Wellcome Trust Centre for Cell Biology, Edinburgh EH9 3BF, United Kingdom.
National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, United States.
ACS Synth Biol. 2020 Dec 18;9(12):3267-3287. doi: 10.1021/acssynbio.0c00326. Epub 2020 Dec 8.
Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.
人源人工染色体（HACs）是表观遗传学工程、测量染色体不稳定性（CIN）以及可能的基因治疗的重要工具。然而，由于输入的 HAC 播种 DNA 在 HAC 形成过程中可能经历不可预测的一系列重排，因此它们在后者中的应用可能受到限制。结果，在转染和 HAC 形成后，每个细胞克隆都包含具有独特结构的 HAC，无法从 HAC 播种 DNA 的结构中精确预测。尽管已经报道了这些重排的发生，但它们的形成时间和机制尚未描述。在这里，我们合成了具有两个不同结构域的 HAC 播种 DNA，并将其引入 HT1080 细胞。我们对许多含有 HAC 的克隆和亚克隆进行了表征，以跟踪 HAC 建立过程中的 DNA 重排。我们证明重排可以在 HAC 形成的早期发生。随后，建立的 HAC 基因组组织在许多细胞世代中稳定维持。因此，HAC 形成的早期阶段似乎至少偶尔涉及类似于染色体重排的 DNA 破碎和洗牌过程，这是许多癌症类型的重要标志之一。了解 HAC 形成过程中的这些事件对于未来旨在合成和利用合成人类染色体的努力具有关键意义。
