Combination of CENP-B Box Positive and Negative Synthetic Alpha Satellite Repeats Improves De Novo Human Artificial Chromosome Formation

Human artificial chromosomes (HACs) can be formed de novo by introducing large (>30 kb) centromeric sequences consisting of highly repeated 171-bp alpha satellite (alphoid) DNA into HT1080 cells. However, only a subset of transformed cells successfully establishes HACs. CENP-A chromatin and heterochromatin assemble on the HACs and play crucial roles in chromosome segregation. The CENP-B protein, which binds a 17-bp motif (CENP-B box) in the alphoid DNA, functions in the formation of alternative CENP-A chromatin or heterochromatin states. A balance in the coordinated assembly of these chromatin states on the introduced alphoid DNA is important for HAC formation. To obtain information about the relationship between chromatin architecture and de novo HAC formation efficiency, we tested combinations of two 60-kb synthetic alphoid sequences containing either tetO or lacO plus a functional or mutated CENP-B box combined with a multiple fusion protein tethering system. The combination of mutated and wild-type CENP-B box alphoid repeats significantly enhanced HAC formation. Both CENP-A and HP1α were enriched in the wild-type alphoid DNA, whereas H3K27me3 was enriched on the mutant alphoid array. The presence or absence of CENP-B binding resulted in differences in the assembly of CENP-A chromatin on alphoid arrays and the formation of H3K9me3 or H3K27me3 heterochromatin.