Visually Tracking Translocations in Living Cells
Chromosomal translocations, the fusion of pieces of DNA from different chromosomes, are often observed in cancer cells and can even cause cancer. However, little is known about the dynamics and regulation of translocation formation. To investigate this critical process, Tom Misteli, Ph.D., in CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleague Vassilis Roukos, Ph.D., developed a novel experimental system that allowed the researchers to see, for the first time, translocations form in individual, live cells.
The system consists of cells with copies of the bacterial Lac operator sequence adjacent to a cleavage site recognized by the yeast restriction enzyme ISceI (LacO-ISceI) on chromosome 7 and copies of the bacterial Tet operator sequence flanking another ISceI recognition site (TetO-ISceI-TetO) on chromosome 1 and both copies of chromosome 10. The cells express fluorescently-tagged Lac repressor (GFP-LacR) and Tet repressor (mCherry-TetR), proteins which bind the LacO and TetO sequences, respectively, and allow the researchers to monitor visually these DNA regions. The investigators caused DNA double-strand breaks (DSBs) by expressing the ISceI enzyme.
Because the formation of translocations is a rare event, the scientists used ultrahigh-throughput imaging to find translocations in single cells. Following ISceI expression, they identified cells in which the GFP-LacR and mCherry-TetR colocalized, indicative of translocations. The percentage of such colocalizations increased from approximately 2 percent to around 7.5 percent after 24 hours and plateaued at about 12 percent at 36 hours. Likewise, the translocation frequency measured by PCR increased from 1 in 2000 cells after 12 hours of ISceI expression to 1 in 400 after 24 hours and 1 in 300 after 36 hours. These results suggest that after DSB formation, a significant number of ends pair up, but only a fraction of those pairs actually become translocations.
The researchers then asked whether there were differences in translocation formation throughout the cell cycle. They saw no difference in pairing frequency in fixed individual cells at different stages of the cell cycle, as measured by DAPI nuclear staining intensity. PCR analysis of LacO-TetO translocations in cells sorted by cell cycle stage and in cells arrested in the G1 or G2/M stage confirmed the lack of translocation cell cycle dependence. This is an important result as the cell-cycle behavior of translocations had been a much debated and speculated about topic.
To ultimately follow individual translocations in living cells, the investigators monitored GFP-LacR and mCherry-TetR signals in thousands of cells over 24 hours using time-lapse ultrahigh-throughput imaging. They found that the fraction of cells with paired LacR and TetR increased with time following ISceI expression. When the scientists examined individual cells over time, they saw the DSBs move randomly around the nucleus. Some DSBs moved close together and then underwent rounds of pairing and unpairing. A subset of pairs persisted and showed highly coordinated movements over several hours, suggesting a translocation had formed.
The researchers also saw that TetR chromosome ends generated by ISceI moved together toward LacR and that the number of TetR-bound sequences per cell was higher in cells with LacR-TetR pairs. These observations suggest that the TetR-bound ends do not separate until after a translocation forms and that the choice of a partner to repair the DSB occurs after the ends are close together.
To examine the role of spatial orientation on translocation further, the scientists determined the relative positions of DSBs just before persistent pairing. They saw that the increase in LacR-TetR pairs correlated with a decrease in cells with DSB separations of 2-6μm, suggesting that most pairs come from associations within this range. Reverse tracking of persistent pairs revealed that over 80 percent were located within 2.5μm in the 5 hours before translocation. A small fraction of pairs was further apart before joining, but the results indicate that the vast majority of translocations occur with proximally positioned ends. Again, this observation settles a long-standing debate in the field.
Finally, the investigators examined the role of the DNA repair machinery in DSB pairing and translocation formation. Reducing the activity of the ATM and ATR kinases, which are key players in the DNA damage response, via inhibitors or RNA interference, had no effect on the proportion of cells with LacR-TetR pairs. Inhibiting DNAPK activity, however, led to a nearly 10-fold increase in translocations, and simultaneously inhibiting DNAPK and ATM synergistically enhanced the number of translocations. When the researchers blocked the activity of Mre11, part of a DNA damage sensing complex, they found a reduced number of LacR-TetR pairs as well as a reduced frequency of translocations. The Mre11 inhibitor also prevented the increased translocation frequency from the DNAPK inhibitor, suggesting that Mre11 acts upstream of DNAPK to affect DSB pairing while DNAPK regulates translocation formation.
Together, these studies provide new insights into the regulation of events during the formation of translocations and pave the way for detailed mechanistic studies.Summary Posted: 09/2013
Roukos V, Voss TC, Schmidt CK, Lee S, Wangsa D, Misteli T. Spatial Dynamics of Chromosome Translocations in Living Cells. Science. August 9, 2013 PubMed Link