A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors

Article

A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors Graphical Abstract

Authors Ari Itoh-Nakadai, Mitsuyo Matsumoto, Hiroki Kato, ..., Kyoko Ochiai, Akihiko Muto, Kazuhiko Igarashi

Correspondence [email protected]

In Brief Microbes skew the balance of innate and acquired immune cells. Itoh-Nakadai et al. report that Bach2 and C/EBP form a gene regulatory network with mutual repression and antagonistic, feedforward regulation of myeloid genes. Bach2 tunes the commitment of multipotent progenitors to myeloid and lymphoid lineages under both normal and infectious conditions.

Highlights d

Bach2 and C/EBP families share target genes in hematopoietic progenitor cells

d

Bach2 and C/EBP families oppose each other, regulating super enhancers of myeloid genes

d

Bach2 directly promotes lymphoid gene expression besides its repressor function

d

Lipopolysaccharide skews progenitors toward myeloid fate by reducing Bach2 expression

Itoh-Nakadai et al., 2017, Cell Reports 18, 2401–2414 March 7, 2017 ª 2017 The Author(s). http://dx.doi.org/10.1016/j.celrep.2017.02.029

Accession Numbers GSE87503 GSE80954

Cell Reports

Article A Bach2-Cebp Gene Regulatory Network for the Commitment of Multipotent Hematopoietic Progenitors Ari Itoh-Nakadai,1,2,3 Mitsuyo Matsumoto,1,4 Hiroki Kato,1 Junichi Sasaki,1 Yukihiro Uehara,1 Yuki Sato,1 Risa Ebina-Shibuya,1,6 Mizuho Morooka,1 Ryo Funayama,4,5 Keiko Nakayama,4,5 Kyoko Ochiai,1,2,4 Akihiko Muto,1,2 and Kazuhiko Igarashi1,2,4,7,* 1Department

of Biochemistry, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan Japan Agency for Medical Research and Development, Tokyo 100-0004, Japan 3Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan 4Center for Regulatory Epigenome and Diseases, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 5Department of Cell Proliferation, United Center for Advanced Research and Translational Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 6Department of Respiratory Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan 7Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.celrep.2017.02.029 2AMED-CREST,

SUMMARY

Hematopoietic stem cell and multipotent progenitor (MPP) commitment can be tuned in response to an infection so that their differentiation is biased toward myeloid cells. Here, we find that Bach2, which inhibits myeloid differentiation in common lymphoid progenitors, represses a cohort of myeloid genes and activates those linked to lymphoid function. Bach2 repressed both Cebpb and its target Csf1r, encoding C/EBPb and macrophage colony-stimulating factor receptor (M-CSFr), respectively, whereas C/EBPb repressed Bach2 and activated Csf1r. Bach2 and C/EBPb further bound to overlapping regulatory regions at their myeloid target genes, suggesting the presence of a gene regulatory network (GRN) with mutual repression between these factors and a feedforward loop leading to myeloid gene regulation. Lipopolysaccharide reduced the expression of Bach2, resulting in enhanced myeloid differentiation. The Bach2-C/EBPb GRN pathway thus tunes MPP commitment to myeloid and lymphoid lineages both under normal conditions and after infection. INTRODUCTION Hematopoietic stem cells (HSCs) in the bone marrow maintain homeostasis by appropriately balancing the numbers of lymphoid cells, monocytes, granulocytes, and erythrocytes (Abramson et al., 1977; Busch et al., 2015). HSCs and multipotent progenitors (MPPs) are lineage marker-negative (Lin–), Sca-1+, and c-Kit+ (LSK). Recent studies have shown that various types of lineage-restricted progenitors exist in the LSK compartment (Miyawaki et al., 2015; Pietras et al., 2015; Yama-

moto et al., 2013). The determination of cell fate (i.e., commitment) in HSCs and MPPs has been suggested to involve a gradual change in gene expression, regulated by combinations of multiple transcription factors (Enver et al., 2009). These transcription factors are presumed to form gene regulatory networks (GRNs) with mutually activating and repressing functions, resulting in positive feedback loops that resolve lineage-specific gene expression programs. The reciprocal inhibition of genes encoding Gata1 and PU.1 primarily organizes the bifurcation of the hematopoietic lineage fate to isolatable myeloerythroid and myelolymphoid progenitor populations (Gata1highPU.1low and Gata1lowPU.1high) in MPPs, comprising the earliest branchpoint of hematopoietic cell fates (Arinobu et al., 2007). Following this branchpoint, some of the lymphoid-specific genes are upregulated in the myelolymphoid population, giving rise to lymphoid-primed multipotent progenitors (LMPPs). LMPPs can differentiate toward lymphoid cells as well as granulocytes and monocyte, but lose any megakaryocyte-erythroid potential (Adolfsson et al., 2005; Ma˚nsson et al., 2007). LMPPs give rise to common lymphoid progenitors (CLPs), which are the precursors of B and natural killer (NK) cells (Kondo et al., 1997). Important transcription factors for the development of LMPPs include E2A, PU.1, and Ikaros (Arinobu et al., 2007; Dias et al., 2008; Yoshida et al., 2006). E2A is known to promote the gene expression of Dntt and Rag1 in LMPPs (Dias et al., 2008). However, the expression of these transcription factors is not initiated anew in LMPPs but is sustained at high levels from HSCs to LMPPs (A.I.-N. and K.I., unpublished data) (Lin et al., 2010). How the expression of lymphoid-specific genes is first activated at the LMPP stage is unclear and is one of the central questions behind a deeper understanding of the mechanism of lineage commitment. The output of myeloid and lymphoid cells is rapidly tailored in the bone marrow according to peripheral demand, such as infection and inflammation. In the initial phase of infection, myeloid cells are expanded at the expense of lymphoid cells

Cell Reports 18, 2401–2414, March 7, 2017 ª 2017 The Author(s). 2401 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

in 1x106 BM cells (1x10)

20

20

15

15

15

10

10

10

5

5

5

0

0

0

in 1x106 BM cells (1x102)

CMP

GMP

12 10 6 8 4

in 1x106 BM cells (1x102)

P = 0.1

0 MEP

1.0

1

0.5

50

0

0

0

200 150 P = 0.02

immatureB

pre-B 5 4 3

3 P = 0.04

P < 0.01

5

1

2

P < 0.01

1

0

0

0 +

+

CD4 CD8 DP

CD8+ SP

CD4 SP

400

40

300

30

200

20

10 8 6

P < 0.01

100

P = 0.02

10

0 Bach2–/–

P = 0.03

4 2 0

0 WT

myeloid

1.5

10

2

5

2

15

4

P = 0.2

3

5

+

in 1x106 Thymus cells (1x102)

10

2.0

pro-B 6

15

4

2 0

CLP

LMPP

ST-HSC

LT-HSC 20

WT

Bach2–/–

WT

Bach2–/–

(Esplin et al., 2011; Medzhitov and Horng, 2009; Nagai et al., 2006). This response is due in part to HSC expression of sensors of pathogen-associated molecular patterns (PAMPs), which can detect lipopolysaccharide (LPS). LSK cells containing HSCs and MPPs, and LMPPs all express TLR4 and its co-receptors MD-2 and CD14 (Nagai et al., 2006). Toll-like receptor (TLR) signaling drives the myeloid differentiation of LSK (Nagai et al., 2006) and elicits a loss of common lymphoid progenitors (CLPs) in vivo (Esplin et al., 2011). Furthermore, macrophage colony-stimulating factor (M-CSF), the levels of which are increased in the serum in response to LPS (Mossadegh-Keller et al., 2013), directly induces the expression of the myeloid master regulator PU.1 in mouse HSCs (Mossadegh-Keller et al., 2013). Collectively, these observations suggest that infectious conditions initially promote myeloid development from the stem and progenitor cells at the expense of lymphocyte development. HSCs and progenitors also show substantial alterations in developmental potential under conditions of hematopoietic stress, such as those imposed by regeneration and inflammation (Busch et al., 2015; Guo et al., 2013; King and Goodell, 2011; Mossadegh-Keller et al., 2013). However, the mechanisms underlying the transcriptional regulation of myeloid and lymphoid cell differentiation of HSCs and early pro-

