Linker Histone H1 subtypes specifically regulate neutrophil differentiation

Neutrophils are important innate immune cells that tackle invading pathogens with different effector mechanisms. They acquire this antimicrobial potential during their maturation in the bone marrow, where they differentiate from hematopoietic stem cells in a process called granulopoiesis. Mature neutrophils are terminally differentiated and short-lived with a high turnover rate. Here, we show a critical role for linker histone H1 on the differentiation and function of neutrophils using a genome-wide CRISPR/Cas9 screen in the human cell line PLB-985. We systematically disrupted expression of somatic H1 subtypes to show that individual H1 subtypes affect PLB-985 maturation in opposite ways. Importantly, H1 subtypes also affect neutrophil differentiation of murine bone marrow stem cells, demonstrating an unexpected subtype-specific role for H1 in granulopoiesis.


Introduction
Because multicellular organisms often face disturbances in their homeostasis, they evolved complex immune responses to deal with sterile and infectious insults.Like all cells in the human body, immune cells differentiate from multipotent precursors, which in the immune context are primarily hematopoietic stem cells in the bone marrow.One of these differentiation programs, called granulopoiesis, leads to the development of granulocytes -basophils, eosinophils and neutrophils.
Neutrophils are the most abundant leukocytes in humans.In homeostatic conditions, up to 2x10 11 neutrophils enter the blood stream per day and patrol the host's body until they sense signs of infection, which triggers them to leave the blood stream and migrate to the inflammatory site where they ensure pathogen removal.An efficient neutrophil response is crucial for human antimicrobial defense and, correspondingly, neutropenia is associated with severe infections 1 .
The three best described defense mechanisms of neutrophils are phagocytosis (engulfment of pathogenic microorganisms and subsequent destruction in phagosomes), degranulation (the release of antimicrobial proteins from granules to the extracellular space) and the formation of neutrophil extracellular traps (NETs).NETs are chromatin structures studded with antimicrobial proteins derived mostly from neutrophil granules, which can trap pathogens 2 .Neutrophils release NETs via various mechanisms, which in most cases lead to neutrophil death 3 ; thus, NETs are an extreme form of antimicrobial defense, as their release also eliminates the cell casting the NET.NET formation occurs in mature neutrophils and therefore we propose that specific cues during neutrophil differentiation shape their ability to form NETs.
Neutrophils differentiate from granulocyte-macrophage precursors.The first progenitor with a clear neutrophil lineage preference during granulopoiesis is the promyelocyte 4 .During maturation, neutrophils acquire a lobulated nuclear shape and produce many of their antimicrobial effector proteins, stored in granules 5 .Granulopoiesis requires balanced activity of various transcription factors 4 .The C/EBP family, especially C/EBP- and -, are key regulators of neutrophil differentiation 6,7 .Other transcription factors can antagonize C/EBP-dependent neutrophil development.

