Ferroptosis is a regulated cell death due to the iron-dependent accumulation of lipid peroxide. F... more Ferroptosis is a regulated cell death due to the iron-dependent accumulation of lipid peroxide. Ferroptosis is known to constitute the pathology of ischemic diseases, neurodegenerative diseases, and steatohepatitis and also works as a suppressing mechanism against cancer. However, how ferroptotic cells affect surrounding cells remains elusive. We herein report the transfer phenomenon of lipid peroxidation and cell death from ferroptotic cells to nearby cells that are not exposed to ferroptotic inducers (FINs). While primary mouse embryonic fibroblasts (MEFs) and NIH3T3 cells contained senescence-associated β-galactosidase (SA-β-gal)-positive cells, they were decreased upon induction of ferroptosis with FINs. The SA-β-gal decrease was inhibited by ferroptotic inhibitors and knockdown of Atg7, pointing to the involvement of lipid peroxidation and activated autophagosome formation during ferroptosis. A transfer of cell culture medium of cells treated with FINs, type 1 or 2, caused the ...
The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, inc... more The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in -deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but...
S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone ... more S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N-methyladenosine (mA) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the mA reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these mAs were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2018
BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation ... more BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from -deficient () mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from mice revealed reduced expression of the antiapoptotic gene encoding Bcl-x and elevated expression of cyclin-dependent kinase inhibitor...
Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea cau... more Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea caused by accumulation of surfactant protein. Dysfunction of alveolar macrophages (AMs), which regulate the homeostasis of surfactant protein, leads to the development of PAP; for example, in mice lacking BTB and CNC homology 2 (Bach2). However, how Bach2 helps prevent PAP is unknown, and the cell-specific effects of Bach2 are undefined. Using mice lacking Bach2 in specific cell types, we found that the PAP phenotype of Bach2-deficient mice is due to Bach2 deficiency in more than two types of immune cells. Depletion of hyperactivated T cells in Bach2-deficient mice restored normal function of AMs and ameliorated PAP. We also found that, in Bach2-deficient mice, hyperactivated T cells induced gene expression patterns that are specific to other tissue-resident macrophages and dendritic cells. Moreover, Bach2-deficient AMs exhibited a reduction in cell cycle progression. IFN-␥ released from T cells induced Bach2 expression in AMs, in which Bach2 then bound to regulatory regions of inflammationassociated genes in myeloid cells. Of note, in AMs, Bach2 restricted aberrant responses to excessive T cell-induced inflammation, whereas, in T cells, Bach2 puts a brake on T cell activation. Moreover, Bach2 stimulated the expression of multiple histone genes in AMs, suggesting a role of Bach2 in proper histone expression. We conclude that Bach2 is critical for the maintenance of AM identity and self-renewal in inflammatory environments. Treatments targeting T cells may offer new therapeutic strategies for managing secondary PAP. This work was supported by Grants-in-aid 15H02506, 24390066, 23116003, and 21249014 from the Japan Society for the Promotion of Science. Additional initiative supports were from the Uehara Foundation, Takeda Foundation, and Astellas Foundation for Research on Metabolic Disorders. The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Fig. S1. The microarray, RNA sequencing, and ChIP sequencing data have been deposited at the Gene Expression Omnibus database under accession codes GSE79139 and GSE79558.
Iron plays the central role in the oxygen transport by the erythrocyte as a constituent of heme a... more Iron plays the central role in the oxygen transport by the erythrocyte as a constituent of heme and hemoglobin. The importance of iron and heme also resides in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is inactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1 mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses thes...
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiatio... more T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Genes to cells : devoted to molecular & cellular mechanisms, 2016
H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone v... more H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene. H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we used the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a nonacetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. In contrast, each one of these three forms of H2A.Z complemented the transcri...
B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of pla... more B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histone H3 and H4 around the Prdm1 intron 5 MARE were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3 lysine 9 was lower in X63/0 cells than BAL17 cells. Purification of Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin beta-like 1X-linked (Tbl1x), and RAP1 interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prd...
The transcription factor Bach2 regulates the immune system at multiple points including class swi... more The transcription factor Bach2 regulates the immune system at multiple points including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells is still unclear. Here we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in mature B cell line BAL17. Among the sites identified, serine...
BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, ®ve (20%) s... more BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, ®ve (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.
Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. S... more Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stressinduced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.
Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), ch... more Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), chiefly due to disruptions in the signaling of granulocyte macrophage colony–stimulating factor (GM-CSF). We found that mice deficient for the B lymphoid transcription repressor BTB and CNC homology 2 (Bach2) developed PAP-like accumulation of surfactant proteins in the lungs. Bach2 was expressed in AMs, and Bach2-deficient AMs showed alterations in lipid handling in comparison with wild-type (WT) cells. Although Bach2-deficient AMs showed a normal expression of the genes involved in the GM-CSF signaling, they showed an altered expression of the genes involved in chemotaxis, lipid metabolism, and alternative M2 macrophage activation with increased expression of Ym1 and arginase-1, and the M2 regulator Irf4. Peritoneal Bach2-deficient macrophages showed increased Ym1 expression when stimulated with interleukin-4. More eosinophils were present in the lung and peritoneal cavity of Bach2-defic...
The oxidative stress response operates by inducing the expression of genes that counteract the st... more The oxidative stress response operates by inducing the expression of genes that counteract the stress. We show here that the oxidative stress-responsive transcription factor Bach2 is a generic inhibitor of gene expression directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. The Bach2-enhanced green fluorescent protein bicistronic retrovirus was used to monitor the fate of Bach2-expressing cells at the single cell level. Bach2 exerted an inhibitory effect on NIH3T3 cell proliferation and caused massive apoptosis upon mild oxidative stress in both NIH3T3 and Raji B-lymphoid cells. Interestingly, Bach1, a highly homologous protein, could not induce cell death, demonstrating the specificity for the apoptosis induction. Although both oxidative stress and leptomycin B, an inhibitor of nuclear export, induce nuclear accumulation of Bach2, the leptomycin B-induced nuclear accumulation of Bach2 was not sufficient to elicit apoptosis. Upon oxidative stress, Bach2 formed nuclear foci that associated with promyelocytic leukemia nuclear bodies. Our results suggest that Bach2 constitutes a cell lineage-specific system that couples oxidative stress and cell death and that inhibition of 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element upon oxidative stress may be critical determinants for apoptosis.
Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced clas... more Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced class switch recombination and a reduced somatic hypermutation of immunoglobulin genes. Little is known about the direct target genes of Bach2 in B cells. By analyzing various B cell and plasma cell lines, we showed that the expression patterns of Bach2 and Blimp-1 (B lymphocyteinduced maturation protein 1), a master regulator of plasma cell differentiation, are mutually exclusive. The reporter gene of the Blimp-1 gene (Prdm1) was repressed by the overexpression of Bach2 in B cell lines. The heterodimer of Bach2/MafK bound to the Maf recognition element located upstream of the Prdm1 promoter in an electrophoretic mobility shift assay. The binding of MafK in B cells to the Prdm1 Maf recognition element was confirmed by chromatin immunoprecipitation assays. When MafK was purified from the BAL17 B cell line, a significant portion of it was present as a heterodimer with Bach2, with no apparent formation of MafK homodimer. These results strongly suggest that Bach2 represses the expression of Blimp-1 together with MafK in B cells prior to plasma cell differentiation. Accordingly, the knockdown of Bach2 mRNA using short hairpin RNA in BAL17 cells resulted in higher levels of Prdm1 expression after the stimulation of B cell receptor by surface IgM crosslinking. Induction of Prdm1 was more robust and faster in primary Bach2-deficient B cells than in wild-type control B cells upon lipopolysaccharide stimulation. Therefore, the Prdm1 regulation in B cells involves the repression by Bach2, which may be cancelled upon terminal plasma cell differentiation.
B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differenti... more B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.
†In two cases sIL-2R was not assessed. ‡In one case MIB-1 index was not assessed. IPI, internatio... more †In two cases sIL-2R was not assessed. ‡In one case MIB-1 index was not assessed. IPI, international prognostic index; LDH, lactate dehydrogenase; OS, overall survival; PFS, progression-free survival; sIL-2R, soluble inteleukin-2 receptor.