2402 Cell Reports 18, 2401–2414, March 7, 2017

100

Figure 1. Competitive Bone Marrow Transplantation Assay Showed a Lower Contribution of Bach2 –/– Cells in Lymphocyte Differentiation CD45.1+/ CD45.2+ heterozygous wild-type (control: left) (n = 4 or 5) and CD45.2 Bach2–/– (right) (n = 4 or 5) total bone marrow cells were transplanted at a 1:1 ratio into lethally irradiated host CD45.1+/ CD45.2+ heterozygous mice. Recipient mice were scarified and analyzed 16 weeks after the transplantation. Absolute numbers of various cell populations (above plots) and p values are shown in each graph (unpaired two-tailed Student’s t test). Double positive (DP), single positive (SP).

genitors under the conditions of infection and other stressors are not yet fully understood. 6 Bach2 and Bach1 are transcription 5 repressors that belong to the basic re4 3 gion-leucine zipper family and bind to 2 Maf-recognition element (MARE) (Oyake P < 0.01 1 et al., 1996). Bach2 is necessary for immu0 noglobulin class-switch recombination and –/– WT Bach2 the somatic hypermutation of immunoglobulin-encoding genes (Muto et al., 2004) and the pre-B cell receptor check point (Swaminathan et al., 2013). Bach2 and Bach1 promote B cell development by repressing genes important for myeloid cells (myeloid genes) in CLPs (Itoh-Nakadai et al., 2014). Bach2 directly represses Cebpb and other myeloid genes in CLPs, and Bach1 partially compensates for the Bach2 function (Itoh-Nakadai et al., 2014). Considering the function of Bach2 in CLPs, we wondered whether Bach2 might also be involved in the homeostasis and commitment of earlier progenitors, including HSCs, and their reprograming in response to environmental perturbations. Here, we examine GRN in MPPs involving direct cross-antagonism between C/EBPb and Bach2 as well as antagonistic, feedforward regulation of myeloid genes. We suggest that Bach2 is a key cell-intrinsic factor for lineage commitment and tuning. matureB

RESULTS Impaired Lymphoid Development Due to Bach2 Deficiency To clarify the role of Bach2 in progenitor cells under regenerative stress, we performed competitive transplantation assays. Bone marrow cells (1 3 106 each) from CD45.1+/ CD45.2+ heterozygous wild-type mice (control mice) and CD45.2 Bach2–/– mice were transplanted into lethally irradiated congenic mice. Flow cytometry of the bone marrow cells from the recipient mice beginning at four months after transplantation showed roughly equal contribution of control and Bach2–/– cells to the HSC compartments (Figure 1). The contribution to LMPPs and CLPs tended to be lower in the Bach2–/– cells than in the wild-type

cells. Significant impairments were observed in the contribution of Bach2–/– cells to the B and T lineage cells (Figure 1). In contrast, myeloid progenitors (common myeloid progenitors [CMPs], granulocyte-monocyte progenitors [GMPs]) and myeloid cells similarly developed from control and Bach2–/– cells. The numbers of megakaryocyte-erythroid progenitors (MEPs) were reduced in the absence of Bach2, raising the possibility that Bach2 is involved in the differentiation of either or both erythroid or megakaryocyte cells as well. Since red blood cells do not express CD45, we could not further explore this possibility. Given that early B and T cell development is not affected by Bach2 deficiency under normal conditions (Itoh-Nakadai et al., 2014), these results suggested that the function of Bach2 in the development of lymphoid cells, including lymphoid progenitors such as LMPPs and CLPs, is essential to hematopoiesis under regeneration stress. Influence of Bach Deficiency on the Development of LMPPs and CLPs To test whether Bach2 is redundant with Bach1 in progenitor cells under normal conditions, we compared wild-type (WT), Bach1-deficient (Bach1–/–), Bach2-deficient (Bach2–/–), and double-deficient (Bach1–/–Bach2–/–) mice. The frequencies and total numbers of HSCs and MPPs were unaltered in the bone marrow of Bach1–/–Bach2–/–, Bach1–/–, Bach2–/–, and wild-type mice (Figure 2A). The numbers of LMPPs and CLPs were significantly lower in the bone marrow of Bach1–/–Bach2–/– mice than in the bone marrow of wild-type mice (51% and 37% lower, respectively) (Figures 2A, S1A, and S1B). The number of interleukin-7 (IL-7) receptor positive cells in the LSK cells did not change between wild-type and Bach1–/–Bach2–/– (Figure S1C), indicating that the observed reduction of LMPPs was not due to a contamination of CLPs. In addition, the surface expression of Sca-1 was markedly increased in the CLPs in Bach1–/–Bach2–/– mice compared with that in the CLPs of the other mice (Figures 2B and S1B). Since CLPs are conventionally defined using Sca-1 (Kondo et al., 1997), we monitored the CLPs in Bach1–/–Bach2–/– mice by another method without using Sca-1. Multilineage progenitors (MLPs), CLPs, and Fraction A (Fr. A, pre-proB cells) can be detected by this method (Rumfelt et al., 2006) (Figures 2C and S1D). The numbers of MLPs were similar for all mice, irrespective of the genotypes (Figure 2C). In contrast, the numbers of CLPs were markedly decreased in the bone marrow of Bach1–/– (38%), Bach2–/– (26%), and Bach1–/–Bach2–/– (21%) mice compared with wild-type mice (Figure 2C). Taken together, these observations indicate that Bach1 and Bach2 are required for the proper development of B cell progenitors, such as LMPPs and CLPs. Inhibition of the Expression of Myeloid Genes in LSK Cells by Bach1 and Bach2 To clarify the role of Bach1 and Bach2 in gene expression in HSCs and progenitors, we performed a microarray analysis of LSK cells isolated from wild-type and Bach1–/–Bach2–/– mice. A gene set enrichment analysis (GSEA) (Subramanian et al., 2005) revealed that Bach1–/–Bach2–/– LSK cells showed increased expression of genes that are normally expressed in early myeloid progenitors (preGMs) and decreased expression