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The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint For example, expression of GATA-1 8 and GATA-2 9 induce eosinophil, basophil or mast cell differentiation 10 . Differentiation programs rely both on transcription factor activation and on epigenetic chromatin alterations.DNA wraps around an octamer of core histones forming a nucleosome.The linker histone H1 binds to nucleosomes and further compacts chromatin 11 .There are 11 different H1 subtypes in mice and humans.Five of them (H1.1 -H1.5) are somatic replication-dependent subtypes, two (H1.0 and H1X) are somatic and replication-independent and four (H1oo, H1t, H1T2 and HILS1) are germline-specific 12 .All H1 subtypes consist of a highly conserved globular domain and two more variable regions: the N-and the C-terminal tails.Individual H1 subtypes are more conserved between species than H1 subtypes are within one species, suggesting that the conservation of different subtypes has functional relevance 12 .Still, some functions of H1, such as global shaping of chromatin structure, are redundant between subtypes.This is reflected by the fact that mice deficient for one or even two H1 subtypes are viable and fertile without striking morphological abnormalities 13 .However, mice lacking three H1 subtypes die in utero, with total H1 levels reduced to about 50% 13 .Appropriate H1 expression is therefore essential for development.Despite some redundancy, H1 subtypes have specific functions.H1 subtypes bind to chromatin with different affinities and knock down of specific H1 subtypes affects gene transcription in distinct ways [14][15][16] .
The role of H1 and H1 subtypes in immunity is poorly understood.H1 contributes to the silencing of pro-inflammatory cytokines after endotoxin challenge 17 , as well as to the silencing of interferonstimulated genes 18 .Interestingly, mice deficient in H1.0 have fewer dendritic cells, but normal numbers of granulocytes, macrophages and lymphocytes, showing a subtype-specific role for H1 in dendritic cell differentiation 19 .However, to date, no studies have systematically addressed H1 subtype involvement in the maturation and function of human immune cells Here we used the human neutrophil-like cell line, PLB-985, to study neutrophil maturation and function.We performed a genome-wide CRISPR/Cas9 screen using survival to a NET-inducing stimulus, phorbol 12-myristate 13-acetate (PMA), as a readout.Surprisingly, we found that depletion 3 was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint of either H1.2 or H1.4 strongly reduced the differentiation of PLB-985 and thereby their ability to form NETs. Notably, deficiency of the other somatic H1 subtypes, H1.1, H1.3 and H1.5, accelerated PLB-985 maturation.RNA-seq analysis showed that H1.2 and H1.4 deficiency leads to an upregulation of eosinophil genes in PLB-985.Mirroring these results, murine bone marrow stem cells from H1.2/H1.4double deficient mice shifted their differentiation profile from neutrophil towards eosinophil cell fate.We further demonstrate that the subtype-specific function of H1 in neutrophil and eosinophil differentiation depends -at least in part -on the transcription factor GATA-2.We uncovered that, unexpectedly, H1 subtypes affect lineage specification during granulopoiesis.

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Cell lines
PLB-985 were kindly provided by Prof. Mary Dinauer, Washington University School of Medicine 20 .
Lentiviral particles were produced using psPAX and pMD2.G packaging plasmids in HEK293T cells.

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The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint All sgRNA sequences and primer sequences are listed in Supplementary Table 3.

Donor consent
Human primary neutrophils, PBMCs and monocytes were isolated from blood samples of healthy volunteers according to the declaration of Helsinki.All donors provided written informed consent and all blood samples were collected with approval from the local ethics committee.

Mice
Breeding of mice and isolation of blood and bone marrow were approved by the Berlin state authority Landesamt für Gesundheit und Soziales.All mice were bred at the Max Planck Insitute for Infection Biology under specific pathogen-free conditions.Animals were maintained on a 12-hour light/12-hour dark cycle and fed ad libitum.H1.2/H1.4-deficientanimals were provided by Prof. Arthur Skoultchi 13 .

Primary cell isolation
Neutrophils were purified by centrifugation of whole blood over Histopaque-1119 (Sigma), followed by centrifugation over a discontinuous Percoll gradient 23 .PBMCs were collected from the upper phase after Histopaque-1119 centrifugation, washed and either used for RNA extraction or for isolation of monocytes by positive selection (CD14) with magnetic beads (130-050-201, Miltenyi Biotec).

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The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint (RPMI supplemented with 2.5 % FCS, 0.5% dimethylformamide (DMF, D4551, Sigma), L-glutamine and antibiotics), usually in 6 well plates in a total volume of 3ml.At d4 of differentiation, 2ml of differentiation medium were added per 6 well.At d7, cells were washed and passed over an equal volume of Histopaque-1077 (Sigma) (centrifugation for 20min at 800 x g, brakes off) to remove debris and subsequently seeded at the required density for experiments in assay medium (RPMI (without phenol red) containing 1 % FCS, 0.5 % DMF and L-glutamine).

Transmission electron microscopy
Cell suspensions, fixed in 2.5% glutaraldehyde, were sedimented and embedded in low melting agarose (2% in PBS).Drops of agarose containing cells were then postfixed in 0,5% osmiumtetroxide, contrasted with tannic acid and 2% uranyl acetate, dehydrated in a graded ethanol series and embedded in epoxy resin.After polymerization, sections were cut at 60 nm and contrasted with lead citrate.Specimens were analyzed in a Leo 906E transmission electron microscope at 100KV (Zeiss, Oberkochen, DE) using a side mounted digital camera (Morada; SIS-Olympus Münster DE).