American Journal of Physiology-Endocrinology and Metabolism, 2013
BTB and CNC homology 1 (Bach1) is a transcriptional repressor of antioxidative enzymes, such as h... more BTB and CNC homology 1 (Bach1) is a transcriptional repressor of antioxidative enzymes, such as heme oxygenase-1 (HO-1). Oxidative stress is reportedly involved in insulin secretion impairment and obesity-associated insulin resistance. However, the role of Bach1 in the development of diabetes is unclear. HO-1 expression in the liver, white adipose tissue, and pancreatic islets was markedly upregulated in Bach1-deficient mice. Unexpectedly, glucose and insulin tolerance tests showed no differences in obese wild-type (WT) and obese Bach1-deficient mice after high-fat diet loading for 6 wk, suggesting minimal roles of Bach1 in the development of insulin resistance. In contrast, Bach1 deficiency significantly suppressed alloxan-induced pancreatic insulin content reduction and the resultant glucose elevation. Furthermore, TUNEL-positive cells in pancreatic islets of Bach1-deficient mice were markedly decreased, by 60%, compared with those in WT mice. HO-1 expression in islets was signifi...
Proceedings of the National Academy of Sciences, 2013
The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in... more The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in B cells with specific functions such as class switch recombination and somatic hypermutation, but its function in T cells is not known. We found equal Bach2 expression in T cells and analyzed its function using Bach2-deficient (−/−) mice. Although T-cell development was normal, numbers of peripheral naive T cells were decreased, which rapidly produced Th2 cytokines after TCR stimulation. Bach2 −/− naive T cells highly expressed genes related to effector-memory T cells such as CCR4, ST-2 and Blimp-1. Enhanced expression of these genes induced Bach2 −/− naive T cells to migrate toward CCR4-ligand and respond to IL33. Forced expression of Bach2 restored the expression of these genes. Using Chromatin Immunoprecipitation (ChIP)-seq analysis, we identified S100 calcium binding protein a, Heme oxigenase 1, and prolyl hydroxylase 3 as Bach2 direct target genes, which are highly expressed in eff...
Ferroptosis is a regulated cell death due to the iron-dependent accumulation of lipid peroxide. F... more Ferroptosis is a regulated cell death due to the iron-dependent accumulation of lipid peroxide. Ferroptosis is known to constitute the pathology of ischemic diseases, neurodegenerative diseases, and steatohepatitis and also works as a suppressing mechanism against cancer. However, how ferroptotic cells affect surrounding cells remains elusive. We herein report the transfer phenomenon of lipid peroxidation and cell death from ferroptotic cells to nearby cells that are not exposed to ferroptotic inducers (FINs). While primary mouse embryonic fibroblasts (MEFs) and NIH3T3 cells contained senescence-associated β-galactosidase (SA-β-gal)-positive cells, they were decreased upon induction of ferroptosis with FINs. The SA-β-gal decrease was inhibited by ferroptotic inhibitors and knockdown of Atg7, pointing to the involvement of lipid peroxidation and activated autophagosome formation during ferroptosis. A transfer of cell culture medium of cells treated with FINs, type 1 or 2, caused the ...
The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, inc... more The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in -deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but...
S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone ... more S-adenosylmethionine (SAM) is an important metabolite as a methyl-group donor in DNA and histone methylation, tuning regulation of gene expression. Appropriate intracellular SAM levels must be maintained, because methyltransferase reaction rates can be limited by SAM availability. In response to SAM depletion, MAT2A, which encodes a ubiquitous mammalian methionine adenosyltransferase isozyme, was upregulated through mRNA stabilization. SAM-depletion reduced N-methyladenosine (mA) in the 3' UTR of MAT2A. In vitro reactions using recombinant METTL16 revealed multiple, conserved methylation targets in the 3' UTR. Knockdown of METTL16 and the mA reader YTHDC1 abolished SAM-responsive regulation of MAT2A. Mutations of the target adenine sites of METTL16 within the 3' UTR revealed that these mAs were redundantly required for regulation. MAT2A mRNA methylation by METTL16 is read by YTHDC1, and we suggest that this allows cells to monitor and maintain intracellular SAM levels.