of genes that are normally expressed in CLPs (Pronk et al., 2007) (Figure 2D). Upregulated genes in Bach1–/–Bach2–/– LSK cells included those important for myeloid differentiation (e.g., Csf1r, Irf8, Cebpa) and the myeloid function (Mpo and Hmox1) (Table S1). To compare the gene expression in LSK cells, we performed a single-cell qPCR analysis using a microfluidic device. The frequencies of cells expressing Csf1r, Irf8, Cebpa, Mpo, Tgfbr2, Gfi1b, or Hmox1 were higher in Bach1–/–Bach2–/– LSK cells than in wild-type LSK cells (Figure S2A). The frequencies of cells expressing Hmox1, Tgfb2, Gfi1b, Irf8, Cebpa, and Mpo were also significantly increased in Bach1–/–Bach2–/– LSK cells (Fisher’s exact test, Figures 2E and S2A). Of the examined genes that are important for B cell commitment (Lin et al., 2010; Mansson et al., 2012; Welinder et al., 2011), the expression frequency of Foxo1 tended to be lower in Bach1–/–Bach2–/– LSK cells than in wild-type cells (Figures 2E and S2A) but did not reach the level of statistically significant difference. LSK cells contain LMPPs (Adolfsson et al., 2005), which were decreased in Bach1–/– Bach2–/– mice. This alteration in the LMPP population might have resulted in the above observations. To address this issue, we used Dntt gene to further stratify the cells. We found that Dntt was expressed in LMPPs and CLPs but not in long-term HSC (LT-HSC) and short-term HSC (ST-HSC) (Figure S2B), enabling us to exclude LMPP and CLP from the single-cell populations. We performed reanalysis of the single-cell PCR experiments using cells not expressing Dntt (Dntt– LSK cells). The mRNA levels of Hmox1, Cebpa, Tgfbr2, and Irf8 were increased in Bach1–/–Bach2–/– Dntt– LSK cells compared wild-type Dntt– LSK cells (Figure S2C). Therefore, HSCs and/or MPPs in the LSK fraction were altered in gene expression in the absence of both Bach2 and Bach1. Taken together, these results suggested that Bach2 and Bach1 promoted the differentiation of LSK cells into lymphocyte progenitors under the normal steady state by repressing myeloid genes. Given our previous observations (Itoh-Nakadai et al., 2014) and the essential role of Bach2 in the lymphoid cell differentiation of HSCs during bone marrow transplantation, these results suggest that Bach2, and not Bach1, plays the main role in the differentiation of LSK cells. To evaluate the role of Bach2 in LSK cells for lineage specification, we sorted the LSK cells and CLPs from Bach2–/– mice and then cultured them on the TSt-4 stroma cells (Ikawa et al., 2010) with SCF, Flt3, and IL-7, which support the generation of both myeloid and B cells. Bach2–/– LSK cells were impaired for the differentiation of B220+CD19– pre-pro-B cells and B220+CD19+ pre-B cells (Figure 2F). In contrast, Bach2–/– CLPs showed milder defects. The myeloid cell differentiation of CLPs was increased in the absence of Bach2, with LSK cells showing a similar trend (Figure 2F). These results indicated that Bach2 plays an important role not only in CLPs, but also in LSK cells for supporting the B cell progenitor development, which involves the repression of myeloid genes. Downregulation of Bach2 Gene Expression in Myeloid Progenitors To clarify the expression of Bach2 in progenitor cells, we compared its expression in HSCs and the progenitor cells using

Cell Reports 18, 2401–2414, March 7, 2017 2403

P = 1.0

B

8 WT

Bone marrow cells (1x104)

7 6

Bach1–/–

Relative fluoresent intensity of Sca-1

A

Bach1–/–Bach2–/–

Bach2–/–

P = 0.1

5 4

P = 0.02

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P = 0.03

2 1 0

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P < 0.05

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Bach1–/–

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Bach2–/– Bach1–/– Bach2–/–

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Enrichment score

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WT Bach1–/–Bach2–/– Bach1–/–

Bach2–/–

0.5 0.4 0.3 0.2 0.1 0.0

0.05 0.00 -0.10 -0.20 -0.30 -0.40

CLP

NES = 1.31 ES = 0.52 P < 1x10-3

preGM

NES = –1.49 ES = –0.4 P < 1x10-3

Low in Bach1–/–Bach2–/–

E

P = 4.8E-07 P = 0.002 Hmox1 Tgfbr2

Expression (log2)

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P = 0.01 Gfi1b

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P = 0.2 Foxo1

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Bach1–/–Bach2–/–

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P = 0.01 2.0 P = 0.002 1.5 1.0 0.5 0

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P = 0.4

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B220+ CD19+

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B220+ CD19–

B220+ CD19+

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LSK

Cells (1x105)

2.5

Cells (1x106)

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F

High in Bach1–/–Bach2–/–

4 WT

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2 1

CD11b+

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CD11b+

Figure 2. Differentiation of the Lymphoid Progenitor Is Defected by Bach2 Gene Deficiency (A) Absolute numbers of total LSK cells (Lineage–Sca-1+c-Kit+), HSCs (Lineage–Sca-1+c-Kit+Flt3–), MPPs (Lineage–Sca-1+c-Kit+Flt3mid), LMPPs (Lineage–Sca1+c-Kit+Flt3High), CLPs (Lineage–Sca-1midc-KitmidFlt3+ IL7Ra+) in the bone marrow of wild-type (n = 8), Bach1–/– (n = 3), Bach2–/– (n = 3), Bach1–/–Bach2–/– (n = 8). p values are shown in each graph (mean ± SEM unpaired two-tailed Student’s t test). (B) The relative MFI (mean fluorescent intensity) of Sca-1 in CLPs of wild-type, Bach1–/–, Bach2–/–, and Bach1–/–Bach2–/– mice. p values are shown in each graph (mean ± SEM of biological quintuplicate: unpaired two-tailed Student’s t test).

(legend continued on next page)

2404 Cell Reports 18, 2401–2414, March 7, 2017

A

B

Figure 3. Bach2 Expression Is High in LSK Cells Compared to Myeloid Progenitors (A) RT-PCR analysis of gene expression of Bach2 (n = 2), Bach1 (n = 2), Ebf1 (n = 1), and Pax5 (n = 1) in each population. (B) Sorting of myeloid-progenitors (mye-prog: Lineage–Sca-1–c-Kit+) and LSK cells in Bach2-RFP mice (left). A histogram of the expression intensity of RFP in wild-type LSK cells, Bach2-RFP LSK cells, Bach2-RFP myeloid progenitors, and Bach2-RFP CLPs. The data shown are representative data of three independent experiments.

RT-qPCR (Figure 3A). The pattern of the Bach2 expression was similar to that of Ebf1 and Pax5; it gradually increased from HSCs to CLPs, was highly expressed in B cells, and downregulated in plasma cells (Figure 3A). In contrast, the expression of Bach1 was high in HSCs and the progenitor fractions (Figure 3A). To further define the expression pattern of Bach2 in progenitors, we analyzed Bach2-red fluorescent protein (RFP) reporter mice (Itoh-Nakadai et al., 2014). The RFP expressions were higher in LSK cells and CLPs than in myeloid progenitors containing CMPs, GMPs, and MEPs (Figure 3B). These results suggest that Bach2 regulates gene expression in the early MPP and stem cells. To clarify the correlation in expression of Bach2 and other lineage-specific genes in HSCs and early progenitors, we analyzed the LSK compartment, including LT-HSCs (CD150+Flt3– in LSK) (Pronk et al., 2007), ST-HSCs (MPPs) (CD150–Flt3– in LSK), and LMPPs (CD150–Flt3+ in LSK), as well as the CLPs from wild-type

mice using a microfluidic single-cell qPCR analysis. We judged that cells had expressed Bach2 (Bach2+ cells) when its expression was detected by both of two primer sets. Foxo1-expressing cells were significantly enriched in the Bach2+ LT-HSCs and STHSCs (Figure S3). Although the frequencies of Foxo1-expressing cells were similar between Bach2+ and Bach2– LMPP cells, the frequency of LMPPs expressing myeloid genes (Csf1r and Cebpa) tended to be higher in Bach2– cells than Bach2+ cells (Figure S3). Interestingly, in the LT-HSCs, Bach2– cells more frequently expressed Gata2 and less frequently Cd34 (Figure S3). To compare the expression pattern of Bach2, Foxo1, and myeloid genes on a ‘‘per-cell’’ basis during the development from LT-HSCs to CLPs, we ranked individual cells according to their expressions (cycling threshold [Ct]) value) (Figure S4A). We found that myeloid genes such as Cebpa, Csf1r, Mpo, and Irf8 exhibited increased expression per cell in ST-HSCs (MPPs) and LMPPs, whereas Bach2 and Foxo1 expression levels did

(C) Relative cell numbers of total MLPs (Lineage–HSA–c-Kit+IL7Ra–B220–), CLPs (Lineage–HSA–c-KitmidIL7Ra+B220–), Fr.A (Lineage–HSA–c-KitlowIL7Ra+B220+) in the bone marrow of wild-type, Bach1–/–, Bach2–/–, and Bach1–/–Bach2–/– mice. p values are shown in each graph (mean ± SEM of biological triplicate: unpaired two-tailed Student’s t test). (D) Gene set enrichment analysis (GSEA) comparing Bach1–/–Bach2–/– LSK cells and wild-type LSK cells. Upper and lower panels show CLP and preGM-specific gene sets, respectively. Normalized enrichment score (NES), enrichment score (ES), and nominal p value are implemented in GSEA. (E) Violin plots showing the expression pattern of different gene of expression pattern in wild-type (red) and Bach1–/–Bach2–/– (green) cells. p values, Fisher’s exact test. (F) Quantification of pre-pro-B cells (B220+CD19–), pre-B cells (B220+CD19+), and myeloid cells (CD11b+) in wells of in vitro culture of wild-type (black) and Bach2–/– (white) LSK cells under B cell development-condition for 12 days. p values are shown in each graph (mean ± SEM of biological triplicate: unpaired twotailed Student’s t test).

