A RUNX–CBFβ-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes

Abstract

The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. It is unclear how Irf8 expression is regulated and how this single transcription factor supports the generation of both monocytes and DCs. Here, we identified a RUNX–CBFβ-driven enhancer 56 kb downstream of the Irf8 transcription start site. Deletion of this enhancer in vivo significantly decreased Irf8 expression throughout the myeloid lineage from the progenitor stages, thus resulting in loss of common DC progenitors and overproduction of Ly6C+ monocytes. We demonstrated that high, low or null expression of IRF8 in hematopoietic progenitor cells promotes differentiation toward type 1 conventional DCs, Ly6C+ monocytes or neutrophils, respectively, via epigenetic regulation of distinct sets of enhancers in cooperation with other transcription factors. Our results illustrate the mechanism through which IRF8 controls the lineage choice in a dose-dependent manner within the myeloid cell system.

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Fig. 1: Identification of a putative enhancer 56 kb downstream of the Irf8 TSS.
Fig. 2: The +56 kb Irf8 enhancer is required for the development of cDC1s but not monocytes.
Fig. 3: The dose-dependent impact of IRF8 on the generation of cDCs, monocytes and neutrophils.
Fig. 4: Enhancer landscapes in myeloid progenitors are determined in an Irf8 dose-dependent manner.
Fig. 5: Comparison of IRF8-bound enhancers between WT monocytes and cDC1s.
Fig. 6: Clustering of active enhancers indicates RUNXs as the TFs acting on the +56 kb Irf8 enhancer.
Fig. 7: RUNX–CBFβ induces Irf8 expression via the +56 kb Irf8 enhancer.

Data availability

The data supporting the findings of this study are available from the corresponding authors (T.T. and A.N.) upon reasonable request. The sequencing data generated in this study were deposited in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/). RNA-seq data, ChIP-seq data and ATAC-seq data are available at GSE149762. The following data were retrieved from Gene Expression Omnibus database: RNA-seq for WT pDCs (GSE121446); RNA-seq for WT B cells, WT CD4+ T cells and WT CD8+ T cells (GSE127267); RNA-seq for WT CLPs (GSE109805); RUNX1 ChIP-seq for Hoxb8-FL cells (GSE84328); RUNX1 ChIP-seq for FDC-P1 cells (GSE81179); RUNX2 ChIP-seq for MA9CL cells (GSE120063); and microarray for IRF8 and IRF8+ LMPPs (GSE113748). The following data were retrieved from DNA Data Bank of Japan Sequence Read Archive (https://www.ddbj.nig.ac.jp/): H3K27ac ChIP-seq for WT GMP, WT MDP, WT cMoP, WT Ly6C+ monocyte, WT CDP, WT neutrophil, Irf8–/– GMP, Irf8–/– MDP and Irf8–/– cMoP (PRJDB3411); and IRF8 ChIP-seq for WT MDP (PRJDB3411). The sequencing data and public data used in this study are listed in Supplementary Table 2. Additionally, the table contains information on the figures associated with these data. Source data are provided with this paper.

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Acknowledgements

The authors thank M. Ichino, I. Harada, M. Yoshinari, S. Honma, H. Sato, G. R. Sato and M. Tachikawa at Yokohama City University for their help with the experiments. This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science/Ministry of Education, Culture, Sports, Science and Technology (MEXT; grant nos. 18K19345 and 15H04860 to T.T. and 19K07372 to A.N.); a Uehara Memorial Foundation Research Grant (to T.T.); a Japanese Society of Hematology Research Grant (to T.T.); and the MEXT Joint Usage/Research Center Program at the Advanced Medical Research Center, Yokohama City University (funding for Y.S., T.K. and T.T.).

Author information

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Authors

Contributions

K.M., H.S., A.N. and T.T. designed the study. K.M., H.S., A.N., D.K., W.K., T.B., S.K. and Y.S. conducted the experiments; K.M., H.S., A.N., J.N. and T.T. analyzed the data; K.M., A.N. and T.T. wrote the manuscript; K.O. provided key resources; H.N., K.O. and T.K. provided intellectual input; and T.T. supervised the project. K.M., H.S. and A.N. contributed equally to this work.

