Fig. 3

H1 depletion has a strong impact on euchromatin organization with increased dispersion of nanoscopic domains, altered distribution of nucleosome coverage, and increased mobility. a, b H1 is abundant in euchromatin distributed as discrete foci partially colocalizing with H2B. a H1 immunostaining and propidium iodide (PI) counterstaining as in Fig. 1 (fixed, spread nuclei; scale bar, 2 μm). b Representative distribution of GFP- and RFP-tagged H1.1 and H2B imaging in the nuclei from fresh root tissue (epidermis, single plane, confocal laser scanning microscopy). The inset shows a close up (1 × 1 μm) in euchromatin displaying H1-enriched regions (dominant green signals) at the nanoscale, but below optical resolution. Scale bar, 1 μm. c–f Ultrastructural analysis of euchromatin organization in wt vs 3h1. c Typical TEM image of the nuclei stained with uranylacetate on 7-nm cryosection (root epidermis, see the “Methods” section). Scale bar, 1 μm. d Representative region of interest (ROI) in euchromatin of wt and 3h1 nuclei used for spatial autocorrelation function (ACF) analyses. Scale bar, 500 nm. e, f Spatial chromatin density analyses show decreased regularity in the spatial chromatin distribution pattern in 3h1 revealed by a less shallow ACF curve within length scales of 20–60 nm (gray zone, graph, e) and higher dispersion of length scales as shown by a bigger range of the estimate D characterizing the spatial autocorrelation fit (f). These differences in 3h1 are restored upon complementation with an H1.1 expressing construct. ***Unpaired t test, p < 0.001, see also Additional file 1: Figure S8. g Nucleosome coverage but not qualitative distribution is altered in H1-depleted euchromatin. Antagonist effects are seen for regions of chromatin states CS1, 3, and 7 (CS1 only is shown here) and CS4 (CS according to [40]), see also Additional file 1: Figure S6 for nucleosome occupancy in 3h1 and wt over regions from all chromatin states. h H2B-RFP fluorescence recovery is ~ 2.5-fold faster in 3h1 compared to in wild-type as measured in FRAP experiments, see the “Methods” section. i Histone acetylation levels are lower in the 3h1 leaf nuclei compared to wt; t test, p < 0.001, see also Additional file 2: Table S1. Scale bar, 2 μm