Cell Reports 18, 2401–2414, March 7, 2017 2405

GFP-EBF1 GFP+CD11b–

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Figure 4. Bach2 Promotes Lymphoid Progenitor Development of MPPs

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Bach2TG down genes : Fold change Il6st: 2.3 Acta1: 2 Acta2: 6.3 Lilrb4: 8.0 Adam8: 13.3 Ly96: 2.3 Arg2: 16.6 Mmp8: 68.9 Pf4: 5.4 Ccl9: 5.6 Cebpd: 3.3 Ptgs2: 4.2 Csf2rb2: 2.3 Tgfbi: 30.4 Fcgr2b: 6.9 Tlr4: 4.0 Fcgr3: 5.7 Gfi1b: 2.7

Xcl1 Irf4 Irf8 Igll1 Blnk Ly6d Tcf7 Spib Il7r Flt3 Flt3 Cd19 Dntt Tcf3 Cd79a Cd79b Pax5 Flt3 Pax5 Id2 Bcl6 Id3 Tcf3 Tcf7 Sfpi1 Ly96 Spic Rag2 Rag2 Rag1 Vpreb3 Ebf1 Cd79a Vpreb2 Vpreb1 Pou2af1 Rag1 Bach2TG B220+

MPP

Bach2TG up genes : Fold change Bcl11a: 2.3 Il2lb: 3.1 Caspl: 4.3 Il7: 2.5 Ccr7: 12.0 Irf4: 7.4 Cd72: 5.4 Xcl1: 67.7 Cd96: 6.2 Zap70: 2.6 Gata3: 9.2 Fasl: 7.1 Flt3: 2.0 Foxo1: 2.8 Igh-vj558: 18.7

EV

Bach2

Normalized value (log2 fold) –4.7

0

Bach2 to promote B cell differentiation against myeloid differentiation. As previously reported (Pongubala et al., 2008), CD19+B220+ pre-B cells appeared in MPPs expressing Ebf1-GFP but did not appear in MPPs transduced with a control virus. Bach2-GFP-transduced MPPs produced many CD19–B220+ pre-pro-B cells Normalized value (log fold) (21.4%) but no CD19+ pre-B cells (Figures –3.3 0 3.3 4A and 4B), suggesting the potential of Bach2 to induce the development of prepro-B cells but nothing further. We analyzed the gene expression profile of B220+ B cells generated from MPPs by Bach2 transduction using a microarray and compared the profile with those of freshly isolated MPPs and CLPs (Figure 4C). We focused on the genes characterizing CLPs, including those for cytokine receptors and recombination of immunoglobulin genes. Il7r and Ly6d were upregulated in the GFP+B220+ generated by Bach2 transduction, whereas Flt3, Rag1, Rag2, and Dntt were not altered as compared with MPPs (Figure 4C). B cell-specific genes such as Ebf1, Vpreb1, Vpreb2, Vpreb3, and Pou2af1 were not induced in the GFP+B220+ Bach2-transduced cells. However, Pax5, Igll1, and other B cells genes were upregulated in these cells compared with MPPs (Figure 4C). Therefore, when overexpressed in MPPs, Bach2 induced a fraction of B cell genes that are normally induced in CLPs. This pattern of gene expression suggests that the B220+ B cells generated from MPPs by Bach2 transduction were pre-pro-B cells, which express only a portion of B cell-specific genes. These results suggest that Bach2 promotes the development of pre-pro-B cells from MPPs and that EBF1 and Bach2 play distinct roles in the development of B cells. Bach2 promotes the differentiation of B220+ pre-pro-B cells, while EBF1 promotes CD19+ pre-B cells. Bach2 may promote B cell lineage commitment in MPPs and CLPs rather than the subsequent B cell differentiation, which is dependent on EBF1. In the preceding experiment, MPPs were cultured serially with two sets of cytokines. Therefore, the properties of the obtained B cells might have represented secondary responses. To clarify the direct function of Bach2 in MPPs, we revisited the previously 2

CLP

4.7

not change among the populations. Importantly, the expression levels of Cebpa and Cebpb became lower in CLPs than in other preceding cell populations. To confirm the increased expression of myeloid genes in Bach1–/–Bach2–/– progenitors, we sorted wild-type and Bach1–/–Bach2–/– MPPs and LMPPs and analyzed using qPCR. The gene expressions of Cebpa and Hmox1 were upregulated in Bach1–/–Bach2–/– MPPs (Figure S4B). We also found gene expressions of Cebpa, Cebpb, Hmox1, Csf1r, and Sfpi1 (encoding PU.1) were upregulated in Bach1–/–Bach2–/– LMPPs (Figure S4B). These observations indicate that myeloid differentiation program is initiated between ST-HSCs (MPPs) and LMPPs and suggest that Bach2 represses myeloid gene expression during the transition of ST-HSCs (MPPs) to LMPPs. Considering that the expression frequencies of Cebpa and Cebpb were reduced in CLPs, myeloid and lymphoid programs are well resolved at this stage. Regulation of Lymphoid Genes by Bach2 To clarify whether or not Bach2 promotes lymphocyte progenitor commitment, we transduced wild-type MPPs with either Ebf1GFP or Bach2-GFP bi-cistronic genes using retrovirus and cultured the cells under conditions that support myeloid cell development for 24 hr and then under conditions that support pre-B cell development for 6 days (Pongubala et al., 2008; Williams et al., 1990) (Figures 4A and 4B). By combining these culture conditions, we were able to assess the potential of

2406 Cell Reports 18, 2401–2414, March 7, 2017

(A and B) Flow cytometry of wild-type MPPs infected with indicated viruses, sorted for GFP+ (infected) and cultured for 24 hr under multilineagedevelopment condition, followed by culture for 6 days under B lineage-development condition (A). B220 and CD19 expressions are shown (B). (C) The clustering analysis of Bach2-infected GFP+B220+ cells induced from MPPs and MPPs and CLPs of wild-type mice. (D) Clustering analysis of wild-type MPPs infected with indicated retroviruses and then cultured for 2 days under the multilineage-development condition. A partial list of genes and fold-change values of gene expression are shown at the right of column.

