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. 2022 Jan 12;6(1):010902.
doi: 10.1063/5.0069286. eCollection 2022 Mar.

Evaluation of chromatin mesoscale organization

Dana Lorber  1 , Talila Volk  1
Affiliations

Evaluation of chromatin mesoscale organization

Dana Lorber et al. APL Bioeng. .

Abstract

Chromatin organization in the nucleus represents an important aspect of transcription regulation. Most of the studies so far focused on the chromatin structure in cultured cells or in fixed tissue preparations. Here, we discuss the various approaches for deciphering chromatin 3D organization with an emphasis on the advantages of live imaging approaches.

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Figures

FIG. 1.
FIG. 1.
Comparison between images of chromatin in live vs fixed conditions. (a), (b)—(a) scheme of live (a) or fixed (b) nuclei and their corresponding optical sections at the Z-axis ilustrates the different outcome in terms of chromatin organization in fixed vs live conditions. (c)—a scheme ilustrates the locations of two LacO insertion sites in chromosome 2. For labeling the DNA, we used a fly line that contains two insertions of multiple (256) repeats of LacO sequences in the right arm of chromosome 2 at genomic locations 57 A and 60AB, which are 3.45 Mbp apart (FBst0025375). In the case of linear DNA fiber, this distance would extend to roughly 1173 μm from point to point. Labeling of the LacO sequences is performed by crossing this fly line with a fly line expressing LacI-GFP under the control of heat shock promoter, enabling visualization of the two lacO sequences with GFP in the progeny larvae following a short exposure to heat shock. (d), (g), and (j)—schemes show the plane of the optical sections through the nuclei relative to the muscle sarcomeres and correspond to the images shown in (e), (f), (h), and (i), and (k), (l). (e), (f), (h), and (i) are distinct X-Y planes from confocal z-stacks. (k) and (l) illustrate confocal Z-stacks of the corresponding nuclei shown in (e), (f), (h), and (i), taken in a plane perpendicular to the XY plane. Note the peripheral chromatin organization observed in the live larvae relative to the homogenous localization in the fixed nucleus. The estimated height of the live nucleus is 9.1 μm with an aspect ratio (length of short axis divided by the length of long axis) of 0.74. The height of the fixed nucleus is 6.5 μm, and its aspect ratio is 0.41. Movies of these nuclei are shown in the supplementary material. The live and fixed larval muscle nuclei were imaged using a similar imaging methodology as described previously.
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