Journal of immunology (Baltimore, Md. : 1950), Jan 15, 2018
BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation ... more BTB and CNC homology 2 (Bach2) is a transcriptional repressor that is required for the formation of the germinal center (GC) and reactions, including class switch recombination and somatic hypermutation of Ig genes in B cells, within the GC. Although BCR-induced proliferation is essential for GC reactions, the function of Bach2 in regulating B cell proliferation has not been elucidated. In this study, we demonstrate that Bach2 is required to sustain high levels of B cell proliferation in response to BCR signaling. Following BCR engagement in vitro, B cells from -deficient () mice showed lower incorporation of BrdU and reduced cell cycle progression compared with wild-type cells. B cells also underwent increased apoptosis, as evidenced by an elevated frequency of sub-G cells and early apoptotic cells. Transcriptome analysis of BCR-engaged B cells from mice revealed reduced expression of the antiapoptotic gene encoding Bcl-x and elevated expression of cyclin-dependent kinase inhibitor...
Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea cau... more Pulmonary alveolar proteinosis (PAP) is a severe respiratory disease characterized by dyspnea caused by accumulation of surfactant protein. Dysfunction of alveolar macrophages (AMs), which regulate the homeostasis of surfactant protein, leads to the development of PAP; for example, in mice lacking BTB and CNC homology 2 (Bach2). However, how Bach2 helps prevent PAP is unknown, and the cell-specific effects of Bach2 are undefined. Using mice lacking Bach2 in specific cell types, we found that the PAP phenotype of Bach2-deficient mice is due to Bach2 deficiency in more than two types of immune cells. Depletion of hyperactivated T cells in Bach2-deficient mice restored normal function of AMs and ameliorated PAP. We also found that, in Bach2-deficient mice, hyperactivated T cells induced gene expression patterns that are specific to other tissue-resident macrophages and dendritic cells. Moreover, Bach2-deficient AMs exhibited a reduction in cell cycle progression. IFN-␥ released from T cells induced Bach2 expression in AMs, in which Bach2 then bound to regulatory regions of inflammationassociated genes in myeloid cells. Of note, in AMs, Bach2 restricted aberrant responses to excessive T cell-induced inflammation, whereas, in T cells, Bach2 puts a brake on T cell activation. Moreover, Bach2 stimulated the expression of multiple histone genes in AMs, suggesting a role of Bach2 in proper histone expression. We conclude that Bach2 is critical for the maintenance of AM identity and self-renewal in inflammatory environments. Treatments targeting T cells may offer new therapeutic strategies for managing secondary PAP. This work was supported by Grants-in-aid 15H02506, 24390066, 23116003, and 21249014 from the Japan Society for the Promotion of Science. Additional initiative supports were from the Uehara Foundation, Takeda Foundation, and Astellas Foundation for Research on Metabolic Disorders. The authors declare that they have no conflicts of interest with the contents of this article. This article contains supplemental Fig. S1. The microarray, RNA sequencing, and ChIP sequencing data have been deposited at the Gene Expression Omnibus database under accession codes GSE79139 and GSE79558.
Iron plays the central role in the oxygen transport by the erythrocyte as a constituent of heme a... more Iron plays the central role in the oxygen transport by the erythrocyte as a constituent of heme and hemoglobin. The importance of iron and heme also resides in their regulatory roles during erythroblast maturation. The transcription factor Bach1 may be involved in their regulatory roles since it is inactivated by direct binding of heme. To address whether Bach1 is involved in the responses of erythroblasts to iron status, low iron conditions that induced severe iron deficiency in mice were established. Under iron deficiency, extensive gene expression changes and mitophagy disorder were induced during maturation of erythroblasts. Bach1 mice showed more severe iron deficiency anemia in the developmental phase of mice and a retarded recovery once iron was replenished when compared with wild-type mice. In the absence of Bach1, the expression of globin genes and Hmox1 (encoding heme oxygenase-1) was de-repressed in erythroblasts under iron deficiency, suggesting that Bach1 represses thes...
T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiatio... more T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.
Genes to cells : devoted to molecular & cellular mechanisms, 2016
H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone v... more H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene. H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we used the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a nonacetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. In contrast, each one of these three forms of H2A.Z complemented the transcri...
B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of pla... more B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histone H3 and H4 around the Prdm1 intron 5 MARE were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3 lysine 9 was lower in X63/0 cells than BAL17 cells. Purification of Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin beta-like 1X-linked (Tbl1x), and RAP1 interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prd...