published gene-expression data of MPPs infected with a control retrovirus or a retrovirus expressing Bach2 and then cultured the cells under multilineage conditions for 2 days (Itoh-Nakadai et al., 2014) (Figure 4D). The genes with a more than 2-fold greater expression in Bach2-expressing MPPs than in control MPPs were clustered using an unsupervised method (Figure 4D, Tables S2 and S3). A gene ontology (GO) analysis of the genes downregulated by Bach2 revealed the enrichment of categories such as ‘‘inflammatory response’’ and ‘‘response to wounding’’ (Figure S5A; Table S4). Terms such as ‘‘response to interferongamma’’ and ‘‘T cell activation’’ were enriched in the gene cluster upregulated by Bach2 (Figure S5A; Table S5). We next analyzed the expression patterns of the genes downregulated by Bach2 in MPPs using the database of the Immunological Genome Project (Figure S5B). Coarse modules 41, 43, and 24, which are upregulated with differentiation and/or in myeloid cells, were significantly repressed by the overexpression of Bach2. The clusters 65, 61, 18, and 19, which contain genes highly expressed in T, B, and NK cells, were induced by Bach2. Bach2 altered the gene expression of transcription factors, cytokines, and cytokine receptors, which are all important for the differentiation of myeloid (Cebpd, Fcgr2b, and Tlr4) and lymphoid (Gata3, Foxo1, Irf4, Flt3, and Il7) cells (Tables S2 and S3). We also found that ‘‘stemness’’ genes, such as Gfi1b and Mmp8, were repressed by Bach2 (Table S2). A mutant Bach2 lacking its basic-leucine zipper domain failed to exert the effects observed with wild-type Bach2 in MPPs (Figure S5C). These results suggested that Bach2 directly repressed the myeloid and stem cell genes in MPPs, promoting their differentiation toward lymphoid progenitors. Bach2 and C/EBP Factors Share Their Binding Sites of Myeloid Genes To identify the direct target genes of Bach2 in progenitor cells, we performed chromatin immunoprecipitation followed by deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) with a Bach2 antibody in Ebf1–/– cells, which represent pre-pro-B cells. It was previously reported that master transcription factors bind super enhancers at key cell identity genes (Whyte et al., 2013). Therefore, we first focused on the intersection of four gene sets: genes downregulated (344 genes, Table S2) or upregulated (434 genes, Table S3) more than 2-fold in MPPs transduced with Bach2 retrovirus (Figure 5A); genes with macrophage super enhancer (Mac-SE) identified by C/EBPa binding (901 genes) (Whyte et al., 2013); and the gene set of nearby Bach2 binding in Ebf1–/– cells (9,781 genes) (Figure 5A). Roughly two-thirds of genes with Mac-SE were bound by Bach2. Among the genes whose expressions were altered by Bach2 overexpression, greater enrichment of Mac-SE was observed with downregulated genes (16.0%, 57 out of 344 genes) than upregulated genes (4.4%, 19 out of 434 genes) (Figure 5A). We detected binding peaks of Bach2 around not only genes repressed by Bach2 (64.5%, 222 out of 344 genes) but also those upregulated by Bach2 (65.6%, 285 out of 434 genes) (Figure 5A). These observations suggest that Bach2 possesses the potential to promote gene expression as well, and a substantial proportion of the genes responding to Bach2 were direct targets of Bach2.

To clarify whether or not Bach2 binding sites are important for the regulation of myeloid gene expression, we used the datasets for binding of C/EBPb and Polymerase II (PolII) in dendritic cells (Garber et al., 2012), C/EBPa in macrophages (Whyte et al., 2013), and those of histone modifications (H3K27ac and H3K4me3) in ten developmental stages of hematopoietic cells (Lin et al., 2010). We then integrated these datasets with those of Bach2 and MafK binding in Ebf1–/– cells (Figures 5B, S6A, and S6B). First, we analyzed the genes of transcription factors that are known to be important for myeloid development. Consistent with our previous report (Itoh-Nakadai et al., 2014), Bach2 bound the Cebpb locus together with MafK (Figure 5B). Bach2 was also found to bind to the enhancers upstream of the Sfpi1, Cebpa, and Irf8 loci (Figure 5B). Bach2 and MafK also bound to the upstream region of Bach2, which appeared to be a regulatory region with H3K27ac and K4me3 modifications, suggesting a self-regulatory loop of Bach2. We found that some of these binding sites of Bach2 were close to C/EBPb and C/EBPa binding sites (for example, Sfpi1, Cebpa, and Bach2: shown with red boxes). These observations indicated that Bach2 and C/EBP factors regulate the expression of the overlapping set of genes by binding to neighboring regulatory DNA regions. Next, we focused on the genes encoding the receptors for macrophage differentiation in LSK cells (Mossadegh-Keller et al., 2013; Nagai et al., 2006). Bach2 bound to either or both the enhancer or promoter regions of the M-CSFr (Csf1r), Tolllike receptor 4 (Tlr4), MD2 (ly96, encoding the co-receptor of Tlr4), and Flt3 loci (Figures 5B and S6A). C/EBPb also bound to the nearby positions of the Bach2 binding regions of Ly96 (Figure S6A), suggesting again their regulation of shared target genes by binding to nearby DNA regions. Finally, we analyzed whether or not Bach2 bound to the loci of upstream regulators of lymphoid development in MPPs, such as Foxo1, Gata3, Il7r, Flt3, and Zap70. Bach2 showed a number of binding instances to these gene loci as well (Figures 5B and S6B). We confirmed the binding of Bach2 to the Foxo1 gene locus using ChIP-PCR. We detected Bach2 binding to 70 kilobase pair (kbp) upstream of the transcriptional start site (enhancer region) and intron of Foxo1 gene locus (Figure S6C). Thus, in addition to its repressor function, Bach2 may possess the potential to promote the expression of lymphoid genes directly. Antagonistic Regulation of Myeloid and Lymphoid Genes by Bach2 and C/EBPb The above microarray and the ChIP-seq analysis showed that Bach2 directly repressed myeloid genes and activated lymphoid genes, and that Bach2 and C/EBP factors compete with each other in the lineage specification through the regulation of shared target genes. To test this hypothesis further, we overexpressed Bach2 and C/EBPb in the Ebf1–/– cells. We used PU.1 as a control, since it promotes macrophage differentiation in early progenitors (Mossadegh-Keller et al., 2013). Bach2 and C/EBPb exerted opposite effects on the expression of the myeloid and the lymphoid genes (Figure 6A). For example, myeloid genes (Sfpi1, Csf1r, Irf8) were activated by C/EBPb but repressed by Bach2. Gata3 showed an opposite response to these transcription factors. To confirm the repression of

Cell Reports 18, 2401–2414, March 7, 2017 2407

A

434 B2TG up

344 B2TG down 901 MacSE

124

147

10

9781 B2 binding

268

0

8709

248 0 2

0 0 17

47

175

577

B Cebpb A530013C23Rik Chr7:35877013-35947763

Chr2:167494711-167525722

H3K4me3 H3K27ac

Bach2-1 Bach2-2 MafK C/EBPa C/EBPb PolII

Prog M Lyn Prog M Lyn

Sfpi1

Chr2:90909541-90956194

489 364 139 27 41 32

Prog M Lyn Prog M Lyn

Irf8

Chr8:123205264-123284797 100

21

Prog M Lyn Prog M Lyn

Irf4

79

Prog M Lyn Prog M Lyn

Bach2

Chr4:32222157-32674204

H3K4me3 H3K27ac

H3K4me3 H3K27ac

M Lyn Prog M Lyn

Chr3: 51938169-52532214

H3K4me3 H3K27ac

17

Bach2-1 382 Bach2-2 116 MafK 20 C/EBPa 11 C/EBPb 4 PolII

Prog

Bach2-1 79 Bach2-2 154 MafK 20 C/EBPa 4.7 C/EBPb 5.6 PolII

37

500

Bach2-1 100 Bach2-2 100 MafK 15 C/EBPa 15 C/EBPb 16 PolII

100

40

Bach2-1 102 Bach2-2 64 MafK 8 C/EBPa 9 C/EBPb 54 PolII H3K4me3 H3K27ac

H3K4me3 H3K27ac

Bach2-1 Bach2-2 MafK C/EBPa C/EBPb PolII

79

Chr13:30803982-30904078

Foxo1

Intron1

Prog M Lyn Prog M Lyn

Prog M Lyn Prog M Lyn

Chr18:61245179-61290862 Bach2-1 Bach2-2 MafK C/EBPa C/EBPb PolII H3K4me3 H3K27ac

H3K4me3 H3K27ac

Bach2-1 271 Bach2-2 107 MafK 9 C/EBPa 11 C/EBPb 96 PolII

Cebpa

107

Prog M Lyn Prog M Lyn

Csf1r

100 82 63 25 15 11

LT-HSC ST-HSC MPP GMP CLP monocyte Macrophage GN B CD4

(legend on next page)

2408 Cell Reports 18, 2401–2414, March 7, 2017

A Cebpb

Sfpi1

Tlr4

Csf1r

Tet2

Irf8

Figure 6. Bach2 Represses the Myeloid Cell Differentiation in LSK Stimulated with LPS or M-CSF