Analysis of ROS production from cells
Cells were seeded at 10 5 /well of a white 96 well plate (Nunc) in triplicates in a final volume of 100 l in assay medium.HRP (1.2 U/ml) and luminol (50 M) were added 10 minutes after seeding and 10 minutes later cells were stimulated with 100 nM PMA.Luminescence signal was measured using a VICTOR XLight Multimode Plate Reader (Perkin Elmer).

Analysis of cell death
Cells were seeded at 10 5 /well of a 96 well plate in duplicates or triplicates in a final volume of 100 l.
The cell impermeable DNA dye SYTOX Green (50 nM) was added and 10 minutes later cells were 7 was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint stimulated with 100 nM PMA or with 5 M A23187.SYTOX Green fluorescence signal corresponding to NET formation and cell death was measured over time using excitation/emission of 485/518 nm.Alternatively, microscopy pictures were acquired using brightfield and GFP channels (for SYTOX Green) in order to visualize the morphology of dead cells.

Analysis of surface marker expression
PLB-985 and murine ex vivo differentiated bone marrow cells were stained at various time points of differentiation with indicated surface marker antibodies for 20 min at 4 °C in PBS with 2% FCS, washed and analyzed by flow cytometry using a MACS Quant Analyzer (Miltenyi Biotec).DAPI was added just before acquisition to exclude dead cells.
Murine blood was stained the same way as described above, but after staining, samples were incubated with eBioscience 1 Step Fix/Lyse Solution (invitrogen, 00-5333-54) for 1h to lyse erythrocytes before acquisition.

CRISPR/Cas9 screen
PLB-985 were transduced with a lentiviral library containing CRISPR/Cas9 sgRNAs 21 at an estimated MOI of <1.Cells were selected with puromycin (Sigma, 2.5 g/ml), differentiation was performed as described for wt PLB-985.At d7 of differentiation, cells were stimulated with 100 nM PMA for 16h and surviving cells were sorted by FACS.DNA extraction was performed by DNeasy Blood & Tissue Kit (Qiagen) and PCR amplification was performed as described 21 .Reads were mapped to the library and abundance of sgRNAs was calculated per condition.Genes with greater than 50 % of their sgRNAs identified at values of at least 2-fold overrepresentation in the PMA-treated group versus the control group were defined as hits.

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Generation of CRISPR/Cas9 knockout lines
PLB-985 were transduced with lentiviral particles containing the sgRNA of interest cloned into lentiCRISPRv2, cells were selected with puromycin (2.5 g/ml) and, if single clones were analyzed, seeded at a density of 0.8 cells/well in 96 wells for limited dilution cloning.Clones were sequenced using the OutKnocker protocol and software 24 (Supplementary Table 3).Clones with out of frame indels leading to efficient gene disruption were used for subsequent experiments.
For GATA-1 and GATA-2 disruption in scr., H1.2 and H1.4-deficient cells, sgRNAs targeting GATA were cloned into a modified lentiCRISPRv2, in which the Cas9 sequence was exchanged for a blasticidin resistance cassette.After transduction, cells were selected with blasticidin (50 g/ml) and used in batch populations for experiments.

RNA extraction and qRT-PCR
Cell samples were pelleted, resuspended and lysed in TRIzol Reagent (life technologies).RNA was isolated by chloroform extraction and isopropanol precipitation.Similar amounts of total RNA were used for RT-PCR (High Capacity cDNA Reverse Transcription Kit, 4368814, Thermo Fisher Scientific).qRT-PCR was performed using SYBR Green (4385612, Thermo Fisher Scientific) on a QuantStudio 3(Thermo Fisher Scientific).

