Epigenetic engineering shows H3K4me2 is required for HJURP targeting and CENP-A assembly on a synthetic human kinetochoreHere, centromeric histone marks on a human artificial chromosome are found to resemble the chromatin landscape in transcribed genes, and selective manipulation shows them to govern the incorporation of the centromere-specifying CENP-A histone variant.

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.

Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We further show that the key CENP-A assembly factor Mis18BP1(HsKNL2) is phosphorylated in a cell cycle-dependent manner that controls its centromere localization during mitotic exit. These results strongly support a model in which the CENP-A assembly machinery is poised for activation throughout the cell cycle but kept in an inactive noncentromeric state by Cdk activity during S, G2, and M phases. Alleviation of this inhibition in G1 phase ensures tight coupling between DNA replication, cell division, and subsequent centromere maturation.