Lymphiod genes

Myeloid genes Ly96

Irf4

Cebpa Runx2

Bach2 Gata3

Foxo1

Contorl

C/EBP

PU.1

Expression (Fold) Bach2 0 Cebpb

Sfpi1

Tlr4

Csf1r

Tet2

Irf8

Ly96

Cebpa Runx2

Irf4

Bach2

Gata3

1

C/EBP

2.0E-02 1.0E-04 1.8E-02 4.7E-03 2.9E-04 7.7E-02 1.2E-01 2.2E-02 2.0E-02 6.0E-01 9.3E-06 1.5E-02 4.2E-01

PU.1

3.3E-02 2.3E-06 6.9E-01 1.8E-03 4.9E-02 3.0E-02 7.4E-02 1.1E-01 2.5E-02 2.2E-02 4.9E-01 4.6E-01 7.8E-01

Bach2

1.6E-02 7.3E-03 4.5E-01 4.7E-02 3.2E-01 5.3E-02 7.2E-02 1.9E-02 3.3E-01 4.7E-03 4.3E-05 4.0E-02 6.6E-02

p-value 0

Control Bach1 Bach2

GFP+

Cells

80

Relative fluoresent intensity of M-Csf1r

100

60 40 20 0 100

103

102

0.1

P = 0.003 P = 0.01 14

M1 cells

B

3

Foxo1

104

12 10 GFP-Control GFP-Bach2 GFP-Bach1

8 6 4 2 0

105

(A) qRT-PCR analysis of Ebf1–/– cells infected with indicated retroviruses, then cultured for 2 days under B cell-developmental conditions and sorted as GFP+ cells. Results are presented relative to those of cells infected with control retrovirus. p values are shown in the lower panel (unpaired two-tailed Student’s t test). (B) M-CSF receptor expression determined by a flow cytometry analysis of M1 cells. These cells were infected with indicated retroviruses, cultured for 2 days, and analyzed using GFP+ cells. Results are presented as histogram (left) and bar chart (right) with p values (mean ± SEM of triplicate: unpaired two-tailed Student’s t test). (C) Myeloid development of LSK cells infected with indicated retroviruses and then cultured for 5 days with LPS. (D and E) Myeloid (D) and B220+ (E) cells development of LSK cells infected with indicated retroviruses and cultured for 5 days with M-CSF. (F) Myeloid development of Ebf1–/– cells infected with indicated retroviruses. Dotplots of GFP+ cells are shown.

M-CSFr

C

GFP-Bach2

GFP-EV 105

GFP+

E

5%

20%

100

104

LSK 6%

18%

60

Cells

Gr-1

EV: 10.2% Bach2: 35.8%

80

0

LSK

40 20

0 102 103 104 105

0

0

CD11b

D

4

B220

GFP+-EV

GFP+-Bach2

F

105

GFP-EV 250k

7.0%

104

1.3%

200k

FSC

Gr1

103

150k

LSK

0

6.5% 102

CD11b

84.7% 103

104

105

54.6%

38.4%

5

-/-

4.1%

100k

0

(Bach2low C/EBPbhigh) versus lymphoid cells (Bach2high C/EBPblow) (see Discussion). Consistent with this model, the over10 10 expression of C/EBPb in pro-B cells has been reported to promote their differentiaGFP-Cebpb tion into myeloid cells (Xie et al., 2004). LSK cells can be induced to differentiate into myeloid cells in vitro using LPS or Ebf1 91.4% M-CSF (Nagai et al., 2006). Using this system, we tested whether or not Bach2 altered the capacity for myeloid differentiation. LPS induced the differentiation of Gr-1+CD11b+ myeloid cells from LSK, which was repressed by overexpression of Bach2 (Figure 6C). M-CSF induced the differentiation of CD11b-positive cells from LSK cells (Figure 6D), which was inhibited by Bach2 (Figure 6D). Surprisingly, Bach2 promoted the differentiation of B220+ cells from LSK cells in the presence of M-CSF without adding any lymphocyte-promoting cytokine (Figure 6E). On the other hand, C/EBPb promoted the differentiation of CD11b-positive cells starting from Ebf1–/– cells without cytokines (Figure 6F). Taken together, these results suggest that Bach2 and C/EBPb form a GRN, in which they mutually

50k 0k 100 102

103

104

105

CD11b

Csf1r by Bach2, we overexpressed Bach2 in the myeloblastic M1 cells. Cell-surface expression of the M-CSF receptor was decreased in M1 cells by Bach2 (Figure 6B). Consistent with our previous report (Itoh-Nakadai et al., 2014), Bach2 repressed Cebpb gene expression in Ebf1–/– cells (Figure 6A). Surprisingly, we also found that C/EBPb strongly repressed Bach2 gene expression (Figure 6A). These findings suggest that Bach2 and C/EBPb may form positive antagonistic, double feedback loops in progenitors to regulate the fate of progenitors toward myeloid

Figure 5. Bach2 Bound to Myeloid Super Enhancers and Lymphoid Genes in Progenitors (A) Set analysis of macrophage super-enhancer-associated genes (MacSE) (Whyte et al., 2013), upregulated (B2TGup) and downregulated (B2TGdown) genes in MPPs infected with Bach2-expressing virus, and genes with Bach2 binding (B2 binding). (B) ChIP-seq binding profiles for the Bach2 (duplicate) and MafK in Ebf1–/– cells. Also shown are C/EBPb and RNA polymerase II (PolII) in dendritic cells (Garber et al., 2012), C/EBPb in macrophage (Whyte et al., 2013), and H3K27Ac and H3K4me3 in HSCs and progenitors (Prog: long-term HSC [LT-HSC], short-term HSC [ST-HSC], MPP, GMP, CLP), myeloid cells (Mye: monocyte [mono], macrophage [mac], granulocyte [GN]), and lymphocytes (Lyn: B cell [B] and CD4 positive T cells). Gene name and location were presented above the each gene locus (Lin et al., 2010). Red boxes show co-binding of Bach2 and C/EBPb in nearby positions.

Cell Reports 18, 2401–2414, March 7, 2017 2409

A

B

Bach2–/– Bach2–/–

Bach2–/–

Bach2–/–

C

D

E

(legend on next page)

2410 Cell Reports 18, 2401–2414, March 7, 2017

and directly repress their expression. Furthermore, they antagonistically regulate their downstream target genes for lymphoid and myeloid differentiation. Above results suggest that Bach2 instructs B cell fate by inducing B cell genes including Foxo1 and repressing myeloid genes. There is also a possibility that survival of cells that are already committed to B cells is supported by Bach2. To further investigate these possibilities, we used rescue experiments of Bach2–/– LSK cells in vitro. In addition to Bach2, we also used Foxo1 as a downstream target gene and Bclxl as a repressor of apoptosis in B cells (Eeva et al., 2009). Bach2–/– LSK cells were infected with viruses expressing these genes or control virus and were cultured under the B cell developmental condition (Figure S7A). Bach2 showed a small but reproducible induction of B220+CD19– pre-pro B cells. Foxo1 failed to rescue the Bach2 function. Bclxl also enhanced B220+ cells development, but the effect was less than that of Bach2. Since Bclxl showed a mild effect, these results suggest that Bach2 promotes B cell development not only by myeloid gene repression, but also by supporting survival of cells becoming B cells. Among target genes of Bach2, Foxo1 did not suffice to support B220+ cells development in LSK cells. Influence of Bach2 on the Myeloid Differentiation of LSK Cells in Infectious Conditions To clarify the role of Bach2-Cebpb GRN under infectious conditions, we mimicked these conditions by injecting LPS into wildtype and Bach2–/– mice. The wild-type mice injected with LPS showed an increased frequency of Gr-1+CD11b+ myeloid cells in the bone marrow compared with the control mice (Figure 7A). Notably, Bach2–/– mice showed higher frequencies of myeloid cells (83.7%) than wild-type mice (63.0%) in response to LPS (Figure 7A). By contrast, the B cell population decreased more severely in Bach2–/– mice than in wild-type mice when treated with LPS (Figure 7A). Concentrations of M-CSF and IL-6 protein in the serum of wild-type and Bach2–/– mice were similar (Figure 7B), suggesting that the skewed outputs of lineages observed in Bach2–/– mice in response to LPS stimulation were due to a cell-intrinsic effect of Bach2 deficiency. We next conducted a gene expression analysis of wild-type LSK cells treated with LPS or M-CSF in vitro using RT-PCR. After LPS stimulation for 48 hr, we detected decreases in the expression of Bach2 and Bach1 and an increase in the Cebpb expression (Figure 7C). Interestingly, the expression of Bach2 and Cebpb was not altered by M-CSF. Instead, Cebpa was induced by M-CSF. M-CSF induces Sfpi1 expression in LT-HSC (Mossadegh-Keller et al., 2013). We obtained similar findings to that report, as the expression of Sfpi1 was increased by both LPS

