Identification of Bach2 as a B-cell-specific partner for small Maf proteins that negatively regulate the immunoglobulin heavy chain gene 3' enhancer
1998; Springer Nature; Volume: 17; Issue: 19 Linguagem: Inglês
10.1093/emboj/17.19.5734
ISSN1460-2075
Autores Tópico(s)NF-κB Signaling Pathways
ResumoArticle1 October 1998free access Identification of Bach2 as a B-cell-specific partner for small Maf proteins that negatively regulate the immunoglobulin heavy chain gene 3′ enhancer Akihiko Muto Akihiko Muto Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Hideto Hoshino Hideto Hoshino Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Linda Madisen Linda Madisen Fred Hutchinson Cancer Center, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Nobuaki Yanai Nobuaki Yanai Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, 980-8575 Japan Search for more papers by this author Masuo Obinata Masuo Obinata Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, 980-8575 Japan Search for more papers by this author Hajime Karasuyama Hajime Karasuyama Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Norio Hayashi Norio Hayashi Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Search for more papers by this author Hiromitsu Nakauchi Hiromitsu Nakauchi Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Masayuki Yamamoto Masayuki Yamamoto Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Mark Groudine Mark Groudine Fred Hutchinson Cancer Center, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Kazuhiko Igarashi Corresponding Author Kazuhiko Igarashi Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Search for more papers by this author Akihiko Muto Akihiko Muto Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Hideto Hoshino Hideto Hoshino Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Linda Madisen Linda Madisen Fred Hutchinson Cancer Center, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Nobuaki Yanai Nobuaki Yanai Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, 980-8575 Japan Search for more papers by this author Masuo Obinata Masuo Obinata Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, 980-8575 Japan Search for more papers by this author Hajime Karasuyama Hajime Karasuyama Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113 Japan Search for more papers by this author Norio Hayashi Norio Hayashi Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Search for more papers by this author Hiromitsu Nakauchi Hiromitsu Nakauchi Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Masayuki Yamamoto Masayuki Yamamoto Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan Search for more papers by this author Mark Groudine Mark Groudine Fred Hutchinson Cancer Center, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA Search for more papers by this author Kazuhiko Igarashi Corresponding Author Kazuhiko Igarashi Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan Search for more papers by this author Author Information Akihiko Muto1,2, Hideto Hoshino1,2, Linda Madisen3, Nobuaki Yanai4, Masuo Obinata4, Hajime Karasuyama5, Norio Hayashi1, Hiromitsu Nakauchi2, Masayuki Yamamoto2, Mark Groudine3 and Kazuhiko Igarashi 1 1Department of Biochemistry, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, 980-8575 Japan 2Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba, 305 Japan 3Fred Hutchinson Cancer Center, 1100 Fairview Avenue North, Seattle, WA, 98109-1024 USA 4Department of Cell Biology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi, Sendai, 980-8575 Japan 5Department of Immunology, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5734-5743https://doi.org/10.1093/emboj/17.19.5734 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Maf family transcription factors are important regulators in various differentiation systems. Putative Maf recognition elements (MAREs) are found in the 3′ enhancer region of the immunoglobulin heavy chain (IgH) gene. These elements are bound in B-cell extracts by a heterodimeric protein complex containing both Bach2 and a small Maf protein. Analysis of normal hematopoietic cells revealed that Bach2 is specifically expressed in B cells. Bach2 is abundantly expressed in the early stages of B-cell differentiation and turned off in terminally differentiated cells. Bach2 acts together with MafK as a negative effector of the IgH 3′ enhancer and binds to the co-repressor SMRT (silencing mediator of retinoid and thyroid receptor). Hence the Bach2–small-Maf heterodimer may represent the first example of a B-cell lineage, and of a developmental stage-restricted negative effector of the MARE in the IgH 3′ enhancer region. Introduction Maf family transcription factors possess a conserved basic-region leucine zipper (bZip) domain which mediates protein–protein interactions and DNA binding (Nishizawa