Department of Biochemistry and Biophysics, Graduate Program in Biochemistry and Molecular Biophysics, and Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Developmental Therapeutics Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Cell. 2019 Jul 25;178(3):624-639.e19. 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.
近年来，合成芽殖酵母染色体方面的突破加速了合成哺乳动物染色体和基因组的进程。与芽殖酵母不同，哺乳动物依赖组蛋白 H3 变体 CENP-A 来在表观遗传上指定着丝粒的位置——这是染色体分离所必需的基因座。先前的人类人工染色体（HAC）需要含有 DNA 序列特异性结合蛋白 CENP-B 结合位点的大量着丝粒α-卫星重复序列阵列。我们报告了一种 HAC 的开发，它独立于这些限制。通过初始的 CENP-A 核小体播种策略形成，缺乏重复的着丝粒 DNA 的构建体形成了几个自给自足的 HAC，这些 HAC 没有摄取基因组 DNA。与传统的α-卫星 HAC 形成相比，非重复的构建体可以在没有 CENP-B 或初始 CENP-A 核小体播种的情况下形成功能性 HAC，揭示了不同 DNA 序列类型形成着丝粒的不同途径。我们的发展简化了 HAC 的构建和表征，以促进哺乳动物合成基因组的努力。
