Fig. 2

Loss of H1 variants leads to global chromatin decondensation but is dispensable for heterochromatin identity. Cytogenetic (a–e, h-i) and nucleosome profile (f, g) analyses of chromatin organization in 3h1 triple mutant and wild-type segregant (wt) seedlings. a H1 depletion induces a significant reduction of the relative heterochromatin fraction (RHF) and in the number of chromocenters (CCs) as well as an increase in nuclear size (area). ***two-sided t test, p < 0.001; error bars, standard error to the mean (SEM). Cytological analyses on isolated, spread leaf nuclei: b Typical wt and 3h1 nuclei as used in a, stained with DAPI. Scale bar, 2 μm. c H1 depletion induces a spatial dispersion of the centromeric repeats (CEN, purple) but not of the 45S rDNA, nucleolar organization region repeats (NOR, green) as shown by fluorescence in situ hybridization (FISH). d 3D image segmentation of the CEN signals shows that the preferentially peripheral localization of CEN repeats is unaffected in 3h1 nuclei despite their lack of condensation. e High-resolution imaging and deconvolution-based reconstruction of 3h1 and wt nuclei. Nanoscopic bodies of condensed chromatin are dispersed throughout the nucleus in 3h1 instead of conspicuous chromocenters as in wt. f Nucleosome occupancy is lower in 3h1 heterochromatin, as defined by the chromatin states (CS) 8 and 9 [40]. g Distribution of nucleosomal repeat lengths (NRLs) in wt and 3h1, chi-square test, ***p < 0.0001. h, i The heterochromatic marks H3K9me1 and H3K27me1 are not reduced but redistributed in 3h1 nuclei. Scale bar, 2 μm. Isolated leaf nuclei were flow-sorted according to their 2C DNA content (a–e, h, i)