The transcription factor Bach2 regulates the immune system at multiple points including class swi... more The transcription factor Bach2 regulates the immune system at multiple points including class switch recombination (CSR) in activated B cells and the function of T cells in part by restricting their terminal differentiation. However, the regulation of Bach2 expression and its activity in the immune cells is still unclear. Here we demonstrated that Bach2 mRNA expression decreased in Pten-deficient primary B cells. Bach2 was phosphorylated in primary B cells, which was increased upon the activation of B cell receptor by an anti-immunoglobulin M (IgM) antibody or CD40 ligand. Using specific inhibitors of kinases, the phosphorylation of Bach2 in activated B cells was shown to depend on the phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR pathway. The complex of mTOR and Raptor phosphorylated Bach2 in vitro. We identified multiple new phosphorylation sites of Bach2 by mass spectrometry analysis of epitope-tagged Bach2 expressed in mature B cell line BAL17. Among the sites identified, serine...
BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, ®ve (20%) s... more BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, ®ve (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.
Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. S... more Cellular senescence is one of the key strategies to suppress expansion of cells with mutations. Senescence is induced in response to genotoxic and oxidative stress. Here we show that the transcription factor Bach1 (BTB and CNC homology 1, basic leucine zipper transcription factor 1), which inhibits oxidative stress-inducible genes, is a crucial negative regulator of oxidative stressinduced cellular senescence. Bach1-deficient murine embryonic fibroblasts showed a propensity to undergo more rapid and profound p53-dependent premature senescence than control wild-type cells in response to oxidative stress. Bach1 formed a complex that contained p53, histone deacetylase 1 and nuclear co-repressor N-coR. Bach1 was recruited to a subset of p53 target genes and contributed to impeding p53 action by promoting histone deacetylation. Because Bach1 is regulated by oxidative stress and heme, our data show that Bach1 connects oxygen metabolism and cellular senescence as a negative regulator of p53.
Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), ch... more Pulmonary alveolar proteinosis (PAP) results from a dysfunction of alveolar macrophages (AMs), chiefly due to disruptions in the signaling of granulocyte macrophage colony–stimulating factor (GM-CSF). We found that mice deficient for the B lymphoid transcription repressor BTB and CNC homology 2 (Bach2) developed PAP-like accumulation of surfactant proteins in the lungs. Bach2 was expressed in AMs, and Bach2-deficient AMs showed alterations in lipid handling in comparison with wild-type (WT) cells. Although Bach2-deficient AMs showed a normal expression of the genes involved in the GM-CSF signaling, they showed an altered expression of the genes involved in chemotaxis, lipid metabolism, and alternative M2 macrophage activation with increased expression of Ym1 and arginase-1, and the M2 regulator Irf4. Peritoneal Bach2-deficient macrophages showed increased Ym1 expression when stimulated with interleukin-4. More eosinophils were present in the lung and peritoneal cavity of Bach2-defic...
The oxidative stress response operates by inducing the expression of genes that counteract the st... more The oxidative stress response operates by inducing the expression of genes that counteract the stress. We show here that the oxidative stress-responsive transcription factor Bach2 is a generic inhibitor of gene expression directed by the 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element. The Bach2-enhanced green fluorescent protein bicistronic retrovirus was used to monitor the fate of Bach2-expressing cells at the single cell level. Bach2 exerted an inhibitory effect on NIH3T3 cell proliferation and caused massive apoptosis upon mild oxidative stress in both NIH3T3 and Raji B-lymphoid cells. Interestingly, Bach1, a highly homologous protein, could not induce cell death, demonstrating the specificity for the apoptosis induction. Although both oxidative stress and leptomycin B, an inhibitor of nuclear export, induce nuclear accumulation of Bach2, the leptomycin B-induced nuclear accumulation of Bach2 was not sufficient to elicit apoptosis. Upon oxidative stress, Bach2 formed nuclear foci that associated with promyelocytic leukemia nuclear bodies. Our results suggest that Bach2 constitutes a cell lineage-specific system that couples oxidative stress and cell death and that inhibition of 12-O-tetradecanoylphorbol-13-acetate response element, the Maf recognition element, and the antioxidant-responsive element upon oxidative stress may be critical determinants for apoptosis.
Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced clas... more Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced class switch recombination and a reduced somatic hypermutation of immunoglobulin genes. Little is known about the direct target genes of Bach2 in B cells. By analyzing various B cell and plasma cell lines, we showed that the expression patterns of Bach2 and Blimp-1 (B lymphocyteinduced maturation protein 1), a master regulator of plasma cell differentiation, are mutually exclusive. The reporter gene of the Blimp-1 gene (Prdm1) was repressed by the overexpression of Bach2 in B cell lines. The heterodimer of Bach2/MafK bound to the Maf recognition element located upstream of the Prdm1 promoter in an electrophoretic mobility shift assay. The binding of MafK in B cells to the Prdm1 Maf recognition element was confirmed by chromatin immunoprecipitation assays. When MafK was purified from the BAL17 B cell line, a significant portion of it was present as a heterodimer with Bach2, with no apparent formation of MafK homodimer. These results strongly suggest that Bach2 represses the expression of Blimp-1 together with MafK in B cells prior to plasma cell differentiation. Accordingly, the knockdown of Bach2 mRNA using short hairpin RNA in BAL17 cells resulted in higher levels of Prdm1 expression after the stimulation of B cell receptor by surface IgM crosslinking. Induction of Prdm1 was more robust and faster in primary Bach2-deficient B cells than in wild-type control B cells upon lipopolysaccharide stimulation. Therefore, the Prdm1 regulation in B cells involves the repression by Bach2, which may be cancelled upon terminal plasma cell differentiation.
B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differenti... more B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.
†In two cases sIL-2R was not assessed. ‡In one case MIB-1 index was not assessed. IPI, internatio... more †In two cases sIL-2R was not assessed. ‡In one case MIB-1 index was not assessed. IPI, international prognostic index; LDH, lactate dehydrogenase; OS, overall survival; PFS, progression-free survival; sIL-2R, soluble inteleukin-2 receptor.
American Journal of Physiology-Endocrinology and Metabolism, 2013
BTB and CNC homology 1 (Bach1) is a transcriptional repressor of antioxidative enzymes, such as h... more BTB and CNC homology 1 (Bach1) is a transcriptional repressor of antioxidative enzymes, such as heme oxygenase-1 (HO-1). Oxidative stress is reportedly involved in insulin secretion impairment and obesity-associated insulin resistance. However, the role of Bach1 in the development of diabetes is unclear. HO-1 expression in the liver, white adipose tissue, and pancreatic islets was markedly upregulated in Bach1-deficient mice. Unexpectedly, glucose and insulin tolerance tests showed no differences in obese wild-type (WT) and obese Bach1-deficient mice after high-fat diet loading for 6 wk, suggesting minimal roles of Bach1 in the development of insulin resistance. In contrast, Bach1 deficiency significantly suppressed alloxan-induced pancreatic insulin content reduction and the resultant glucose elevation. Furthermore, TUNEL-positive cells in pancreatic islets of Bach1-deficient mice were markedly decreased, by 60%, compared with those in WT mice. HO-1 expression in islets was signifi...
Proceedings of the National Academy of Sciences, 2013
The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in... more The transcriptional repressor BTB and CNC homology 2 (Bach2) is thought to be mainly expressed in B cells with specific functions such as class switch recombination and somatic hypermutation, but its function in T cells is not known. We found equal Bach2 expression in T cells and analyzed its function using Bach2-deficient (−/−) mice. Although T-cell development was normal, numbers of peripheral naive T cells were decreased, which rapidly produced Th2 cytokines after TCR stimulation. Bach2 −/− naive T cells highly expressed genes related to effector-memory T cells such as CCR4, ST-2 and Blimp-1. Enhanced expression of these genes induced Bach2 −/− naive T cells to migrate toward CCR4-ligand and respond to IL33. Forced expression of Bach2 restored the expression of these genes. Using Chromatin Immunoprecipitation (ChIP)-seq analysis, we identified S100 calcium binding protein a, Heme oxigenase 1, and prolyl hydroxylase 3 as Bach2 direct target genes, which are highly expressed in eff...
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Papers by Akihiko Muto