and M-CSF (Figure 7C). LPS and M-CSF specifically induced Irf4 and Irf8, respectively (Figure 7C), but both reduced the expression of lymphoid transcription factor Foxo1 (Figure 7C). These results suggest that cell-extrinsic stimuli elicited by infection induced the expression of one or more myeloid regulators (Cebpb, Cebpa, Sfpi1, Irf4, and Irf8) but repressed Bach2, Bach1, and/or Foxo1, reprograming the myeloid differentiation potential of LSK cells. Such alterations in the GRN may be further consolidated by changes in the expression of other categories of regulatory molecules. To address this possibility, we examined the expression of Csf1r (M-CSF receptor), a shared target gene of Bach2 and C/EBPb (Figure 6A), in LSK cells. Csf1r mRNA gene expression was markedly increased in LSK cells stimulated with LPS or M-CSF (Figure 7D), indicating that LPS reprograms LSK cells by altering the status of the Bach2-Cebpb GRN so that the expression of transcription factor genes as well as the signaling molecules supports myeloid cell differentiation (Figure S7B). DISCUSSION Based on the effects of Bach2 and C/EBPb overexpression and ChIP-seq data, we propose a model GRN in multipotent cells (Figure 7E). Importantly, we found that C/EBPb repressed the gene expression of Bach2. This mutual repression creates two positive feedback loops, as Bach2 activates its own expression by repressing C/EBPb, and vice versa. Another finding is that critical myeloid genes were co-regulated in an antagonistic manner by Bach2 and C/EBPb. Moreover, whereas Bach2 binds to many of the T cell super enhancers to restrict T cell activation (Vahedi et al., 2015), we found that Bach2 bound to a substantial fraction of myeloid super enhancers targeted by C/EBPa. Interestingly, Bach2 has been identified as one of the C/EBPa target genes regulated by its super enhancers (Whyte et al., 2013). Therefore, the Bach2Cebpb GRN appears to involve a network of super enhancers in multipotent hematopoietic cells. This GRN therefore explains the cell-intrinsic program mediated by super enhancers for the commitment of multipotent cells to balance myeloid and lymphoid cell differentiation as well as its modulation by cellextrinsic signals derived from cytokines and infectious conditions, as discussed below. A strong lineage bias in HSCs has recently been reported, producing several-100-fold larger numbers of myeloid cells than lymphoid cells (Busch et al., 2015). In terms of evolution, it has been postulated that myeloid cells represent a prototype of hematopoietic cells and that erythroid, T, and B cells represent specialized types generated by modifying the differentiation

Figure 7. Modulation of Bach2 GRN by LPS and M-CSF (A) Percentages of myeloid (left) or B cells (right) in bone marrows of wild-type or Bach2–/– mice injected with RPMI or LPS. Each mouse was scarified 5 days after the treatment. Three independent experiments were performed (mean ± SEM, unpaired two-tailed Student’s t test). (B) Concentration of M-CSF and IL-6 in the serum of wild-type and Bach2–/– mice detected by ELISA. (C and D) qRT-PCR analysis of transcription factors (C) and Csf1r encoding M-CSF receptor (D) in control LSK cells and those stimulated with LPS or M-CSF in vitro (mean ± SEM of biological triplicate: unpaired two-tailed Student’s t test). (E) GRN of Bach2 and C/EBPb involving other regulators and M-CSF receptor gene. Myeloid or lymphoid commitment is decided by the balance between Bach2 and Cebp genes. Myeloid fate is consolidated by feedforward inhibition or activation of M-CSF receptor and other myeloid genes, whereas lymphoid fate is fixed by the positive feedback loop involving Bach2 and Foxo1. Infectious conditions such as LPS modulate the commitment process of the GRN.

Cell Reports 18, 2401–2414, March 7, 2017 2411

processes of myeloid cells (Kawamoto and Katsura, 2009). These facts and considerations raise several important questions in hematology: first, how the balance of myeloid and other lineages is maintained under normal conditions and how it is tuned in response to hematopoietic demands, including stress conditions such as infection; and second, how the presumably prototypic myeloid cell differentiation processes are modified by transcription factors and signaling cascades to generate other lineages of cells, the process of silencing inner myeloid (Igarashi and Itoh-Nakadai, 2016). We suggest that the Bach2-Cebpb GRN tunes the balance between myeloid and lymphoid cell fates in MPPs. Importantly, Bach2 represses not only Cebpb but also other myeloid genes, including the myeloid transcription factor genes Cebpa and Gfi1b, Irf8, and the genes involved in receiving extracellular signals, such as Csf1r. Together with the activation of these genes, this GRN contains a pair of coherent negative feedforward loops that are expected to efficiently inhibit (i.e., via Bach2) or activate (i.e., via C/EBPs) myeloid cell differentiation once one of these factors exceeds the other (Figure 7E). Of particular note, Bach2 and C/EBPb both bind to many myeloid target genes with opposite regulatory effects (i.e., repression versus activation). Therefore, the Bach2-Cebpb GRN may be sensitive to fluctuations in the activities of these two regulatory genes, resulting in the dynamic responses of multipotent cells. Under normal conditions, the balance of Bach2 and Cebpb may not only favor Cebpb-dependent myelopoiesis but also secure a minor fraction of cells for lymphoid fate. Bach2 may have been one of the first evolutionary inventions that had modulated myeloid cell differentiation to generate other types of cells. The regulatory status of the Bach2-Cebpb GRN was altered in response to LPS, which reduced the expression of Bach2 and induced that of Cebpb. Our results also showed that Bach2 was critical for preventing the exhaustion of lymphoid cells under stress. Bach2 may secure a fraction of progenitor cells that eventually undergo lymphoid differentiation as needed under pathological conditions (Figure S7B). It has recently been reported that Irf8 and Gfi1 are key components of counteracting myeloid-GRN, specifying either neutrophils or macrophages (Olsson et al., 2016). The Bach2-Cebpb GRN most likely operates at the upstream of the Irf8-Gfi1 GRN. Together with previous observations that Foxo1 promotes Bach2 gene expression in early B cell development (Kometani et al., 2013; Lin et al., 2010), our observations suggested that Bach2 and Foxo1 formed a positive feedback loop in HSCs and MPPs. We recently reported that Bach2 interacts with the Swi/Snf complex in B cells (Tanaka et al., 2016), suggesting that the Bach2 complex performs chromatin remodeling of Foxo1, Gata3, and other target genes to stimulate their expression. The feedback loop of Bach2 and Foxo1 may consolidate lymphoid commitment once the expression of one or the other genes increases above a threshold level. Foxo1 failed to rescue the developmental defect of Bach2–/– LSK cells in vitro (Figure S7A), suggesting that the major roles of Bach2 in promoting B cell development are borne by both the direct repression of myeloid genes and activation of B cell genes such as Foxo1.

