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The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program

Nature Immunology volume 15, pages 1171–1180 (2014)Cite this article

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Abstract

Mature lymphoid cells express the transcription repressor Bach2, which imposes regulation on humoral and cellular immunity. Here we found critical roles for Bach2 in the development of cells of the B lineage, commencing from the common lymphoid progenitor (CLP) stage, with Bach1 as an auxiliary. Overexpression of Bach2 in pre-pro-B cells deficient in the transcription factor EBF1 and single-cell analysis of CLPs revealed that Bach2 and Bach1 repressed the expression of genes important for myeloid cells ('myeloid genes'). Bach2 and Bach1 bound to presumptive regulatory regions of the myeloid genes. Bach2hi CLPs showed resistance to myeloid differentiation even when cultured under myeloid conditions. Our results suggest that Bach2 functions with Bach1 and EBF1 to promote B cell development by repressing myeloid genes in CLPs.

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Figure 1: Bach1−/−Bach2−/− mice show substantial defects in B cell development and aberrantly enhanced myelopoiesis.
Figure 2: Bach1−/−Bach2−/− mice show a failure in the initiation but not the maintenance of B cell gene expression.
Figure 3: Diminished in vitro B cell–development potency of Bach1−/−Bach2−/− CLPs is accompanied by greater myeloid-development potency.
Figure 4: Bach2 represses genes related to myeloid and inflammatory responses in CLPs and mature B cells.
Figure 5: Bach2 directly represses a subset of myeloid genes.
Figure 6: The expression of myeloid genes is increased in Bach1−/−Bach2−/− CLPs.
Figure 7: Bach2 promotes the B cell development of CLPs.

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Acknowledgements

We thank H. Singh (Cincinnati Children's Hospital Medical Center) for discussions, the Ebf1 retroviral construct and Ebf1−/− cells; M.R. Clark (University of Chicago) for the retroviral construct encoding constitutively active STAT5b; K. Takatsu (Toyama University) for OP9 stroma cells; T. Iino, A. Akashi, A. Brydun and M. Matsumoto for advice on DNA microarray analysis; A. Brydun for the generation of antibody to Bach1; R. Yamashita for advice on the analysis of modules from the Immunological Genome Project; H. Kawamoto and members of the Igarashi laboratory for discussions; A. Arakawa for help in the generation of Bach2 reporter mice; K. Watanabe for help with experiments; M. Satake for discussions about repressors; and the Biomedical Research Core of Tohoku University Graduate School of Medicine for their technical support. Supported by Grants-in-Aid from the Japan Society for the Promotion of Science (09J07369 to A.I.-N.; 21229007 to T.K.; 00250738, 21249014 and 00250738 to K.I.), the Network Medicine Global-COE Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Uehara Foundation, the Takeda Foundation, the NOVARTIS Foundation (Japan) for the Promotion of Science and Astellas Foundation for Research on Metabolic Disorders, and the Japan Society for the Promotion of Science and the Tohoku University Institute for International Advanced Research and Education (A.I.-N.).

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Authors and Affiliations

  1. Department of Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan

    Ari Itoh-Nakadai, Reina Hikota, Akihiko Muto, Miki Watanabe-Matsui, Yuki Sato, Masahiro Kobayashi, Atsushi Nakamura, Yuichi Miura, Kyoko Ochiai & Kazuhiko Igarashi

  2. CREST, Japan Science and Technology Agency, Sendai, Japan

    Ari Itoh-Nakadai, Akihiko Muto, Kyoko Ochiai & Kazuhiko Igarashi

  3. RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Kohei Kometani & Tomohiro Kurosaki

  4. Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Graduate School of Biomedical Science, Hiroshima University, Hiroshima, Japan

    Yoko Yano, Satoshi Tashiro & Jiying Sun

  5. Laboratory for Immune Regeneration RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Tomokatsu Ikawa

  6. Center for Regulatory Epigenome and Diseases, Tohoku University Graduate School of Medicine, Sendai, Japan

    Kyoko Ochiai & Kazuhiko Igarashi

  7. WPI Immunology Frontier Research Center, Osaka University, Suita, Japan

    Tomohiro Kurosaki

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  1. Ari Itoh-Nakadai
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Contributions

A.I.-N. performed all experiments with the help of R.H., A.M., M.W.-M., Y.S., M.K., A.N., Y.M., Y.Y., S.T., J.S., T.I. and K.O.; R.H. performed multiplex-single-cell PCR analysis; M.W.-M. performed electrophoretic mobility-shift assays; K.K. and T.K. generated Bach2 reporter mice; A.I.-N. designed the study and wrote the manuscript; K.I. supervised the project and wrote the manuscript; and all authors discussed the results and their implications and commented on the manuscript.

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Correspondence to Kazuhiko Igarashi.

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Integrated supplementary information

Supplementary Figure 1 CD19+ pro-B cells are less abundant in Bach1–/–Bach2–/– mice.

The data were obtained from mice distinct from those analyzed in Fig. 1b using same analyzer and antibodies of colors. a. Flow cytometry analysis of CD19+ pro-B cells. Bone marrow cells of WT and DD mice were analysed by FACSAriaII for surface expression of PerCP-cy5.5-CD19+ (ID3;BD), FITC-CD43+, (S7;BD) and APC-B220+ (RA3-6B2;TONBO). b. Absolute cell numbers of pro-B cells and CD19+ cells in pro-B cells in bone marrow of WT (n = 3) and DD mice (n = 4). The data were analyzed using unpaired Student’s t-test. The results are presented as the means ± S.E.M. from three individual experiments.