RNA-sequencing
Total RNA of PLB-985 at various time points was isolated using RNeasy kit (Qiagen).Library preparation and illumina sequencing were performed by the Max Planck-Genome-centre Cologne, Germany (https://mpgc.mpipz.mpg.de).The data were mapped to hg38.87 using STAR 2.5.2b 25 and differential expression analysis was performed using edgeR 26 .Gene level read counts from primary human cells at various steps of neutrophil differentiation were retrieved from Blueprint 27 (http://www.blueprint-epigenome.eu, Datasets EGAD00001002446, EGAX00001244028, EGAX00001244022 and EGAD00001002366) and analyzed the same way.Gene module enrichment 9 was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

Ex vivo differentiation of murine bone marrow cells into neutrophils and eosinophils
Murine bone marrow was extracted from the femurs and tibiae of adult age-matched mice.Cells were flushed, erythrocytes were lysed by osmotic lysis, cells were washed and lineage-negative precursor cells were sorted by negative selection using magnetic beads (130-110-470, Miltenyi Biotec).Lineagenegative cells were subsequently propagated analogous to the protocol described 32 .Briefly, cells were seeded in IMDM supplemented with 20% FCS, antibiotics, L-glutamine and 0.1 M 2mercaptoethanol (Sigma), as well as with the recombinant cytokines IL-3 (20 ng/ml), IL-5 (50ng/ml), IL-9 (50 ng/ml), GM-CSF (10 ng/ml) and SCF (20 ng/ml) for 6 days.

Statistical analysis
The descriptive statistics of all figure panels are mentioned in the respective figure legends.
Populations of circulating leukocytes in murine blood were compared by a two-tailed, unpaired t test and p values are indicated above the populations.RNA-seq data were analyzed as described in the respective methods section.

PLB-985: a model for neutrophil maturation and function
We used the human promyelocytic cell line PLB-985 33 .PLB-985 differentiate into neutrophil-like cells that resemble primary human neutrophils in various ways 20,[34][35][36] .PLB-985 changed their nuclear morphology and developed granules, though fewer than primary neutrophils, throughout the 7 days of differentiation (referred as d0 to d7 in this report) (Fig. 1a).Once differentiated, PLB-985 also produced ROS in response to the agonist phorbol 12-myristate 13-acetate (PMA) (Fig. 1b, Supplementary Fig. 1a) and upregulated the surface marker CD11b (Fig. 1c).Furthermore, differentiated PLB-985 phagocytosed Escherichia.coli,a process that could be blocked by the phagocytosis inhibitor cytochalasin B (Supplementary Fig. 1b).Importantly, PMA potently induced cell death in fully differentiated PLB-985, but not in d0 or d3 cells (Fig. 1d).PMA-induced cell death was morphologically similar to NETosis; dying PLB-985 expanded their nuclei and a significant fraction released DNA as shown by SYTOX Green staining (Fig. 1e, Supplementary Fig. 1c).Of note, the calcium ionophore A23187, another stimulant of NET formation, also induced cell death in PLB-985, both at d3 and d7 of differentiation (Supplementary Fig. 1d).Taken together, these experiments show that PLB-985 cells differentiate into neutrophil-like phagocytes that produce ROS and NETs.
