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Abstract Although cancer initiation and progression are generally associated with the accumulation of somatic mutations^1,2, substantial epigenomic alterations underlie many aspects of tumorigenesis and cancer susceptibility^3–6, suggesting that genetic mechanisms might not be the only drivers of malignant transformation⁷. However, whether purely non-genetic mechanisms are sufficient to initiate tumorigenesis irrespective of mutations has been unknown. Here, we show that a transient perturbation of transcriptional silencing mediated by Polycomb group proteins is sufficient to induce an irreversible switch to a cancer cell fate inDrosophila. This is linked to the irreversible derepression of genes that can drive tumorigenesis, including members of the JAK–STAT signalling pathway andzfh1, the fly homologue of theZEB1oncogene, whose aberrant activation is required for Polycomb perturbation-induced tumorigenesis. These data show that a reversible depletion of Polycomb proteins can induce cancer in the absence of driver mutations, suggesting that tumours can emerge through epigenetic dysregulation leading to inheritance of altered cell fates. 

Pericentromeric heterochromatin is mostly composed of repeated DNA sequences, which are prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins that drive directed motions. Understanding this pathway requires the detection of nuclear actin filaments that are significantly less abundant than those in the cytoplasm, and the imaging and tracking of repair sites for long time periods. Here, we describe an optimized protocol for live cell imaging of nuclear F-actin in Drosophila cells, and for repair focus tracking in mouse cells, including: imaging setup, image processing approaches, and analysis methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair. 

Heterochromatin is mostly composed of long stretches of repeated DNA sequences prone to ectopic recombination during double-strand break (DSB) repair. In Drosophila, “safe” homologous recombination (HR) repair of heterochromatic DSBs relies on a striking relocalization of repair sites to the nuclear periphery. Central to understanding heterochromatin repair is the ability to investigate the 4D dynamics (movement in space and time) of repair sites. A specific challenge of these studies is preventing phototoxicity and photobleaching effects while imaging the sample over long periods of time, and with sufficient time points and Z-stacks to track repair foci over time. Here we describe an optimized approach for high-resolution live imaging of heterochromatic DSBs in Drosophila cells, with a specific emphasis on the fluorescent markers and imaging setup used to capture the motion of repair foci over long-time periods. We detail approaches that minimize photobleaching and phototoxicity with a DeltaVision widefield deconvolution microscope, and image processing techniques for signal recovery postimaging using SoftWorX and Imaris software. We present a method to derive mean square displacement curves revealing some of the biophysical properties of the motion. Finally, we describe a method in R to identify tracts of directed motions (DMs) in mixed trajectories. These approaches enable a deeper understanding of the mechanisms of heterochromatin dynamics and genome stability in the three-dimensional context of the nucleus and have broad applicability in the field of nuclear dynamics.