et al., 1989; Kataoka et al., 1993). While c-Maf, MafB and NRL contain putative transcription activation domains (Nishizawa et al., 1989; Swaroop et al., 1992; Kataoka et al., 1994a), MafF, MafK and MafG lack canonical trans-activation domains (Andrews et al., 1993b; Fujiwara et al., 1993; Igarashi et al., 1994, 1995b; Kataoka et al., 1995; Blank et al., 1997). MafF, MafG and MafK are essentially composed of bZip domains and are collectively referred to as the small Maf family proteins. Various dimeric combinations of Maf family proteins bind in vitro to a DNA sequence motif called T-MARE (TGCTGAG/CTCAGCA) containing a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE), TGAG/CTCA (Kataoka et al., 1994a, b, 1995). Maf family proteins are emerging as important regulators of cell differentiation in various systems (Blank and Andrews, 1997; Motohashi et al., 1997). The hematopoietic cell-specific transcription factor NF-E2 is a heterodimer formed between the erythroid- and megakaryocyte-specific bZip protein, p45 NF-E2 and one of the small Maf family proteins (Andrews et al., 1993a, b; Ney et al., 1993; Igarashi et al., 1994). Resulting heterodimers bind to a NF-E2 consensus site (TGCTGAG/C TCAT/C), which is related to T-MARE (Andrews et al., 1993a, b; Igarashi et al., 1994). A simple nomenclature of NF-E2 binding sites as Maf recognition elements (MAREs, including T-MARE and its derivatives) was recently suggested (Motohashi et al., 1997). Besides generating NF-E2, the expression patterns of the small Maf proteins suggest that they also function in other cell lineages and tissues (Fujiwara et al., 1993; Igarashi et al., 1995b; Motohashi et al., 1996). Fos and several p45-related bZip factors such as Nrf1 (LCR-F1/TCF-11), Nrf2 (ECH), Bach1 and Bach2 have been shown to form heterodimers with the small Maf proteins and bind to the MARE in vitro (Chan et al., 1993; Caterina et al., 1994; Luna et al., 1994; Moi et al., 1994; Itoh et al., 1995; Oyake et al., 1996; Toki et al., 1997; Johnsen et al., 1998). However, the precise role of these potential heterodimeric combinations during development and cell differentiation has remained elusive. Among these bZip proteins, Bach proteins may play mechanistically distinct roles as MARE-binding proteins, since they bear a BTB/POZ domain (Oyake et al., 1996). Several lines of evidence suggest that the BTB domain may be involved in the regulation of chromatin structure (Albagli et al., 1995). MARE-like elements are found in the regulatory regions of an increasing number of genes (Kataoka et al., 1994b). Of particular interest, we noticed that TREs within the 3′ enhancer region of the immunoglobulin heavy chain (IgH) gene (Pettersson et al., 1990; Matthias and Baltimore, 1993; Madisen and Groudine, 1994) resemble MAREs. Since immunoglobulin genes are regulated by B-cell-specific transcription factors (Fitzsimmons and Hagman, 1996; Arulampalam et al., 1997), the presence of MARE-like elements in the IgH enhancer region suggests the presence of B-cell-specific MARE effector proteins. In this study, we examined MARE-binding activity in B cells. The results presented here indicate that Bach2 functions as a B-cell- and developmental-stage-specific partner for a small Maf protein and that the heterodimer interacts with the MAREs. Interestingly, co-expression of Bach2 and MafK repressed activity of the IgH 3′ enhancer. Hence, the Bach2–small-Maf heterodimer may represent the first example of a cell-lineage-restricted negative effector of MAREs, and might be involved in the repression of immunoglobulin genes at earlier stages of B-cell differentiation. Results Bach2 binds to MARE in IgH 3′ enhancer regions Several TREs have been identified in the 3′ enhancer/locus control region (LCR) of the immunoglobulin heavy chain (IgH) gene (Pettersson et al., 1990; Lieberson et al., 1991; Matthias and Baltimore, 1993; Madisen and Groudine, 1994; Grant et al., 1995; Chauveau and Cogne, 1996). At least two of them, located in the Cα3′E and HS3, are identical to the MARE consensus sequence (Figure 1). It should be noted that Cα3′E is composed of an inverted repeat of HS3 (Chauveau and Cogne, 1996), and hence it contains an identical MARE. To examine whether Maf dimers bind to these elements in B cells, we carried out electrophoretic mobility shift assays (EMSAs) using nuclear extracts from various B cell lines (Figure 2). The DNA probe which we used is a 29 bp fragment that was derived from IgH HS3 and contains one putative MARE. Figure 1.Structure of mouse IgH locus. (A) Schematic representation of mouse IgH gene after recombination of the variable region. Boxes and circles indicate exons and enhancers, respectively. Cα3′E and HS3 contain identical putative MAREs. (B) Comparison of the putative MAREs in the IgH Cα3′E and HS3 with NF-E2-type