To show that PLB-985 can be genetically modified by CRISPR/Cas9, we disrupted expression of NOX2 (CYBB), a component of the NADPH oxidase complex, which is required for PMA-induced NETosis 37 .We also transduced PLB-985 with a non-target scrambled single-guide RNAs (sgRNA) as a control (subsequently called scr).Differentiated PLB-985 cells lacking NOX2 failed to produce an oxidative burst and did not undergo cell death in response to PMA (Supplementary Fig. 1e, f).To inactivate NADPH oxidase independently of NOX2, we also disrupted expression of its NCF2 subunit, and showed again that these cells did not die upon PMA stimulation (Supplementary Fig. 1g).
These results demonstrate that PLB-985, which can be genetically modified, are a suitable model to mimic human neutrophils.
A genome-wide screen to identify regulators of neutrophil maturation and function Since we found that PMA-induced NET formation only occurred in fully differentiated PLB-985, we used this readout to perform a genome-wide CRISPR/Cas9 screen to identify genes required for neutrophil maturation and function (Fig. 1f).We reasoned that, besides NET-defective cells, we could also identify differentiation-defective cells by sorting cells that survived PMA treatment.We transduced PLB-985 with a library targeting all human genes with 5-6 sgRNAs per gene 21 .As expected, PMA stimulation reduced the fraction of identified guides in the survivor population (Supplementary Fig. 1h), indicating selection of specific clones.
We defined "hits" as genes for which at least 50 % of the identified sgRNAs were overrepresented (more than two-fold) in our survivor population.Underlining the validity of the screening approach, we identified NOX2, NCF1 and MPO as hits; all three are known to be required for PMA-induced NET formation 37,38 (Fig. 1g, Supplementary Table 1).We also found C/EBP-, a member of the C/EBP family of transcription factors that are crucial for granulopoiesis 39 (Fig. 1g).Unexpectedly, we identified two members of the linker histone H1 family among the 20 highest ranked genes.HIST1H1E (H1.4) was overrepresented with 100 % of sgRNAs and showed the highest median enrichment (Fig. 1g, Supplementary Table 1).Notably, there are no studies showing a functional role of H1 in neutrophil differentiation or NET formation.
We also analyzed a dataset comparing mRNA expression of human primary bone marrow cells undergoing differentiation into neutrophils at various stages of maturation, namely myelocytes, was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint metamyelocytes, band form neutrophils and segmented neutrophils 27 (see methods section for RNAseq).Consistent with our PLB-985 data, we found a similar reduction of H1 mRNA as maturation progressed (Supplementary Fig. 2b).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 (Fig. 2b).Interestingly, peripheral blood mononuclear cells (PBMCs) and monocytes showed a different expression pattern of this gene family than neutrophils (Supplementary Fig. 2c, d).
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 2 sgRNAs) and two clones deficient for H1.4 (from 1 sgRNA) in our analysis.Cell death was reduced in both H1.2 and H1.4-deficient clones, confirming the results of the screen (Fig. 2c).H1.2 and H1.4-deficient clones produced ROS less efficiently than wild type cells upon PMA stimulation (Fig. 2d).Furthermore, cells deficient for either of the two H1 subtypes expressed less MPO (Fig. 2e).These findings suggest that H1.2 and H1.4 are required for PLB-985 to differentiate into mature, neutrophillike cells.