Corresponding authors

Correspondence to Akira Nishiyama or Tomohiko Tamura.

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The authors declare no competing interests.

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Peer review information Nature Immunology thanks Venetia Bigley, Charlotte Scott, Alberto Yáñez and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Models of myeloid cell differentiation in WT and Irf8−/− mice.

Irf8 starts to be expressed at the MPP stage and its expression sharply increases in MDPs. The expression of Irf8 further increases as cells differentiate into the DC lineage, while remaining relatively low or downregulated in the monocytic lineage. Neutrophils do not express Irf8. Irf8–/– mice lack Ly6C+ monocytes, CDPs, pDCs, and cDC1s. Irf8–/– mononuclear phagocyte progenitors accumulate and aberrantly give rise to neutrophils. MPPs include both MPP3s and MPP4s/LMPPs that express low amounts of Irf8. The dashed lines denote disputed pathways. Cell populations affected by the enhancer or gene deletion are highlighted in green.

Extended Data Fig. 2 Flow cytometric analysis of bone marrow and spleen cells.

a-h, Representative FACS plots of HSPCs (a), myeloid progenitors (b), CLPs (c), mononuclear phagocyte progenitors (d), and cMoPs (e) in bone marrow and those of monocytes and neutrophils (f), cDCs (g), and pDCs (h) in spleens.

Extended Data Fig. 3 Creation of Irf8 enhancer-null mice by CRISPR/Cas9 genome editing.

a, The Genome Browser image of the regions deleted in each enhancer-null mouse strain. b, Representative cropped gel images of genomic PCR confirming the deletion. Primer sets are indicated. Data are representative of over 20 independent experiments for each genotype, which yielded similar results. Full scans are shown in Source Data. c, The Genome Browser image of input DNA data at the Irf8 gene locus on ∆+56 cMoP in ChIP-seq analysis. Gray boxes indicate known enhancers at –50, +41, and +32 kb. Source data

Extended Data Fig. 4 The Irf8 +56 kb region regulates Irf8 expression and cell fate.

a, Representative FACS plots of pre-cDC1s, pre-cDC2s, pre-DCs, and Ly6C monocytes analyzed in Fig. 2a. b, IFN-α and IFN-β production by pDCs isolated from WT or ∆+56 mice followed by overnight stimulation with poly(U) (1.0 µg/mL) or CpG-A (10 µM) (n = 3 mice per genotype). Data are representative of two independent experiments, which yielded similar results. c, In vitro culture of MDPs. Ten thousand MDPs from WT, ∆+56, and Irf8–/– were cultured with Flt3L for 5 days. Representative FACS plots of DC subsets (upper panels) and their absolute cell numbers (lower panels) produced in the culture are shown (total n = 4 cell cultures). The data were pooled from two independent experiments. d,e, Bone marrow chimera experiments. WT or ∆+56 HSPCs (c-Kit+, 3.0 × 105 cells) were transplanted into irradiated mice (CD45.1+) together with 2.0 × 105 competitor WT whole bone marrow cells (CD45.1+). Cells were analyzed 2 months after transplantation by FACS and RT-qPCR. In (d), absolute numbers of progenitor populations in bone marrow and differentiated cells in spleens derived from WT or ∆+56 donor cells are shown. The data were pooled from two independent experiments (total n = 6 mice per genotype). In (e), Irf8 mRNA expression in donor-derived bone marrow progenitor populations (total n = 3 mice per genotype). The data were pooled from two independent experiments. Data in b, c (lower panels), d and e are shown as mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t test) with a fold-change greater than 1.5 or less than 0.66. The exact P values are provided in Source Data. N.D., not detected in (b) and not determined in (e). Source data

Extended Data Fig. 5 Phenotypes of the mice devoid of either the –50 kb or +32 kb Irf8 enhancer.

