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. Access through your institution Buy or subscribe This is a

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* Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS PIVOTAL ROLE OF DPYSL2A IN KLF4-MEDIATED MONOCYTIC

DIFFERENTIATION OF ACUTE MYELOID LEUKEMIA CELLS Article Open access 20 November 2020 THE EPIGENETIC PIONEER EGR2 INITIATES DNA DEMETHYLATION IN DIFFERENTIATING MONOCYTES AT BOTH STABLE AND

TRANSIENT BINDING SITES Article Open access 10 March 2021 THPOK IS A CRITICAL MULTIFACETED REGULATOR OF MYELOID LINEAGE DEVELOPMENT Article 20 July 2023 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

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genome-wide expression profiles. _Proc. Natl Acad. Sci. USA_ 102, 15545–15550 (2005). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references 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 Author notes * These authors contributed equally:

Koichi Murakami, Haruka Sasaki, Akira Nishiyama. AUTHORS AND AFFILIATIONS * Department of Immunology, Yokohama City University Graduate School of Medicine, Kanagawa, Japan Koichi Murakami, 

Haruka Sasaki, Akira Nishiyama, Daisuke Kurotaki, Wataru Kawase, Tatsuma Ban & Tomohiko Tamura * Advanced Medical Research Center, Yokohama City University, Kanagawa, Japan Koichi

Murakami, Jun Nakabayashi & Tomohiko Tamura * Laboratory of Stem Cell Biology, Department of Biosciences, Kitasato University School of Science, Kanagawa, Japan Satoko Kanzaki, Yoichi

Sekita & Tohru Kimura * Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School of Medicine, Kanagawa, Japan Hideaki Nakajima * Program in Genomics of

Differentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA Keiko Ozato Authors * Koichi Murakami View author publications You

can also search for this author inPubMed Google Scholar * Haruka Sasaki View author publications You can also search for this author inPubMed Google Scholar * Akira Nishiyama View author

publications You can also search for this author inPubMed Google Scholar * Daisuke Kurotaki View author publications You can also search for this author inPubMed Google Scholar * Wataru

Kawase View author publications You can also search for this author inPubMed Google Scholar * Tatsuma Ban View author publications You can also search for this author inPubMed Google Scholar

* Jun Nakabayashi View author publications You can also search for this author inPubMed Google Scholar * Satoko Kanzaki View author publications You can also search for this author inPubMed

 Google Scholar * Yoichi Sekita View author publications You can also search for this author inPubMed Google Scholar * Hideaki Nakajima View author publications You can also search for this

author inPubMed Google Scholar * Keiko Ozato View author publications You can also search for this author inPubMed Google Scholar * Tohru Kimura View author publications You can also search

for this author inPubMed Google Scholar * Tomohiko Tamura View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION 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. PUBLISHER’S NOTE

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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.

SUPPLEMENTARY INFORMATION REPORTING SUMMARY PEER REVIEW INFORMATION 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. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 22 May 2020 *

Accepted: 11 January 2021 * Published: 18 February 2021 * Issue Date: March 2021 * DOI: https://doi.org/10.1038/s41590-021-00871-y SHARE THIS ARTICLE Anyone you share the following link with

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