Opposing effects of H1 subtypes on neutrophil differentiation
To confirm that deficiency of H1.2 and H1.4 affected PLB-985 maturation, we showed that surface expression of the differentiation marker CD11b is decreased on both d3 and d7 of differentiation (Fig. 3a, b, Supplementary Fig. 3a, b) in cells where these genes were deleted.We also disrupted expression of the other three somatic H1 subtypes.Surprisingly, loss of these subtypes resulted in the opposite phenotype.We observed markedly enhanced expression of CD11b already at d3, especially in clones deficient in H1.1 and H1.5 (Fig. 3a, b, Supplementary Fig. 3a, b).

Differentiation of neutrophils and PLB-985 is terminal -if not activated, these cells die by apoptosis.
The number of wild type and control (scr.)PLB-985 cells increased more from d0 to d3 than from d3 13 was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.

The impact of H1 deficiency manifests before onset of differentiation
To understand the extent of H1 subtype-specific effects on neutrophil differentiation we performed RNA-sequencing of the knockout lines.We sampled wild type cells, scr.cells and two clones per H1 subtype at d0, d3 and d7 of differentiation in 4 independent experiments (Supplementary Table 2).To validate the applicability of our experiment, we compared the transcriptome of control PLB-985 cells (the combination of wild type and scr.samples) to human primary cells at various differentiation stages towards neutrophils (also see methods section).Gene set enrichment analysis demonstrated that PLB-985 and primary cells concordantly regulate processes during differentiation (Fig. 4a, Supplementary Fig. 4a, b.Supplementary Fig. 4a depicts the same analysis as Fig. 4a with the gene set labels).Using principal component analysis (PCA), we saw that these cells follow a similar differentiation trajectory (Supplementary Fig. 4c).As expected, among the most robustly upregulated genes during differentiation of control cells we found several with known functions in neutrophils (Fig. 4b).
We analyzed the H1-deficient clones by PCA and found that H1.2 and H1.4-deficient clones at d7 clustered more closely with control cells at d3, indicating delayed differentiation (Fig. 4c).H1.1, H1. 3   14   was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
Analysis of the enrichment of gene modules confirmed that, during differentiation, control cells upregulated genes related to the innate immune response and downregulated genes corresponding to cell cycle and division, in line with the context of terminal differentiation (Supplementary Fig. 4d).
Interestingly, in H1.2 and H1.4-deficient cells, the modules were less regulated, again demonstrating prolonged proliferation and obstruction of differentiation (Supplementary Fig. 4d).We performed the same analysis for clones deficient in H1.1, H1.3 and H1.5 and confirmed accelerated differentiation (Supplementary Fig. 4d).We subsequently asked whether absence of H1 subtypes also affected the determination and lineage choice of PLB-985 before differentiation.To address this question, we compared differentially expressed genes of H1.2 and H1.4-deficient cells and control cells at d0.We found that both H1.2 and H1.4-deficient cells downregulated neutrophil genes, such as MPO and neutrophil proteases among others (Fig. 4d), indicating that lineage fate was affected in undifferentiated cells.Surprisingly, among the upregulated genes we found a significant number of genes associated with eosinophils and a GATA-1 signature (Fig. 4d, e, see methods section for the source of datasets) We found this upregulation specifically in clones deficient in H1.2 and H1.4 in comparison to all other genotypes (Fig. 4e).This suggests that loss of H1.2 and H1.4 affects lineage determination of PLB-985 cells even before the onset of differentiation by driving them towards a more eosinophil-like fate.
These data indicate that H1 subtype depletion affects both cell fate determination and differentiation per se.

Enhanced eosinophil differentiation of H1.2/H1.4-deficient hematopoietic stem cells
Loss of H1.2 and H1.4 appears to bias the lineage commitment of PLB-985 away from neutrophils via regulation of an eosinophil-like transcriptional program.To test whether this bias also exists in primary cells, we analyzed cells isolated from H1.2/H1.4double-deficient mice 13 .The profile of circulating leukocytes in the blood of adult animals was indistinguishable from wild type animals 15 was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint (Supplementary Fig. 5a-e).Furthermore, wild type and H1.2/H1.4-deficientanimals had the same proportion of neutrophils in circulation (Fig. 5a).We isolated lineage negative cells from murine bone marrow and differentiated them into several lineages 31 .Importantly, H1.2/H1.4-deficientcells expressed elevated levels of surface markers for eosinophils and decreased levels of neutrophil surface markers, as compared to wild type cells.This recapitulates the phenotype we observed in PLB-985 (Fig. 5b-d).These experiments show that H1.2 and H1.4 cooperate to determine the neutrophileosinophil cell fate decision in hematopoietic progenitor cells.Furthermore, this suggests that, in vivo, the H1.2 and H1.4 deficiency is compensated to generate normal amounts of neutrophils during homeostasis.