a-d, ∆–50 (a,b) and ∆+32 mouse (c,d) strains were analyzed by FACS and RT-qPCR. Absolute cell numbers of progenitor populations in bone marrow and differentiated cells in spleens are shown in (a) and (c). The data were pooled from two independent experiments (total n = 3 mice per genotype for pre-cDC1s, pre-cDC2s, and pre-DCs; total n = 4 mice per genotype for CD43+ Ly6C monocytes, CD43 Ly6C cells, and the other cell types of ∆–50 and ∆+32 mice; and total n = 6 mice for the other cell types of WT and Irf8–/– mice). Irf8 mRNA expression in the indicated cell populations are shown in (b) and (d). The data were pooled from two independent experiments (total n = 3 mice per genotype except for B cells of ∆–50 mice; n = 2 mice for B cells of ∆–50 mice). All data in Extended Data Figure 5 are shown as mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t test) with a fold-change greater than 1.5 or less than 0.66. The exact P values are provided in Source Data. N.D., not determined. Source data

Extended Data Fig. 6 Expression of GFP, exogenous IRF8, and endogenous IRF8.

a, Representative FACS plots of immunostaining for IRF8 in Irf8–/– c-Kit+ cells transduced with a bicistronic retrovirus expressing IRF8 and GFP for two days. Cells in the lower and upper quarters were sorted into GFPlow and GFPhi populations (left panels). IRF8 expression concentrations in these populations are shown (right panel, n = 3 cell cultures). Data are representative of two independent experiments, which yielded similar results. b, IRF8 expression in bone marrow progenitor cells and splenic differentiated cells from WT and ∆+56 mice (n = 3 mice per genotype). Data are representative of two independent experiments, which yielded similar results. ΔMFI was calculated by subtracting the background MFI with isotype control IgG1. Data in a (right panel) and b are presented as mean + SD; * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t test). The exact P values are provided in Source Data. N.D., not determined. Source data

Extended Data Fig. 7 Dynamics of MDP enhancers in WT, ∆+56, and Irf8−/− mice.

a, Box plots of the normalized H3K27ac ChIP-seq tag densities within the clusters 2, 3, 4, and 5 identified in Fig. 4a in the indicated cell types from WT, ∆+56, and Irf8–/– mice (horizontal lines within the box, median; the lower and upper ends of the box, 25th [Q1] and 75th [Q3] percentiles; the minimum limit of whiskers, minimum value or Q1 − 1.5× interquartile range [IQR]; the maximum limit of whiskers, maximum value or Q3 + 1.5× IQR). The P values were calculated by the paired two-tailed t test. The exact P values are provided in Source Data. b, A heat map illustrating normalized enrichment scores (NES) of GSEA for the genes nearest to the regions in the clusters 2, 3, 4, and 5. Each box shows a GSEA NES that compares the hematopoietic population on its left side with that on its upper side. mRNA expression data were obtained by RNA-seq. A positive NES means a greater value in the left-hand population. NaN, not-a-number. c, mRNA expression of the representative genes from the clusters 2, 3, 4, and 5 identified in Fig. 4a. The indicated cell types from WT, ∆+56, and Irf8–/– mice were analyzed by RNA-seq (n = 2 biologically independent samples per population). d, Expression of Klf4 mRNA analyzed by RNA-seq in the indicated cell types from WT, ∆+56, and Irf8–/– mice (n = 2 biologically independent samples per population). N.D., not determined. Data in c and d are presented as mean. Source data

Extended Data Fig. 8 Expression of genes encoding TFs co-operating with IRF8.

mRNA expression amounts of the indicated genes in WT Ly6C+ monocytes and cDC1s determined by RNA-seq (n = 2 biologically independent samples per population). Data are shown as mean. Source data

Extended Data Fig. 9 RUNX–CBFβ regulates the development of cDCs.

