Fig 2: Histone H1 represses the deposition of H3K36me3 by SETD2.a Schematic drawing of the nucleosome bound with a linker histone H1. b HMT assays performed with SETD2 toward the 197-bp nucleosomes bound with histone H1.4. The input of the HMT reactions is shown in Supplementary Fig. S5a. c Integrative Genomics Viewer (IGV) tracks representing the distributions of H3K36me3, H1.4 and IgG ChIP-Seq results with two replicates. Rep1, replicate 1, Rep2, replicate 2. d Venn diagram illustration showing the overlap of H3K36me3, H1.4 and IgG peaks. The peaks in independent replicates were combined. e The percentages of H3K36me3, H1.4 and IgG peaks in different genomic elements. ChIP-Seq results in independent replicates were combined. The distributions were analyzed by HOMER with default parameters. f The enrichments of H3K36me3, H1.4 and IgG ChIP-Seq results at H1.4 peak regions. A 100-bp sliding window was used to scan 10-kb regions surrounding the peak center. Heatmaps illustrating ChIP-Seq read densities from 5 kb upstream to 5 kb downstream of the peak regions (in rows) were shown on a per-peak basis (in columns). g The distribution profiles of normalized H3K36me3, H1.4 and IgG read density from 5 kb upstream of the TSS to 5 kb downstream of the TES.

Fig 3: RNA-seq reveals H1 subtype-specific effects on differentiation and an eosinophilic signature of undifferentiated H1.2/H1.4 knockout lines.(a) Concordantly and discordantly regulated genes when comparing PLB-985 differentiation (wt and scr., d0 to d7) and human neutrophil differentiation (Blueprint, neutrophilic myelocyte to segmented neutrophil of bone marrow) using discordance scores and tmod. Most modules are concordantly regulated. (b) Volcano plot of all differentially expressed genes of control cells (wt and scr.) between d7and d0, darker gray depicts p<0.05 and logFC >2. A selection of strongly upregulated genes is labelled and neutrophil markers are highlighted in red. Expression of granule proteins, such as MPO, neutrophil elastase (ELANE), proteinase 3 (PRTN3) and cathepsin G (CTSG) decreases during differentiation. (c) Principal component analysis (PCA) of control and mutant PLB-985 during differentiation shows clustering according to experimental replicates and genotypes and separates samples according to differentiation state. Lines indicate the center-of-mass trajectory for ‘normal’ (wild type and scr. cells), ‘hyper’ (H1 clones with enhanced differentiation) and ‘hypo’ (H1 clones with reduced differentiation) samples. While lines deficient in H1.1, H1.3 and H1.5 already at d3 cluster closely to wt and scr. at d7, indicating faster and more efficient differentiation, H1.2 and H1.4-deficient clones at d7 still only cluster with wt and scr. at d3, indicating a stalled development.(d) Scatter dot plot depicting differentially expressed genes of H1.2 knockouts vs control cells at d0 of differentiation (x axis) and H1.4 knockouts vs control cells at d0 of differentiation (y axis), color (alpha) indicates p-value of differential expression between H1.2 and H1.4 at d0. Neutrophil genes such as neutrophil elastase (ELANE), proteinase 3 (PRTN3), cathepsin G (CTSG), azurocidin (AZU1) and MPO are strongly downregulated in both conditions when compared to wt and scr., whereas eosinophil genes such as galectin 10 (CLC), interleukin-5 receptor alpha (IL5RA), GATA-1 (GATA1), GATA-2 (GATA2) or eosinophil peroxidase (EPX) are upregulated. (e) Gene set enrichment analysis of indicated samples at d0 of differentiation shows enhanced expression of eosinophil and GATA-1 gene sets (see Materials and methods) in H1.2 and H1.4-deficient lines, but not in other knockout conditions.Figure 4—source data 1.RNA-seq expression tables.The individual sheets display expression values of indicated conditions (Cont. is wild type and scr. combined, for the respective H1 subtypes values of 2 clones are combined).

Fig 4: H1 effects on PLB-985 differentiation are subtype-specific.(a, b) Expression of the surface marker CD11b at d3 (a) or d7 (b) of differentiation. Depicted is the percentage of CD11b-positive cells out of all living singlets for wt, scrambled (scr.) and two knockout clones per H1 subtype. (c, d) Growth curves of PLB-985 during differentiation (c) and cell counts at d7 of differentiation (d) show restricted growth of H1.1, H1.3 and H1.5-deficient cells and enhanced growth of H1.2 and H1.4-deficient clones. (a–d) Depicted are five independent experiments, error bars are SEM. (e, f) Viability, as determined by DAPI negative cells, at d8 (e) and d12 (f) of differentiation of 3 independent experiments. Error bars are SEM.

Fig 5: H1.2 and H1.4 are required for PLB-985 differentiation.(a) mRNA expression levels of indicated H1 subtypes (nomenclature refers to protein names) in PLB-985 cells at d0, d3 and d7 of differentiation, relative to the housekeeping gene ß-microglobulin, depicted is the mean -/+ SEM of 3 independent experiments. (b) mRNA expression levels of indicated H1 subtypes in human primary neutrophils, relative to ß-microglobulin. Data points are from four different donors, error bars are mean -/+ SEM. (c) PMA-induced cell death of wild type (wt) scrambled sgRNA (non-target, scr.) and two clones of either H1.2 or H1.4 over time, measured by SYTOX Green fluorescence. Depicted are mean -/+ SEM of 5 independent experiments of cells at d7 of differentiation. (d) Measurement of PMA-induced ROS production from wt, scr. and H1.2 or H1.4 deficient PLB-985, depicted are mean -/+ SEM of 5 independent experiments at d7 of differentiation. (c, d) wt and scr. values are the same in the left and right panels, respectively. (e) Western blot of lysates of PLB-985 at d3 of differentiation, showing efficient disruption of H1.2 and H1.4 (both antibodies recognize the other subtype, which is marked by a red asterisk for H1.2 and a blue asterisk for H1.4) as well as reduced MPO expression, the core histone H3 served as loading control.