Loss of H1.2 and H1.4 affect neutrophil cell fate determination via GATA-2
Two transcription factors required for eosinophil differentiation, GATA-1 and GATA-2, were upregulated in H1.2 and H1.4 knockout lines (Fig. 4e).GATA-1 transcripts were very low in all lines sequenced (Supplementary Fig. 6a).GATA-2 transcript levels at d0 inversely correlated with the potential of PLB-985 to adopt a neutrophil-like cell fate (Supplementary Fig. 6b).We speculated that H1 subtypes affect neutrophil differentiation through either GATA-1 or GATA-2.To test this hypothesis, we disrupted expression of GATA-1 and GATA-2 by CRISPR/Cas9 and analyzed batch populations of various mutations at d4 of differentiation.We chose d4 because we assumed that cells still expressing GATA could outgrow GATA-deficient cells at later time points.Interestingly, GATA-2 deficiency enhanced maturation of PLB-985, as indicated by an increased ability to produce ROS in response to PMA (Supplementary Fig. 6c).In contrast, GATA-1 knockout lines behaved as scr.cells (Supplementary Fig. 6c).
To test whether the differentiation phenotype of H1.2 and H1.4-deficient lines relied on GATA-2, we performed a rescue experiment by generating double-deficient lines.We transduced scr.cells and H1.2 or H1.4-deficient cells with GATA-1 and GATA-2 sgRNAs or with a scr.sgRNA as a control and analyzed these populations at d4. Importantly, disruption of GATA-2 expression rescued ROS production (Fig. 6a-c) and CD11b expression (Fig. 6d) in H1.2 and H1.4-deficient PLB-985.Again, was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint disruption of GATA-1 had no effect (Fig. 6a-d).These experiments demonstrate that the differentiation phenotype resulting from H1.2 and H1.4 deficiency depends on upregulation of GATA-2 and, importantly, that loss of GATA-2 is sufficient to allow differentiation in the absence of these H1 subtypes.
In a second approach, we treated PLB-985 with inhibitors for GATA-1 and GATA-2, starting treatment one day before differentiation.Inhibition of GATA-2 enhanced CD11b expression of H1.2 or H1.4deficient lines (Fig. 6e).To confirm the effect of GATA-2 inhibition on differentiation, we measured mRNA expression of two genes, one that increased (aquaporin-9, AQP9) and one that decreased (MPO) during differentiation (Supplementary Table 2).As expected, H1.2 and H1.4-deficient lines expressed less AQP9 and more MPO than scr.cells at d7 of differentiation (Supplementary Fig. 6d, e).GATA-2 inhibition upregulated expression of AQP9 and downregulated expression of MPO in H1.2 and H1.4 knockout lines at d7 of differentiation, indicating a rescue of maturation (Supplementary Fig. 6d, e).In line with our results with CRISPR/Cas9-mediated gene disruption, GATA-2 inhibition restored the capacity of H1.2 and H1.4-deficient clones to mount an oxidative burst (Fig. 6f-h).As expected, treatment with the GATA-1 inhibitor did not affect maturation and function of PLB-985 of either genotype (Fig. 6e, i-k).The combination of GATA-1 and GATA-2 inhibitors did not have an additive effect on PLB-985 maturation, confirming that GATA-2 is the relevant transcription factor for the phenotype observed in H1.2 and H1.4-deficient clones (Supplementary Fig. 6f).Importantly, the response to either GATA-2 deficiency or inhibition was stronger in H1-deficient PLB-985 than in scr.cells.This is in line with enhanced baseline expression of the transcription factor in H1.2 or H1.4deficient clones.These data show that H1.2 and H1.4 affect fate determination and subsequent neutrophil differentiation and function at least partly through the regulation of GATA-2.

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Discussion
We identified an unexpected role of H1 subtypes in neutrophil development; loss of H1.2 and H1.4 inhibits differentiation while deficiency in H1.1, H1.3 and H1.5 enhances it.We propose that in neutrophil precursors, H1 subtypes control the expression or activity of GATA-2, which determines cell fate via transcription of neutrophil or eosinophil-specific genes.Enhanced expression of eosinophil genes -or reduced expression of neutrophil-specific genes -disrupts normal maturation, leading to prolonged proliferation and viability and reduced neutrophil function.Consistent with this, genetic ablation or inhibition of GATA-2, but not GATA-1, rescued the differentiation of H1.2 and H1.4-deficient cells.Interestingly, another study found that overexpression of GATA-2 in murine progenitor cells switched their lineage determination from macrophages to a megakaryocyte state 40 .
GATA-2 therefore acts as a key molecule for fate determination of immune cells.Importantly, we observed a preference for eosinophil development in hematopoietic stem cells isolated from the bone marrow of H1.2/H1.4-deficientmice.However, adult H1.2/H1.4-deficientanimals had normal numbers of circulating neutrophils.It is possible that the turnover rate or the longevity of neutrophils is affected in these mice.Indeed, neutropenia in peripheral tissues triggers de novo production of neutrophils in the bone marrow 41 , demonstrating that granulopoiesis in vivo is regulated by feedback loops.This suggests that initial neutrophil deficiency in adult H1.2/H1.4-deficientmice could be compensated by a different cytokine milieu.Interestingly, in human breast cancer cells, combined loss of H1.2 and H1.4 leads to an upregulation of an interferon signature 42 .Although we did not observe an obvious interferon signature in H1.2 or H1.4-deficient PLB-985, it is possible that in H1.2/H1.4-deficientmice some cell types contribute to immune cell differentiation via interferonstimulated genes.H1 can act in redundant and subtype-specific ways 12 .Mice lacking one or two H1 subtypes are viable and fertile 13 .Analogously, PLB-985 deficient in any of the five H1 subtypes that we disrupted were viable and did not show growth defects.This is in contrast to another study, where shRNA-mediated knock down of H1.2 and H1.4 reduced cell growth and viability in various non-immune cell lines 16 .

