Fig. 4.

H1 is necessary to secure transcriptional state-specific nucleosomal and epigenetic profiles yet influence only a moderate gene fraction. a Nucleosome distribution profiles clearly define distinct gene classes according to expression levels in wild type but no longer in 3h1. Quintiles 5 to 1 represent categories of genes with expression levels ranked from the highest to the lowest level, respectively, as previously described [28]. b H1 depletion induces moderate changes in the transcriptional profile, yet a subset of 701 genes (p < 0.05 and fold change > 2) is misregulated. The volcano plot was cropped around the denser part of the dataset. The full plot is presented in Additional file 1: Figure S10. c Upregulated loci show a characteristic nucleosome occupancy with high periodicity and a higher coverage in 3h1 downstream the TSS. TSS, transcription start site. d, e Decreased abundance of H3K27 and H3K4 trimethylation (green, immunosignals (Ab); red, propidium iodide (PI) counterstaining) in 3h1 measured by quantitative immunostaining on isolated leaf nuclei. The graphs shows the relative abundance of these respective histone modifications (Ab/PI ratio relative to wt). Scale bar, 2 μm. f Genes which are upregulated (p < 0.05) in 3h1 share a significant overlap with H3K27me3 targets defined by [52] (p = 0.0007, Fisher exact test) but remain distinct from those affected by the clf-29 mutation [53]. g A twofold reduction of H3K27me3 levels upon H1 depletion is detected by Western blot on chromatin extracts from seedlings yet is less dramatic than in a loss of PRC2 function mutant, clf-29. Left panel: the blots show H3K27me3 and total histone H4 levels (loading control) detected on the same membrane. A dilution series of wild-type samples, together with the absence of signal saturation in luminescence imaging, allows showing proportionality of detection signals with protein amounts. The two original gel blots are presented in Additional file 1: Figure S15. Right panel: the histogram shows mean H3K27me3 relative to H4 signals from three individual biological replicates. The wild-type level has been arbitrarily set to 1. 3h1, triple H1 mutant; 2h1, h1.1h1.2 double mutant