Abstract
.We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in 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 , which simultaneously photoactivate a matrix pattern of GFP-labeled histones, with spots 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 a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent 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.
Highlights
Dynamic motions of chromatin are thought to critically influence genomic processes such as gene expression, DNA replication, DNA repair, and the biogenesis of genomic translocations
Most studies on chromatin mobility rely on tracking artificial DNA arrays integrated in the genome,[2,3,4] or more recently, DNA repeats by CRISPR/dCas[9] imaging.[5,6]
The optical method used for photoactivation is critical, and conventional scanning confocal microscopy commonly used for analysis does not allow to simultaneously illuminate and photoactivate multiple subcellular regions
Summary
Dynamic motions of chromatin are thought to critically influence genomic processes such as gene expression, DNA replication, DNA repair, and the biogenesis of genomic translocations. Most studies on chromatin mobility rely on tracking artificial DNA arrays integrated in the genome,[2,3,4] or more recently, DNA repeats by CRISPR/dCas[9] imaging.[5,6] These tracking approaches have several limitations. Photoactivatable histone probes have been used as an alternative to study chromatin motions in a near-native chromatin context.[8,9,10] With this approach, the optical method used for photoactivation is critical, and conventional scanning confocal microscopy commonly used for analysis does not allow to simultaneously illuminate and photoactivate multiple subcellular regions
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