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was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.
The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint We found that loss of H1.2 and H1.4, in the context of neutrophil differentiation, enhanced viability, demonstrating cell type specific effects of H1 subtypes.
H1 has long been considered a general repressor of transcription 43 .Accordingly, a study in human cells found that genomic regions associated with active transcription were devoid of H1 subtypes 44 .
However, murine embryonic stem cells that are triple-deficient in H1.2, H1.3 and H1.4 show a surprisingly low number of only 38 differentially expressed genes when compared to wild type cells 45 .
Our data show that loss of H1 subtypes in more differentiated cells affects the expression of many genes with clear functional consequences.We postulate that only a fraction of differentially expressed genes in PLB-985 is directly regulated by H1 subtypes.The enhanced expression of lineagedetermining transcription factors, such as GATA-2, leads to further changes in the transcriptome and An important open question is: how do H1 subtypes control expression of GATA-2?It is possible that H1 subtypes directly occupy the promoter or enhancer elements of GATA-2 and silence its expression.
H1 subtype-specific binding to genomic regions has, for example, been shown for human H1.5 46 .Interestingly, H1.5 targeted over 20-fold more regions in differentiated human lung fibroblasts than in embryonic stem cells and H1.5 binding led to chromatin compaction and gene silencing.It is therefore tempting to speculate that similar mechanisms are at play during neutrophil development.In addition to a direct repressive effect of H1.5, its binding to chromatin was also associated with an enrichment of demethylation of histone H3 at lysine 9 (H3K9me2) 46 .H3K9me2 is a posttranslational modification of a core histone that is associated with a repressive chromatin state.H1 subtypes are therefore likely to regulate transcription via epigenetic marks on core histones.Furthermore, H1 subtypes themselves are posttranslationally modified in a subtype-specific manner 12 .Although less is known about H1 modifications than about core histones, it is likely that such modifications can determine subtypespecific protein-protein interactions, chromatin binding properties or other features that subsequently cause transcriptional changes.

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The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint It is puzzling that vertebrates express so many different H1 subtypes.Although they are able to compensate each other in "extreme" situations, such as the loss of one or even two subtypes in a whole organism 13 , they may also have more subtle and specific functions.This study contributes to our understanding of how different H1 subtypes initiate distinct transcriptional programs.Importantly, these programs appear to be evolutionarily conserved, since we found similar responses in human and murine cells.
In summary, we identified novel and opposing functions for specific H1 subtypes in granulopoiesis.
H1 subtypes affect the lineage determination between neutrophils and eosinophils.We speculate that inappropriate expression of H1 subtypes in the hematopoietic compartment shapes pathologies with neutrophil or eosinophil involvement, such as asthma.Our findings suggest that linker histones represent another layer of complexity to the fascinating process of immune cell differentiation.

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Fig. 1 .
Fig. 1.A genome-wide screen to identify genes required for PLB-985 cells differentiation and function.

1 NOX2 sgRNA1 clone 2 NOX2 sgRNA2 clone 1 was
was not certified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.notcertified by peer review) is the author/funder.All rights reserved.No reuse allowed without permission.The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.bypeer review) is the author/funder.All rights reserved.No reuse allowed without permission.The copyright holder for this preprint (which this version posted October 29, 2019.; https://doi.org/10.1101/763482doi: bioRxiv preprint Supplementary Fig.

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