); when they do form over genic areas, the affected genes are suppressed or silenced (Ishii et al 2008;Ketel et al 2009;Shang et al 2013). Why does centromeric chromatin avoid actively transcribed genes?…”