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Structural organization of the chromatin is a complex process that involves the association of distinct structural components with specific DNA sequences and contributes to control the accessibility of the DNA to a number of proteins, some of them involved in transcription. Recent studies have provided an insight into the structural determinants of chromatin organization and dynamics (4–7). Here we use live cell microscopy approaches to demonstrate that bidimensional (2D) chromatin organization has a direct contribution to DNA antibiotic-induced chromatin modifications. We observed the formation of chromatin loops, partial uncoupling of chromosomes, and an altered polarity of locus repositioning in the presence of DNA-damaging agents in human fibroblast cells expressing histone H2B-GFP. These data strongly support a view suggesting that 3D chromatin organization is directly controlled by the surrounding environment. These chromatin features are not limited to the fibroblasts, since we also observed similar changes in the satellite cell nucleus of the somitic muscle precursor cells in the live zebrafish embryo (8).
To establish a link between DNA repair dynamics and chromatin modifications, we followed in live cells the change in accessibility of DNA to the endonucleases HaeIII or PshAI after selectable and specific double-strand breaks (dsbs) induced at defined positions in the 293T cell line, stably expressing the human RAD51 recombination mediator protein (hRAD51.GFP) (9). Using the ClonoTrack system we observed the formation of chromatin loops surrounding or protruding from the dsbs, as demonstrated by the heteromorphic, non-uniform loading of the Alexa Fluor 488–labeled HaeIII and PshAI molecules (Fig. 2a,b). During the repair process, PshAI-labeled DNA was relocated to the nuclear periphery, where it remained for a short period of time. In contrast, HaeIII-marked DNA rapidly migrated from inside the loop to the perinuclear region. We also performed a time-lapse analysis of the induction and dissociation of RPA-coated DNA domains in response to DNA damage or inactivation of DNA-PKcs. This analysis indicated that the RPA-rich areas are highly dynamic and their organization is sensitive to modulation by DNA repair dynamics (Fig. 2c,d). d2c66b5586