2412 Cell Reports 18, 2401–2414, March 7, 2017

EXPERIMENTAL PROCEDURES Mice The generation of Bach1–/–, Bach2–/–, Bach1–/–Bach2–/–, and RFP-Bach2 reporter mice were generated by breeding these mice in the C57BL/6J background (Itoh-Nakadai et al., 2014; Muto et al., 2004; Sun et al., 2002). Mice were sacrificed between 7 and 10 weeks old in all experiments. Both male and female mice were used, but same-sex mice were used for each independent experiment. All experiments involving mice were approved by the Institutional Animal Care and Use Committee of the Tohoku University Environmental & Safety Committee. LPS Injections The solution of 4 mg LPS (O111:B4; Sigma-Aldrich) in 100 mL RPMI (Sigma) or RPMI alone was injected into intraperitoneal of wild-type or Bach2–/– mice at concentration of 0.2 mg LPS per body weight (g). Analysis of bone marrow was performed after 5 days of injection. Assay of Lineage Potential For B cell development condition, cells were cultured for 12 day with TSt4 cell and the cytokines including SCF (10 ng/mL), Flt3 (10 ng/mL), and IL-7 (5 ng/mL). Every 3 days, new cytokines were added to the cell-culture medium. For multilineage-development conditions, cells were cultured in medium containing the cytokines SCF (10 ng/mL), IL-7 (5 ng/mL), GM-CSF (10 ng/mL), M-CSF (10 ng/mL), G-CSF (10 ng/mL), and IL-3 (10 ng/mL). Cultured medium was RPMI 1640 (Sigma) with 10% fetal bovine serum (FBS) supplemented with 500 nM 2-mercaptoethanol (2-ME), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin. All cytokines were from R&D Systems. Single-Cell PCR Single-cell RT-PCR with the Fluidigm platform. Single-cell qPCR with an RT analysis was done as described (Itoh-Nakadai et al., 2014). A single progenitor was sorted into each well of a 96-well plate containing 5 mL of Cell Direct reaction mix (Cells Direct One step RT-qPCR kit, Invitrogen). Retroviral Transduction and In Vitro Differentiation Assay ‘‘Spin infection’’ and culture for multilineage differentiation were done as described (Itoh-Nakadai et al., 2014; Pongubala et al., 2008) with retroviral supernatants generated by the transfection of Plat-E retroviral packaging cells with the retroviral construct EGFP (mouse stem cell cirus [MSCV]), Ebf1-GFP (Mig-EBF), Bach2-EGFP (MSCV-Bach2), Cebpb-GFP (MigRCebpb), PU.1-GFP (MigR-PU.1), Foxo1-GFP (MigR-Foxo1) (Ochiai et al., 2012), and Bclxl (MSCV-Bclxl). MPPs or LSK cells were stimulated overnight with SCF (50 ng/mL), IL-3 (5 ng/mL), and IL-6 (10 ng/mL) and were spin infected the next day. Infected cells were cultured under the appropriate condition. Statistical Methods Data were analyzed with F-test for compare two population variances and a two-tailed Student’s t test or Welch’s t test. Single-cell data were analyzed with Fisher’s exact test. Enrichment of Immunological Genome Project modules was evaluated with a hypergeometric test as described (Itoh-Nakadai et al., 2014). p values of less than 0.05 were considered statistically significant. ACCESSION NUMBERS The accession numbers for the ChIP-sequencing dataset and the microarray reported in this paper are GEO: GSE87503 and GSE80954, respectively. The accession number for the dotplot data of flow cytometry analysis reported in this paper is FlowRepository: FR-FCM-ZYZV. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, seven figures, and five tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2017.02.029.

AUTHOR CONTRIBUTIONS

chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810–822.

A.I.-N. performed most experiments with the help of J.S., Y.U., H.K., Y.S., R.E.-S., and A.M. A.M. performed ChIP experiment and Figure S6C. A.I.-N, and Y.S. performed experiments in Figure 1, A.I.-N. and J.S. performed experiments in Figures 6B–6D. Y.U. performed experiments in Figures 6F and 7A. A.I.-N., H.K., and J.S. performed experiments in Figures 7B and 7C. K.O. performed experiments in Figure S7A. H.K. performed experiments in Figures 7B, S1C, and S4B. R.F. and K.N. performed ChIP sequencing, and M.M. analyzed the data. A.I.-N. designed the study and wrote the manuscript; K.I. supervised the project and wrote the manuscript.

Guo, G., Luc, S., Marco, E., Lin, T.W., Peng, C., Kerenyi, M.A., Beyaz, S., Kim, W., Xu, J., Das, P.P., et al. (2013). Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire. Cell Stem Cell 13, 492–505.

ACKNOWLEDGMENTS We thank H. Singh (Cincinnati Children’s Hospital Medical Center) for the Ebf1 and PU.1 retroviral construct and Ebf1–/– cells. We thank T. Ikawa (RIKEN) for the TSt-4 cells. We thank K. Watanabe, M. Tsuda, M. Kikuchi, M. Nakagawa, and K. Kuroda for technical assistance. We thank R. Young and B. Abraham (Cambridge) for C/EBPa ChIP-seq data . We thank R. Yamashita for advice on the analysis of gene expression. We also acknowledge the technical support of the Biomedical Research Core of Tohoku University Graduate School of Medicine. We were supported by Grants-in-Aid from the Japan Society for the Promotion of Science (15K18998 to A.I.-N., 15H02506, 24390066, 21249014 to K.I.) and the Agency for Medical Research and DevelopmentCore Research for Evolutional Science and Technology (AMED-CREST; no. 15gm0510001h0005). Received: June 11, 2016 Revised: November 23, 2016 Accepted: February 8, 2017 Published: March 7, 2017 REFERENCES Abramson, S., Miller, R.G., and Phillips, R.A. (1977). The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J. Exp. Med. 145, 1567–1579. Adolfsson, J., Ma˚nsson, R., Buza-Vidas, N., Hultquist, A., Liuba, K., Jensen, C.T., Bryder, D., Yang, L., Borge, O.J., Thoren, L.A., et al. (2005). Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295–306. Arinobu, Y., Mizuno, S., Chong, Y., Shigematsu, H., Iino, T., Iwasaki, H., Graf, T., Mayfield, R., Chan, S., Kastner, P., and Akashi, K. (2007). Reciprocal activation of GATA-1 and PU.1 marks initial specification of hematopoietic stem cells into myeloerythroid and myelolymphoid lineages. Cell Stem Cell 1, 416–427. Busch, K., Klapproth, K., Barile, M., Flossdorf, M., Holland-Letz, T., Schlenner, S.M., Reth, M., Ho¨fer, T., and Rodewald, H.R. (2015). Fundamental properties of unperturbed haematopoiesis from stem cells in vivo. Nature 518, 542–546. Dias, S., Ma˚nsson, R., Gurbuxani, S., Sigvardsson, M., and Kee, B.L. (2008). E2A proteins promote development of lymphoid-primed multipotent progenitors. Immunity 29, 217–227. Eeva, J., Nuutinen, U., Ropponen, A., Ma¨tto¨, M., Eray, M., Pellinen, R., Wahlfors, J., and Pelkonen, J. (2009). Feedback regulation of mitochondria by caspase-9 in the B cell receptor-mediated apoptosis. Scand. J. Immunol. 70, 574–583. Enver, T., Pera, M., Peterson, C., and Andrews, P.W. (2009). Stem cell states, fates, and the rules of attraction. Cell Stem Cell 4, 387–397. Esplin, B.L., Shimazu, T., Welner, R.S., Garrett, K.P., Nie, L., Zhang, Q., Humphrey, M.B., Yang, Q., Borghesi, L.A., and Kincade, P.W. (2011). Chronic exposure to a TLR ligand injures hematopoietic stem cells. J. Immunol. 186, 5367–5375. Garber, M., Yosef, N., Goren, A., Raychowdhury, R., Thielke, A., Guttman, M., Robinson, J., Minie, B., Chevrier, N., Itzhaki, Z., et al. (2012). A high-throughput

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