Chromatin structure exhibits spatio-temporal heterogeneity within the cell nucleus

Abstract

Local chromatin compaction undergoes dynamic perturbations to regulate genetic processes. To address this, the direct measurement of the fluidity of chromatin structure is carried out in single live cells using steady-state anisotropy imaging and polarization modulation microscopy. Fluorescently tagged core and linker histones are used to probe different structural aspects of chromatin compaction. A graded spatial heterogeneity in compaction is observed for the chromatin besides the distinct positional ordering of core and linker histones. These spatio-temporal features are maintained by active processes and perturbed during death. With cell cycle, the distribution in compaction heterogeneity continually changes maximizing during M-G1 transition where it displays bimodal behavior. Such measurements of spatio-temporal chromatin fluidity could have broader implications in understanding chromatin remodeling within living cells.

FIGURE 1

(a) Color-coded anisotropy map of polytene chromosomes extracted from salivary glands of Drosophila larvae; (inset) polytene chromosomes in 600 mM NaCl; bar, 10 μm. (b) Anisotropy distribution for polytene chromosomes; (inset) anisotropy distribution of polytene chromosomes treated with 600 mM NaCl. (c) Color-coded anisotropy map of interphase HeLa cell nucleus; bar, 10 μm. (d) Anisotropy distribution of interphase HeLa cell nucleus; (inset) line scan of anisotropy values across the HeLa cell nucleus shown as the dashed line in Fig. 1 c. (e) Anisotropy distributions of normal cells as well as cells treated with TSA. (Inset) Bar graph for mean anisotropy (r) and standard deviation (sd(r)) of anisotropy across single nucleus averaged over 20 cells for normal and TSA-treated cells: TSA50 (50 ng/ml TSA, 4 h) and TSA150 (150 ng/ml TSA, 4 h) cells; representative intensity images of normal and TSA-treated (150 ng/ml, 4 h) cells.

FIGURE 2

(a) Color-coded anisotropy map of HeLa-EGFP, HeLa-H1.1-EGFP, and HeLa-H2B-EGFP cells when untreated (WT), ATP depleted (ATP dep), and treated with staurosporine (Stau). (b) Bar graph representation of mean anisotropy (r) and standard deviation of anisotropy (sd(r)) of all pixels inside a single nucleus averaged over 45 different cells in each case. Bar, 10 μm.

FIGURE 3

(a) Schematic of polarization modulation experiment. (b) Mean peak height and standard deviation (error bars) for untreated (WT) HeLa-H2B-EGFP, HeLa-H1.1-EGFP, HeLa-EGFP, and on ATP depletion (ATP dep) as well as staurosporine (Stau) treatment.

FIGURE 4

(a) Anisotropy distributions of H2B-EGFP from metaphase to interphase in a single HeLa cell nucleus. Anaphase (t = 0:17 min) onwards the anisotropy distributions of the left daughter cell has been plotted. (b) Panel shows color-coded anisotropy snapshots at corresponding time points. Bar, 10 μm.