Analysis of complex DNA rearrangements during early stages of HAC formation

Elisa Pesenti, Mikhail Liskovykh, Koei Okazaki, Alessio Mallozzi, Caitlin Reid, Maria Alba Abad, A. Arockia Jeyaprakash, Natalay Kouprina, Vladimir Larionov, Hiroshi Masumoto, William C. Earnshaw

Research output: Contribution to journalArticlepeer-review

Abstract

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.
Original languageEnglish
Pages (from-to)3267-3287
Number of pages20
JournalACS Synthetic Biology
Volume9
Issue number12
Early online date8 Dec 2020
DOIs
Publication statusPublished - 18 Dec 2020

Keywords

  • centromere
  • human artificial chromosome
  • epigenetic engineering
  • mitosis
  • kinetchore
  • CENP-A

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