Abstract
A method allowing real-time visualization of translocations reveals properties of their formation.
Major finding: A method allowing real-time visualization of translocations reveals properties of their formation.
Approach: Cells were engineered to contain binding site arrays adjacent to inducible double-strand break sites.
Impact: This system can provide insight into the dynamics and cellular requirements of chromosomal translocations.
Chromosomal translocations are a common feature of human cancers and have been implicated in tumorigenesis, but the mechanisms underlying their formation remain poorly understood. Roukos and colleagues developed a system to visualize chromosomal translocation formation in mammalian cells by generating NIH3T3 cells that contained ISceI restriction endonuclease sites adjacent to arrays of multiple copies of either the Lac operator (LacO) or Tet operator (TetO) DNA sequences. Such arrays allow the visualization of the chromosomal loci where they are integrated in live cells upon stable expression of fluorescently labeled Lac repressor (LacR) or Tet repressor (TetR) proteins, respectively. DNA double-strand breaks (DSB) were introduced by transfection of ISceI, leading to the formation of DNA damage foci that colocalized with the LacR- and TetR-bound arrays. The formation of translocations in individual cells, identified by colocalization between LacR and TetR, was visualized with ultrahigh-throughput imaging and confirmed by real-time PCR and sequencing. Translocation formation was cell cycle independent and peaked 36 hours after introduction of ISceI. Time-lapse microscopy of several thousand cells showed that upon DSB formation, the DSBs (marked by LacR or TetR) moved in a random, discontinuous manner, with some LacO-TetO array pairs transiently moving into spatial proximity and others persistently pairing, indicative of a translocation. The DSBs did not separate before translocation, and DSBs in close proximity were more likely to pair. Using this system, the authors found that inhibition of DNA-dependent protein kinase (DNAPK) specifically and dramatically increased translocation frequency. In contrast, meiotic recombination 11 homolog (MRE11) inhibition reduced DSB pairing efficiency and thus prevented translocations. Real-time detection of chromosomal translocations with this experimental system has the potential to provide new insights into the spatiotemporal dynamics of chromosomal translocations as well as the factors directly required for their formation.