Human Artificial Chromosomes that Bypass Centromeric DNA

• Published in: Cell 2019-07, Vol.178 (3), p.624-639.e19
• Main Authors: Logsdon, Glennis A., Gambogi, Craig W., Liskovykh, Mikhail A., Barrey, Evelyne J., Larionov, Vladimir, Miga, Karen H., Heun, Patrick, Black, Ben E.
• Format: Article
• Language: English
• Subjects: Binding Sites; Cell Line, Tumor; centromere; Centromere - genetics; Centromere - metabolism; Centromere Protein A - genetics; Centromere Protein A - metabolism; Centromere Protein B - deficiency; Centromere Protein B - genetics; Centromere Protein B - metabolism; chromatin; Chromosomes, Artificial, Human - metabolism; DNA, Satellite - metabolism; Epigenesis, Genetic; epigenetics; HAC; histone; human artificial chromosome; Humans; kinetochore; mitosis; nucleosome; Nucleosomes - chemistry; Nucleosomes - metabolism; Plasmids - genetics; Plasmids - metabolism; synthetic chromosome
• ISSN: 0092-8674; 1097-4172; 1097-4172
• DOI: 10.1016/j.cell.2019.06.006

Recent breakthroughs with synthetic budding yeast chromosomes expedite the creation of synthetic mammalian chromosomes and genomes. Mammals, unlike budding yeast, depend on the histone H3 variant, CENP-A, to epigenetically specify the location of the centromere—the locus essential for chromosome segregation. Prior human artificial chromosomes (HACs) required large arrays of centromeric α-satellite repeats harboring binding sites for the DNA sequence-specific binding protein, CENP-B. We report the development of a type of HAC that functions independently of these constraints. Formed by an initial CENP-A nucleosome seeding strategy, a construct lacking repetitive centromeric DNA formed several self-sufficient HACs that showed no uptake of genomic DNA. In contrast to traditional α-satellite HAC formation, the non-repetitive construct can form functional HACs without CENP-B or initial CENP-A nucleosome seeding, revealing distinct paths to centromere formation for different DNA sequence types. Our developments streamline the construction and characterization of HACs to facilitate mammalian synthetic genome efforts. [Display omitted] •Development of human artificial chromosomes (HACs) where CENP-A chromatin is seeded•Seeding CENP-A nucleosome assembly induces centromere formation•Seeding centromeric chromatin bypasses sequence elements in repetitive centromere DNA•Non-repetitive HAC templates ease initial construction and downstream genomic analyses Development of human artificial chromosomes that bypass centromeric DNA removes a key barrier limiting mammalian synthetic genome efforts.