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. 2019 Apr 25;133(17):1803-1813.
doi: 10.1182/blood-2018-06-857789. Epub 2019 Feb 22.

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice

Daisuke Kurotaki  1 , Wataru Kawase  1 , Haruka Sasaki  1 , Jun Nakabayashi  2 , Akira Nishiyama  1 , Herbert C Morse 3rd  3 , Keiko Ozato  4 , Yutaka Suzuki  5 , Tomohiko Tamura  1   2
Affiliations

Epigenetic control of early dendritic cell lineage specification by the transcription factor IRF8 in mice

Daisuke Kurotaki et al. Blood. .

Abstract

Dendritic cells (DCs), which are vital for immune responses, are derived from bone marrow hematopoietic stem cells via common DC progenitors (CDPs). DC lineage fate decisions occurring at stages much earlier than CDPs have recently been recognized, yet the mechanism remains elusive. By single-cell RNA-sequencing, in vivo cell transfer experiments, and an assay for transposase-accessible chromatin sequencing using wild-type, IRF8-GFP chimera knock-in or IRF8-knockout mice, we demonstrate that IRF8 regulates chromatin at the lymphoid-primed multipotent progenitor (LMPP) stage to induce early commitment toward DCs. A low but significant expression of IRF8, a transcription factor essential for DC and monocyte development, was initiated in a subpopulation within LMPPs. These IRF8+ LMPPs were derived from IRF8- LMPPs and predominantly produced DCs, especially classical DC1s, potentially via known progenitors, such as monocyte-DC progenitors, CDPs, and preclassical DCs. IRF8+ LMPPs did not generate significant numbers of monocytes, neutrophils, or lymphocytes. Although IRF8- and IRF8+ LMPPs displayed very similar global gene expression patterns, the chromatin of enhancers near DC lineage genes was more accessible in IRF8+ LMPPs than in IRF8- LMPPs, an epigenetic change dependent on IRF8. The majority of the genes epigenetically primed by IRF8 were still transcriptionally inactive at the LMPP stage, but were highly expressed in the downstream DC lineage populations such as CDPs. Therefore, early expression of the key transcription factor IRF8 changes chromatin states in otherwise multipotent progenitors, biasing their fate decision toward DCs.

