Linker histone H1.2 and H1.4 affect the neutrophil lineage determination
- Max Planck Institute for Infection Biology, Department of Cellular Microbiology, Germany
- University of Dundee, School of Life Sciences, Division of Cell Signalling and Immunology, United Kingdom
- Institut für Biologie, Humboldt Universität zu Berlin, Germany
- Max Planck Institute for Molecular Genetics, Germany
- Department of Cell Biology, Albert Einstein College of Medicine, United States
Figures
Figure 1 with 1 supplement
A genome-wide screen to identify genes required for PLB-985 cells differentiation and function.
(a–d) Characterization of PLB-985 differentiation and function at d0, d3 and 7 of differentiation. (a) Electron microscopy images of PLB-985 showing acquisition of granules (green arrows, the black …
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Figure 1—source data 1
List of genes identified as hits in the CRISPR/Cas9 screen.
The table shows gene ID and the fold representation of identified sgRNAs in PMA-treated versus untreated PLB-985. sgRNAS that were not identified in either sample are labelled as NA. The table also shows the median and mean values per gene and the percentage of overrepresented (at least two fold) sgRNAs.
- https://cdn.elifesciences.org/articles/52563/elife-52563-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Characterization of PLB-985 function and morphology.
(a) ROS production of PLB-985 in response to PMA at different time points of differentiation, depicted are mean -/+ SEM of two independent experiments. (b) Phagocytosis assay of differentiated (d7) …
Figure 2 with 1 supplement
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 β-microglobulin, …
Figure 2—figure supplement 1
H1 expression levels in human PBMCs, monocytes and PLB-985.
(a) Relative mRNA expression levels corresponding to Figure 2a, values are relative to d0 of differentiation and indicate reduction of mRNA expression for genes encoding H1.1, H1.2, H1.4 and H1.5 …
Figure 3 with 1 supplement
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 …
Figure 3—figure supplement 1
Subtype-specific H1 impact on PLB-985 maturation and function.
(a, b) Flow cytometry analysis corresponding to Figure 3a,b, depicted is the mean fluorescence intensity (MFI) of CD11b in indicated samples at d3 (a) or d7 (b) of differentiation. Each dot …
Figure 4 with 3 supplements
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 …
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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).
- https://cdn.elifesciences.org/articles/52563/elife-52563-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Transcriptional differences between H1-deficient clones during differentiation.
(a) Concordantly and discordantly regulated modules when comparing PLB-985 differentiation (wt and scr., d0 to d7) and human neutrophil differentiation (Blueprint, neutrophilic myelocyte to …
Figure 4—figure supplement 2
Dysregulation of several transcription factors upon loss of H1 subtypes.
Heatmap showing the expression of mRNAs encoding GATA-1, GATA-2 and the indicated C/EBP family transcription factors identified in the RNA-seq data. Loss of H1.2 and H1.4 induces GATA-1 and GATA-2 …
Figure 4—figure supplement 3
Expression of eosinophil markers in PLB-985 upon loss of H1.2 or H1.4.
(a) Transmission electron microscopy (TEM) pictures of control (scr.) PLB-985 and a clone of H1.4- and H1.2-deficient cells at indicated time points of differentiation. No clear changes in overall …
Figure 5 with 7 supplements
Deletion of H1.2 or H1.4 biases murine hematopoietic cells towards an eosinophil lineage.
(a–c) Analysis of circulating neutrophils and eosinophils in wild type (wt) and H1.2/H1.4-double deficient mice (H1.2/H1.4 -/-). p Values, derived from unpaired two-tailed T tests, are indicated. (a)…
Figure 5—figure supplement 1
Gating strategies and circulating leukocytes.
(a, b) Gating strategy for different subsets of immune cells in whole blood and bone marrow. (a) Single cells are gated for expression of CD45, then for CD3. CD3 positive cells are divided into CD4 …
Figure 5—figure supplement 2
Leukocytes in circulation and bone marrow in homeostasis.
(a–n) Analysis of leukocytes in blood (a–g) and bone marrow (h–n) of wild type and H1.2/H1.4-deficient female mice (n = 4). Animals were sacrificed, bone marrow and blood was isolated and directly …
Figure 5—figure supplement 3
Leukocytes in circulation and bone marrow upon casein injection.
(a–n) Same cell populations and staining procedure as in Figure 5—figure supplement 2, but upon injection of 2 × 1 ml 7% casein into the peritoneal cavity of female mice (n = 4). Animals were …
Figure 5—figure supplement 4
Eosinophil counts in bone marrow upon casein injection and in circulation.
(a) Counts of eosinophils in the blood of casein-injected animals, showing that the enhanced number of eosinophils seen in homeostatic H1.2/H1.4-deficient mice is not seen in inflammatory …
Figure 5—figure supplement 5
Differentially regulated ageing markers in H1.2/H1.4-deficient mice.
a Counts of neutrophils in the bone marrow of wild type and H1.2/H1.4-deficient animals, showing similar numbers of neutrophils in both genotypes. (b) Mean fluorescence intensity (MFI) of CD62L …
Figure 5—figure supplement 6
Differentially regulated cytokines in H1.2/H1.4-deficient mice.
a, b mRNA expression of the chemokine CXCL1 (a) and the inflammatory cytokine IL6 (b) in homeostatic (left panel) and casein-injected (right panel) animals. H1.2/H1.4-deficient bone marrow cells …
Figure 5—figure supplement 7
Enhanced survival and granularity of H1.2/H1.4-deficient bone marrow cells and neutrophils.
(a) Bone marrow of casein-injected wild type and H1.2/H1.4-deficient mice was incubated overnight in the presence or absence of G-CSF (100 ng/ml). G-CSF enhanced the survival of cells, measured by …
Figure 6 with 2 supplements
Deficiency of H1.2 or H1.4 is rescued by inhibition of GATA-2.
(a–c) Deficiency in GATA-2 but not GATA-1 rescues H1.2 and H1.4 deficiency. Populations are batches of PLB-985. scr. (a), H1.4 (b) and H1.2 (c) knockout lines were transduced with an sgRNA against GA…
Figure 6—figure supplement 1
Expression of GATA-1, GATA-2 and C/EBP transcription factors in PLB-985 and murine bone marrow.
(a, b) Transcripts per million (TPM) were calculated from PLB-985 RNA-seq data for GATA1 (a) and GATA2 (b) for the indicated genotypes at all time points of differentiation. GATA1 expression is …
Figure 6—figure supplement 2
H1.2 or H1.4 deficiency can be reverted by inhibition of GATA-2.
(a) PLB-985 were transduced with two sgRNAs against GATA1 and 2 sgRNAs against GATA2. Cells were differentiated in batches and ROS production in response to PMA was measured at d4 of …
Additional files
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Supplementary file 1
List of sgRNA sequences, primer sequences and antibodies.
Individual sheets contain sequences of sgRNAs, sequencing primers, qRT-PCR primers and antibody catalog and lot numbers.
- https://cdn.elifesciences.org/articles/52563/elife-52563-supp1-v3.xlsx
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Supplementary file 2
Key Resources table.
Table of reagents, cell lines, genetically modified organisms, others.
- https://cdn.elifesciences.org/articles/52563/elife-52563-supp2-v3.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52563/elife-52563-transrepform-v3.docx
