Short Exposure to the DNA Intercalator DRAQ5 Dislocates the Transcription Machinery and Induces Cell Death

Richard, Élodie; Causse, Sébastien; Spriet, Corentin; Fourré, Nicolas; Trinel, Dave; Darzacq, Xavier; Vandenbunder, Bernard; Héliot, Laurent

doi:10.1111/j.1751-1097.2010.00852.x

Cited by 6 publications

(5 citation statements)

References 18 publications

(27 reference statements)

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“…DRAQ5 has been used for quantitative DNA labelling of living cells because of its high cell permeability and DNA binding, deep red fluorescence and photostability (Martin et al., ; Edward, ). At the same time, DRAQ5 is known for its effect to irreversibly change DNA‐binding capacity of chromatin‐associated proteins such as polymerases, repair proteins, transcription factors, PcG CBX8 protein and even some histones, and their release from chromatin of live cells (Mari et al., ; Richard et al., ).…”

Section: Resultsmentioning

confidence: 99%

Dynamics of Polycomb chromatin domains under conditions of increased molecular crowding

Šmigová

Jůda

Bártová

et al. 2013

Biology of the Cell

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Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Brno, Czech Republic Background Information. A Polycomb (PcG) body is an orphan nuclear subcompartment characterised by accumulations of Polycomb repressive complex 1 (PRC1) proteins. However, seemingly contradictory reports have appeared that describe the PcG bodies either as protein-based bodies in the interchromatin compartment or chromatin domains. In this respect, molecular crowding is an important factor for the assembly and stability of nuclear subcompartments. In order to settle this contradiction, crowding experiments, that represent a convenient model distinguishing between interchromatin and chromatin compartments, were carried out.Results. In sucrose-hypertonically induced crowding, we observed in U-2 OS cells that PcG bodies disappeared, but persisted as nuclear domains characterised by accumulations of DNA. This phenomenon was also observed in cells hypertonically treated with sorbitol and NaCl. Importantly, the observed changes were quickly reversible after re-incubation of cells in normal medium. We found that the PcG foci disappearance and the dissociation of PRC1 proteins (BMI1 and RING1a proteins) from chromatin were associated with their hyper-phosphorylation. In addition, under hyper-and hypotonic conditions, the behaviour of the PcG bodies differed from that of the typical nucleoplasmic body.Conclusion. PRC1 proteins accumulations do not represent a genuine nuclear subcompartment. The PcG body is a chromosomal domain, rather than a nucleoplasmic body.

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Section: Resultsmentioning

confidence: 99%

Dynamics of Polycomb chromatin domains under conditions of increased molecular crowding

Šmigová

Jůda

Bártová

et al. 2013

Biology of the Cell

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“…As such, ChromEMT provides a facile and universal method to compare the structure of genomic DNA in different kingdoms of life. To offset the possibility that DRAQ5 binding could disrupt DNA interactions (49, 50), we fixed cells with glutaraldehyde before labeling. However, glutaraldehyde cross-linking could induce higher-order structures that are not present in living cells.…”

Section: Discussionmentioning

confidence: 99%

“…S1, D and E). In live cells, DRAQ5 binding to DNA could potentially displace RNA polymerase II–transcription factor complexes (49) and histone H1 (50). Therefore, we fixed cells and DNA-chromatin complexes with glutaraldehyde before staining DNA with DRAQ5.…”

Section: A Cell-based Screen Identifies a Fluorescent Dna-binding Dyementioning

confidence: 99%

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

Phan

Deerinck

et al. 2017

Science

743

665

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The chromatin structure of DNA determines genome compaction and activity in the nucleus. On the basis of in vitro structures and electron microscopy (EM) studies, the hierarchical model is that 11-nanometer DNA-nucleosome polymers fold into 30- and subsequently into 120- and 300-to 700-nanometer fibers and mitotic chromosomes. To visualize chromatin in situ, we identified a fluorescent dye that stains DNA with an osmiophilic polymer and selectively enhances its contrast in EM. Using ChromEMT (ChromEM tomography), we reveal the ultrastructure and three-dimensional (3D) organization of individual chromatin polymers, megabase domains, and mitotic chromosomes. We show that chromatin is a disordered 5- to 24-nanometer-diameter curvilinear chain that is packed together at different 3D concentration distributions in interphase and mitosis. Chromatin chains have many different particle arrangements and bend at various lengths to achieve structural compaction and high packing densities.

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“…However, it is important to note that DRAQ5 exposure has been demonstrated to cause cell damage with modications of the cell cycle progression. 35,36 A promising method for life cell imaging of the nuclear dynamics is the uorescent tagging of proteins of the nucleosome, for instance by expression of an articial histone H2B-GFP gene. 37…”

Section: Mechanical Properties Of the Nucleusmentioning

confidence: 99%

Cell confinement: putting the squeeze on the nucleus

et al. 2013

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The nucleus has long been considered as a passive compartment containing the genetic information.However, recent attention to its structure, mechanical properties and physical connections with other cellular compartments has shown that the nucleus changes dynamically its morphology and internal organization for important cellular processes, especially those associated with cellular confinement. In this paper, we review some recent progress in experimental investigations of nuclear squeezing that lead to a better understanding of the nuclear remodeling in response to various situations of cellular confinement. We will discuss compelling examples of original experiments performed with microsystems that have recently brought new insights into the close relationship between nuclear mechanics and cellular organization. We will show that the study of nuclear confinement with microsystems has opened up new experimental avenues that already offer promising clues for understanding diseases that are associated with defective nuclear mechanics.

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Short Exposure to the DNA Intercalator DRAQ5 Dislocates the Transcription Machinery and Induces Cell Death

Cited by 6 publications

References 18 publications

Dynamics of Polycomb chromatin domains under conditions of increased molecular crowding

Dynamics of Polycomb chromatin domains under conditions of increased molecular crowding

ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells

Cell confinement: putting the squeeze on the nucleus

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