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Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Identification of IRF8-expressing LMPPs. (A) Flow cytometry analysis of HSCs and early progenitors in IRF8-GFP mice. Data are representative of 5 independent experiments. (B) Hierarchical clustering of scRNA-seq data in WT LMPPs. Transcription factor genes upregulated or downregulated more than fivefold in LMPPs compared with CDPs were selected. Red and blue gene symbols denote upregulated and downregulated genes in CDPs, respectively. The Irf8 transcript-expressing cluster is enclosed by a green rectangle. (C) Hierarchical clustering of microarray transcriptome data in hematopoietic populations. Mo, monocyte; neu, neutrophil.
Figure 2.
Figure 2.
Differentiation potential of IRF8+ LMPPs in vivo. Flow cytometric analysis of splenic DC subpopulations, monocytes, and neutrophils 4 days (A), 7 days (B), or 10 days (C) after intravenous transplantation of LMPP subpopulations. A total of 1000 LMPPs (CD45.2+) were transplanted into irradiated Ly5.1 mice (CD45.2−), and donor-derived (CD45.2+) cells were analyzed. Absolute cell numbers (per spleen of a mouse) of the indicated progeny cells derived from transplanted LMPPs are shown in the boxplots. (A-C) Values from 3 independent experiments are shown. Representative FACS plots of cDCs (D, left) and monocytes and neutrophils (D, right) on day 7. (E) Single-cell differentiation analysis of LMPP subpopulations. LMPP subpopulations were single cell-sorted into 96-well plates and cultured with Flt3L for 7 days. Following staining, cells were analyzed by flow cytometry. A total of 192 single cells of each subpopulation were analyzed. cDC1, cDC2, and pDC differentiation potential in single LMPPs was determined from the staining patterns of cells (top). Total cell numbers of indicated DC subsets yielded in 192 wells are calculated (bottom). Data are representative of 2 independent experiments with similar results. *P < .05, **P < .01, ***P < .001 (Student t test). MHC, major histocompatibility complex.
Figure 3.
Figure 3.
IRF8+ LMPPs are derived from IRF8− LMPPs. Flow cytometric analysis of bone marrow progenitors 3 days after intravenous transplantation of LMPP subpopulations. A total of 30 000 LMPPs were transplanted into a nonirradiated Ly5.1 mouse, and donor-derived cells were analyzed. Representative FACS plots of donor-derived LMPP subpopulations (A), MDPs (B), CDPs (B), and pre-cDCs (C) from 3 independent experiments. (D) Absolute cell numbers (per lower mouse limbs) of the indicated progeny cells derived from transplanted LMPPs are calculated. Values in the bar graph are the mean ± standard deviation from 3 independent experiments. *P < .05, **P < .01, ***P < .001 (Student t test).
Figure 4.
Figure 4.
IRF8+ LMPP signature genes are barely expressed in DC lineage cells. (A) Single-cell RT-qPCR analysis of Irf8 and Gapdh in WT and Irf8−/− LMPPs. Expression levels of Irf8 and Gapdh are expressed in −1 × Ct units. (B) A heat map showing z scores for the expression of LMPP subpopulation-specific genes. A total of 151 genes, significantly upregulated or downregulated in WT Irf8-transcript+ LMPPs compared with WT Irf8-transcript− LMPPs, were selected by using the scRNA-seq data (1-way analysis of variance, P < .05). Irf8-transcript+ cells were defined as cells with Irf8 fragments per kilobase million >10. (C) Representative LMPP subpopulation-specific genes. IRF8-dependent or IRF8-independent genes are shown as violin plots. *P < .05 (1-way analysis of variance). (D) GSEA comparing hematopoietic populations for 110 Irf8-transcript+ LMPP signature genes.
Figure 5.
Figure 5.
ATAC-seq analysis of IRF8+ LMPPs. ATAC-seq analyses were performed in IRF8− and IRF8+ LMPPs isolated from IRF8-GFP mice. Distal ATAC-seq peaks within H3K4me1-marked regions were selected. (A) A Venn diagram showing the overlap of ATAC-seq peaks between LMPP subpopulations. (B) Genome browser images of ATAC-seq data at the Jak2 and Irf8 gene loci. Blue rectangles indicate IRF8− LMPP (top) and IRF8+ LMPP (bottom) specific peaks. The orange rectangle indicates a previously reported Irf8 enhancer region. (C) De novo motif analysis of ATAC-seq peak regions specific for each subpopulation and those common to 2 subpopulations. (D) GSEA comparing hematopoietic populations for genes associated with IRF8+ LMPP-specific ATAC-seq peaks. (E) Heat map showing NES of GSEA. NES, normalized enrichment score.
Figure 6.
Figure 6.
IRF8 governs the chromatin accessibility of DC lineage enhancers in IRF8+ LMPPs. ATAC-seq analyses were performed in WT and Irf8−/− LMPPs. IRF8+ LMPP-specific 1434 ATAC-seq peak regions detected in WT LMPPs were analyzed. (A, left) ATAC-seq tag-densities of the peak regions in WT and Irf8−/− LMPPs are shown in violin plots. Student t test was performed for statistical significance. (A, right) The presence or absence of the 1434 ATAC-seq peaks were judged in Irf8−/− LMPPs. (B) De novo motif analysis of IRF8+ LMPP-specific peaks lost in Irf8−/− LMPPs (ie, 846 peaks). (C) Genome browser image of ATAC-seq data at the Ifi44 gene locus. Blue rectangle indicates an IRF8-dependent IRF8+ LMPP-specific peak. (D, left) Overlap between LMPP subpopulation-specific ATAC-seq peaks and IRF8 ChIP-seq peaks in MDPs. (D, right) Heat maps of IRF8 binding in MDPs at the LMPP subpopulation-specific ATAC-seq peak regions. Each horizontal line represents the density of IRF8 ChIP-seq tags in the 4-kb region centered on the ATAC-seq peak summit.
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