Linker Histone H1.2 And H1.4 Affect The Neutrophil Lineage ... - ELife

There are five somatic replication-dependent (H1.1 – H1.5) and two replication-independent H1 subtypes (H1.0 and H1X). PLB-985 expressed mRNA encoding H1.2 and H1.4 more abundantly than the other subtypes (Figure 2a). The expression of replication-dependent H1 subtypes occurs mainly in the S phase of proliferating cells and, accordingly, H1 mRNA levels decreased during PLB-985 differentiation (Figure 2—figure supplement 1a, same experiments as in Figure 2a, but plotted as relative to d0). As expected, this reduction was more evident for subtypes with a higher baseline expression (Figure 2a, Figure 2—figure supplement 1a). When we looked at H1.2 and H1.4 protein expression of PLB-985 during differentiation, we found that protein levels also decreased as the cells matured (Figure 2—figure supplement 1b).

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We also analyzed two datasets comparing mRNA and protein expression of human primary bone marrow cells undergoing differentiation into neutrophils at various stages of maturation, namely myelocytes, metamyelocytes, band form neutrophils and segmented neutrophils (Adams et al., 2012; Hoogendijk et al., 2019) (see Materials and methods section for RNA-seq). Consistent with our PLB-985 data, we found a similar reduction of H1 mRNA as maturation progressed (Figure 2—figure supplement 1c). The protein levels of H1 subtypes in these datasets remained relatively stable during differentiation, despite a reduction in mRNA (Figure 2—figure supplement 1c,d). Hence, all H1 subtypes are expressed in differentiating primary neutrophils and although the mRNA levels decrease over time of differentiation, the protein levels seem to be relatively stable.

mRNA levels of each H1 subtypes were still detectable in differentiated PLB-985 and primary cells. Accordingly, human primary neutrophils isolated from blood of healthy donors expressed detectable amounts of the H1 genes. H1.2 and H1.4, the two hits in our genome wide screen, were the most abundantly expressed subtypes (Figure 2b). Interestingly, peripheral blood mononuclear cells (PBMCs) and monocytes showed a slightly different expression pattern of this gene family than neutrophils (Figure 2—figure supplement 1e,f).

To verify the results from our screen, we disrupted expression of H1.2 and H1.4 in PLB-985 by targeting these genes with CRISPR/Cas9. We used two clones deficient for H1.2 (from two sgRNAs) and two clones deficient for H1.4 (from one sgRNA) in our analysis. Cell death was reduced in both H1.2 and H1.4-deficient clones, confirming the results of the screen (Figure 2c). H1.2 and H1.4-deficient clones produced ROS less efficiently than wild type cells upon PMA stimulation (Figure 2d). Furthermore, cells deficient for either of the two H1 subtypes expressed less MPO (Figure 2e). Cells deficient for H1.2 or H1.4 could still phagocytose, demonstrating that not all effector functions were equally affected by the loss of H1 subtypes (Figure 2—figure supplement 1g,h). These findings suggest that H1.2 and H1.4 are required for PLB-985 to differentiate into mature, neutrophil-like cells.