Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

Liu, Jing; Vidi, Pierre‐Alexandre; Lelièvre, Sophie A.; Irudayaraj, Joseph

doi:10.1242/jcs.161885

Cited by 19 publications

(13 citation statements)

References 39 publications

(51 reference statements)

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“…Importantly, values of D measured with the DOE system in live cells closely matched previous measurements of chromatin diffusion in mammalian cells performed with different methods (20). We reported previously that DNA damage causes a transient decrease in chromatin diffusion in U2OS cells (21). This effect was clearly apparent when comparing D values from cells treated with the radiomimetic drug bleomycin (BLM) to control, untreated cells (Fig.…”

Section: Chromatin Trackingsupporting

confidence: 85%

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Structured illumination to spatially map chromatin motions

Bonin

Smelser

Moreno

et al. 2017

Preprint

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Abstract. We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in the nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340±30 nm, which simultaneously photoactivate a 7x7 matrix pattern of GFP-labeled histones, with spots 1.70 µm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals DNA damage-dependent decrease in chromatin diffusion. The DOE instrumentation can easily and cheaply be implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in non-adherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies.

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Section: Chromatin Trackingsupporting

confidence: 85%

“…Previously, a paired-particle tracking approach was implemented to determine chromatin D, using a confocal microscope for photoactivation and imaging of two PAGFP-H2A spots (21).…”

Section: Discussionmentioning

confidence: 99%

Structured illumination to spatially map chromatin motions

Bonin

Smelser

Moreno

et al. 2017

Preprint

Self Cite

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“…The techniques that have proven to provide the most informative data for the assessment of nucleosome stability include biochemical or biophysical measurements on isolated or reconstituted nucleosomes 2 , 18 – 26 , approaches based on metabolic labeling 27 , 28 , biochemical strategies embedded in genomics approaches 29 – 31 , single-molecule 32 magnetic tweezer or FRET measurements 33 – 37 , proteomic analyses 27 , 28 , 38 , 39 and microscopic studies using transfected histones fused with fluorescent 40 – 42 and photo-activatable proteins 43 , 44 . The above methods assess dissociation of histones from the nucleosomes either in live cells where it occurs spontaneously, or when purified or reconstituted nucleosomes are exposed to different ionic environments, or by evoking changes of superhelicity with the help of mechanical torsion or intercalators.…”

Section: Introductionmentioning

confidence: 99%

“…S11A,B . References: (A): 27 , 28 , 38 , 39 , 43 , (B): 2 , 18 – 26 , 32 – 37 , 45 , (C): 29 – 31 , 46 , (D): 40 – 44 .…”

Section: Introductionmentioning

confidence: 99%

Nucleosome stability measured in situ by automated quantitative imaging

Imre

Simándi

Horváth

et al. 2017

Sci Rep

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Current approaches have limitations in providing insight into the functional properties of particular nucleosomes in their native molecular environment. Here we describe a simple and powerful method involving elution of histones using intercalators or salt, to assess stability features dependent on DNA superhelicity and relying mainly on electrostatic interactions, respectively, and measurement of the fraction of histones remaining chromatin-bound in the individual nuclei using histone type- or posttranslational modification- (PTM-) specific antibodies and automated, quantitative imaging. The method has been validated in H3K4me3 ChIP-seq experiments, by the quantitative assessment of chromatin loop relaxation required for nucleosomal destabilization, and by comparative analyses of the intercalator and salt induced release from the nucleosomes of different histones. The accuracy of the assay allowed us to observe examples of strict association between nucleosome stability and PTMs across cell types, differentiation state and throughout the cell-cycle in close to native chromatin context, and resolve ambiguities regarding the destabilizing effect of H2A.X phosphorylation. The advantages of the in situ measuring scenario are demonstrated via the marked effect of DNA nicking on histone eviction that underscores the powerful potential of topological relaxation in the epigenetic regulation of DNA accessibility.

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“…At great length scales, the movements of genome domains were followed by confocal microscopy using fluorescently tagged histones (55,56), generally performed after partial photo-bleaching or photo-activation. The minimal area that can be studied with this approach is defined by the size of the laser spot used to photo-bleach or photoconvert fluorescent histones (57), which encompasses several Mb to Gb of chromatin. Live cell tracking of telomeres over several minutes corroborated the notion of self-contained chromosome domains in yeast (58) and metazoan cells (59).…”

Section: -Chromosome Labeling Technologiesmentioning

confidence: 99%

Capturing Chromosome Structural Properties From Their Spatial and Temporal Fluctuations

Bancaud

Gadal

Bystricky

2017

Epigenetics and Systems Biology

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Running title: Live cell microscopy to determine chromosome structure Abstract Chromosome folding and nuclear organization are influenced by cellular processes while regulating genome functions. Intense efforts based on interdisciplinary research at the forefront of physics, mathematics, molecular and cellular biology and biochemistry have contributed to a better understanding of the physical properties and mechanisms of chromosome conformation.In this chapter, we focus on technologies for capturing chromosome spatial and temporal fluctuations based on the realization that chromatin conformation is highly dynamic at every length and time scale. We describe state of the art chromatin labeling technologies, focusing on optimizing yield of signal-to-noise ratio, high-throughput automated imaging techniques, and physical modeling. Systems biology approaches to map chromosome conformation using single or multiple labeled loci are illustrated, and implications of chromosome structural fluctuations on biological functions are discussed.

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Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

Cited by 19 publications

References 39 publications

Structured illumination to spatially map chromatin motions

Structured illumination to spatially map chromatin motions

Nucleosome stability measured in situ by automated quantitative imaging

Capturing Chromosome Structural Properties From Their Spatial and Temporal Fluctuations

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