Supplementary Figure 2 Normal early T cell development in the thymus of Bach1–/–Bach2–/– mice.

a. Flow cytometry analysis of T cells in the thymus. ETP from WT, Bach1-/-, Bach2-/-, and DD mice were analysed for surface expression of Lin-, CD25- and c-Kit+. b. Absolute cell numbers of indicated cell populations in the thymus from WT (white columns, n=4), Bach1-/- (line columns, n = 2), Bach2-/-(gray columns, n = 2), and DD (black columns, n=4) mice. Total T cells (CD4+CD8–, CD4–CD8+), ETP (Lin–CD4–CD8–c-Kit+CD25–), DN2 (Lin–CD4–CD8–c-Kit+CD25+) and DN3 (Lin–CD4–CD8–c-Kit–CD25–) in the thymus. The data were analyzed using unpaired Student’s t-test.

Supplementary Figure 3 Methods for the analysis of CLPs and intracellular phosphorylated STAT5.

a. Flow cytometry analysis of CLPs. WT bone marrow cells were analysed for surface expression of Lin–, PI–, IL7Ra+, c-kitmid, Flt3+ and Sca-1mid. The staining reagents are described in online materials and methods. b. Surface marker staining of Lin– cells sorted by MACS. c. The levels of phosphorylated STAT5 (pSTAT5) of CLPs obtained from WT (dotted line) and DD (solid line) mice. The shaded histogram represents the results obtained with an isotype control (gray). High phosphorylation areas (not detected with the isotype control) were gated.

Supplementary Figure 4 Flow cytometry of GFP– cells among infected Ebf1–/– cells.

FACS analysis of GFP–negative, uninfected cells in Ebf1-/- cells treated with retroviruses carrying indicated genes. Contour plots of GFP-negative cells in the experiment depicted in Fig. 3e are shown. Frequency values are mean of three experiments ± s.e.m. The data were analyzed using unpaired two tailed Student’s t-test. There was no statistical significance among groups.

Supplementary Figure 5 Schematic drawings of the preparation of microarray samples in Figure 4.

Supplementary Figure 6 The search for binding locus of Bach factors.

a. Schematic representation of the MARE-like sequences of Cebpb, Ahr, Ly96, Spic, Bach1, Cish and Spi1.The arrows denote MARE-like sequences. b. Specificity of anti-Bach1 monoclonal antibody. Monoclonal antibody N9648 against mouse Bach1 was generated by immunizing mice with recombinant mouse Bach1 fragment (amino acid residues 318-392) and by establishing hybridoma clones from the spleens. The specificity of the antibody was confirmed by western blotting of extracts prepared from wild-type and Bach1-deficient embryonic fibroblasts. c. EMSA showing binding of Bach1 to the putative MAREs. EMSA was carried out using total 100 ng proteins (MBP-Bach1 or MBP-Mafk) with oligonucleotide probe which contained the MARE-like sequences of Ahr or Cebpb. Arrows indicate bindings of Bach1 (homodimer), Bach1-MafK (heterodimer) and MafK (homodimer). WT and Mut indicate competitor DNAs with wild-type and mutated sequences, respectively.

Supplementary Figure 7 Clustering analysis of single-cell analysis of wild-type and Bach1–/–Bach2–/– CLPs.

Gene expression of a single CLP in WT and DD was analysed by 48.48 dynamic arrays on a BioMark system using 218-219 cells for each genotype. Clustering analysis of total CLPs (a) and EBF1+ CLPs (b) population in WT and DD CLPs were carried out using myeloid-related genes.

Supplementary Figure 8 Generation of Bach2-td RFP reporter mice.

a. Generation of Bach2-td RFP reporter mice. Schematic diagram of Bach2-td RFP reporter mice. Arrows indicate primer position for PCR. The length of PCR product for wild type (wt) and knock in allele (KI) is 402bp and 248bp, respectively. To generate Bach2-tandem red fluorescent protein (tdRFP) targeted mice, homology region of Bach2, 6kb long arm (LA) and 2.5kb short arm (SA), were subcloned into a vector containing a diphtheria toxin A (DTA) gene. One tdRFP gene conjugated with neomycin resistant cassette flanked with two FRT sequences was inserted in Bach2 start codon site. The targeting vector was electroporated into C57BL/6-derived Bruce4 embryonic stem cells. After the selection with G418, correctly targeted clones were identified by PCR. The targeted ES clones were injected into blastocysts from BALB/c mice. The obtained chimeric mice were crossed with C57BL/6 mice to obtain germline transmitted animals. To remove the neomycine resistant casette, CAG-Flp transgenic mice were further crossed. b. Sorting of CLPs depending on expression of Bach2 RFP. The bottom panels show re-analysis of sorted Bach2hi (left) and Bach2lo (right) CLPs. c. Regulatory interactions of Bach1, Bach2, and Ebf1 with other genes. The network was assembled using BioTapestry (http://www.biotapestry.org/). Activating and repressive interactions are indicated with arrowheads and stop lines, respectively.

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Single cell PCR Ct value (XLSX 1144 kb)

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Itoh-Nakadai, A., Hikota, R., Muto, A. et al. The transcription repressors Bach2 and Bach1 promote B cell development by repressing the myeloid program. Nat Immunol 15, 1171–1180 (2014). https://doi.org/10.1038/ni.3024

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