a, Genome Browse images of RUNX1 and RUNX2 ChIP-seq tags in HSPC cell lines around the Irf8 gene and enhancers, retrieved from previous reports39,40,41. ATAC-seq data on WT MDPs newly obtained in this study (n = 2 biologically independent samples, each using 1 mouse) and H3K27ac ChIP-seq data on WT MDPs retrieved from our previous publication21 (n = 2 biologically independent samples, each using 20 mice) are shown for reference. b,c, WT LSK cells were transduced with a lentivirus encoding shRNA against Cbfb and cultured with SCF and Flt3L as in Fig. 7. Representative FACS plots on day 7 are shown in (b). The percentages of MDPs and CDPs (day 5, n = 3 cell cultures), cDCs, pDCs, and monocytes/macrophages (day 7, n = 4 cell cultures) among GFP+ cells are shown in (c). Data are representative of two independent experiments, which yielded similar results. The bar graphs are shown as mean + SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t test). The exact P values are provided in Source Data. d, Representative histograms of reporter assays in MDPs shown in Fig. 7f. e, mRNA expression of Runx1, Runx2, Runx3, and Cbfb analyzed by RNA-seq in the indicated cell types from WT mice (n = 2 biologically independent samples). Data are shown as mean. f, Normalized microarray intensities of Runx1, Runx2, Runx3, Cbfb, and Irf8 in IRF8 and IRF8+ LMPPs (n = 2 biologically independent samples). Data are shown as mean. g, A Genome Browser image of sequence conservation at the human IRF8 gene locus against the mouse genome. The region corresponding to mouse +56 kb Irf8 enhancer (dotted line) and the region used for the +56 kb enhancer reporter assay (orange) are indicated. Positions of the two RUNX motifs are shown as blue arrow lines. Mac, macrophage; NC, negative control. Source data

Extended Data Fig. 10 The proposed model of the IRF8-dose dependent myeloid lineage choice.

a, Schematic models of the phenotypes of three mouse strains devoid of the +56, +32 or –50 kb Irf8 enhancer. The models are described as in Extended Data Fig. 1. Phenotypes of WT and Irf8–/– mice are also displayed for comparison. b, Proposed model: The RUNX–CBFβ-driven Irf8 + 56 kb enhancer induces early IRF8 expression in myeloid progenitors. The +56 kb enhancer-mediated high IRF8 expression in myeloid progenitor cells is essential for the development of CDPs and cDC1s, whereas low IRF8 expression in myeloid progenitor cells preferentially induces monopoiesis. The absence of IRF8 expression leads to differentiation into neutrophils (Neu). The lineage choice is epigenetically determined in an IRF8 dose-dependent manner via cooperation or antagonism with other TFs to activate distinct sets of downstream enhancers. TFs, transcription factors.

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Supplementary Table 1

Oligonucleotides used in this study.

Supplementary Table 2

Datasets used in this study.

Source data

Source Data Fig. 1

Statistical source data with exact P values.

Source Data Fig. 2

Statistical source data with exact P values.

Source Data Fig. 3

Statistical source data with exact P values.

Source Data Fig. 4

Statistical source data with exact P values.

Source Data Fig. 5

Statistical source data with exact P values.

Source Data Fig. 7

Statistical source data with exact P values.

Source Data Extended Data Fig. 3

Full scans of gel images for genomic PCR.

Source Data Extended Data Fig. 4

Statistical source data with exact P values.

Source Data Extended Data Fig. 5

Statistical source data with exact P values.

Source Data Extended Data Fig. 6

Statistical source data with exact P values.

Source Data Extended Data Fig. 7

Statistical source data with exact P values.

Source Data Extended Data Fig. 8

Statistical source data with exact P values.

Source Data Extended Data Fig. 9

Statistical source data with exact P values.

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Murakami, K., Sasaki, H., Nishiyama, A. et al. A RUNX–CBFβ-driven enhancer directs the Irf8 dose-dependent lineage choice between DCs and monocytes. Nat Immunol 22, 301–311 (2021). https://doi.org/10.1038/s41590-021-00871-y

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