Alterations induced by the PML-RARα oncogene revealed by image cross correlation spectroscopy

Description

The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. To un-derstand how oncogenes affect chromatin architecture, it is important to visualize fundamental processes such as DNA replica-tion and transcription in intact nuclei and quantify the alterations of their spatiotemporal organization induced by oncogenes. Here, we apply superresolution microscopy in combination with image cross correlation spectroscopy to the U937-PR9 cell line, an in vitro model of acute promyelocytic leukemia that allows us to activate the expression of the PML-RARa oncogene and analyze its effects on the spatiotemporal organization of functional nuclear processes. More specifically, we perform Tau-stimulated emission depletion imaging, a superresolution technique based on the concept of separation of photons by life-time tuning. Tau-stimulated emission depletion imaging is combined with a robust image analysis protocol that quickly produces a value of colocalization fraction on several hundreds of single cells and allows observation of cell-to-cell variability. Upon acti-vation of the oncogene, we detect a significant increase in the fraction of transcription sites colocalized with PML/PML-RARa. This increase of colocalization can be ascribed to oncogene-induced disruption of physiological PML bodies and the abnormal occurrence of a relatively large number of PML-RARa microspeckles. We also detect a significant cell-to-cell variability of this increase of colocalization, which can be ascribed, at least in part, to a heterogeneous response of the cells to the activation of the oncogene. These results prove that our method efficiently reveals oncogene-induced alterations in the spatial organization of nuclear processes and suggest that the abnormal localization of PML-RARa could interfere with the transcription machinery, potentially leading to DNA damage and genomic instability.SIGNIFICANCE The molecular mechanisms that underlie oncogene-induced genomic damage are still poorly understood. Here, we combine image cross correlation spectroscopy with state-of-the-art superresolution microscopy to quantify alterations induced by the PML-RARa oncogene in an in vitro model of acute promyelocytic leukemia. We find that activation of the oncogene induces a significant increase in the fraction of transcription sites colocalized with PML/PML-RARa due to disruption of physiological PML bodies into a large number of PML-RARa microspeckles. We also detect a heterogeneous response of the cells to the activation of the oncogene. These results suggest that our image cross correlation spectroscopy-based approach can be useful for characterizing global alterations in the spatial organization of chromatin, in single cells, in response to oncogene activation, or in other triggering events.

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