MARE and TRE. Download figure Download PowerPoint Figure 2.MARE-binding complex in B-cell extracts. (A) EMSA was carried out with oligonucleotide probe which contains the MARE in the HS3/Cα3′E (lanes 1–7) or its derivative containing mutations in the MARE (lanes 8 and 9). Nuclear extracts were from pro-B cell line 63–12 (lanes 2, 4, 6 and 8) and mature-B cell line BAL17 (lanes 3, 5, 7 and 9). Unlabeled wild-type or mutated competitor DNAs (lanes 4–5 and 6–7, respectively) were added at 100-fold molar excess prior to addition of the radiolabeled probe. The arrow indicates the specific MARE-binding complex in the B-cell extracts. (B) Effects of various antibodies were examined. EMSA was carried out with nuclear extracts of 63-12 cells (lanes 2, 5, 9, 12 and 15), BAL17 cells (lanes 3, 6, 10, 13 and 16) and J558L plasmacytoma cells (lanes 4, 7, 11, 14 and 17) in the presence (lanes 5–7 and 12–17) or absence (lanes 2–4 and 9–11) of various antibodies. Antibodies were anti-Bach2 (lanes 5–7), anti-small-Maf (lanes 12–14) and anti-Fos (lanes 15–17). Bach2–small-Maf complex is indicated with an arrow. (C) The effect of anti-Bach2 monoclonal antibody was examined as above. EMSA was carried out with nuclear extracts of 63-12 cells (lanes 2, 4 and 6) or BAL17 cells (lanes 3, 5 and 7) in the presence of Bach2 monoclonal antibody 2G11-11 (lanes 4–5) or preimmune rabbit serum (lanes 6–7). Download figure Download PowerPoint As shown in Figure 2A, EMSA and competition assays revealed the presence of only one specific DNA-binding protein complex in the nuclear extracts prepared from the pro-B cell line 63-12 and from the mature B cell line BAL17 (Figure 2A, lanes 2–5). An oligonucleotide containing mutations in the MARE failed to compete with the bound complex (Figure 2A, lanes 6 and 7). Furthermore, the mutated DNA did not generate a corresponding protein–DNA complex when it was radiolabeled and incubated with the nuclear extracts (Figure 2A, lanes 8 and 9). These results established specific binding of the complex to the MARE. This MARE-binding complex was not detected using nuclear extracts from the plasmacytoma cell line J558L (Figure 2B, lane 4), indicating its stage-specific activity. To reveal constituents of the complex, we utilized antibodies that recognize various factors that could bind to a MARE. Formation of the specific protein–DNA complex was inhibited by anti-Bach2 as well as by anti-small-Maf antisera (Figure 2B, lanes 5, 6, 12 and 13). On the other hand, preimmune sera did not show any effect on complex formation (Figure 2C, lanes 6–7). Furthermore, anti-Fos antibodies, which react with c-Fos, FosB, Fra-1 and Fra-2, or anti-Jun antibodies, which recognize c-Jun, JunB and JunD, did not have any effects (Figure 2B, lanes 15–16; data not shown). Finally, involvement of Bach2 in the MARE-binding complex was confirmed using an anti-Bach2 monoclonal antibody (Figure 2C): addition of the Bach2 monoclonal antibody supershifted the MARE-binding complex. These results established that the TRE within the 3′ enhancers Cα3′E and HS3 of the IgH gene is actually a MARE and bound by a heterodimer of Bach2 and one or another of the small Maf proteins in B-cell extracts. Expression of Bach2 in hematopoietic cells We have reported previously that expression of bach2 mRNA in mice is restricted to the brain and spleen. To determine the possible role played by Bach2 during hematopoiesis, we isolated total RNA samples from bone marrow, thymus and spleen of adult mice as well as from fetal livers (13.5 days post-coitus embryos), and performed RNA blotting analysis (Figure 3A). Hematopoietic cells in the adult bone marrow revealed a 2.5-fold higher level of bach2 mRNA expression compared with that of other hematopoietic tissues or brain. Figure 3.Expression of bach2 mRNAs in hematopoietic tissues. (A) Total RNAs (20 μg) were hybridized with a bach2-specific DNA probe after separation on 1.0% agarose–formaldehyde gel and transfer onto a nylon membrane. RNAs were from 13.5 days post-coitus fetal liver, adult bone marrow, spleen, thymus and brain. Relative levels of bach2 mRNA, normalized for β-actin mRNA, are 0.5, 2.6, 0.8, 0.7 and 1.0, respectively. Positions of 28S and 18S ribosomal RNAs are indicated with arrows. (B) Hematopoietic cells were fractionated depending on the surface marker expression, and bach2 expression in each fraction was determined by RT–PCR analysis. B220+ (B cell), Gr-1+ (granulocyte), Mac-1+ (mono-macrophage) and TER-119+ (erythroid) fractions were from bone marrow, the Thy-1.2+ (mature T-cell) fraction from spleen, and the CD4/CD8-double positive (immature T-cell) fraction from thymus. (C) The relationship between bach2 mRNA expression and B cell differentiation was examined by RT–PCR. Bone marrow B220+ cells (lane 1) were further fractionated into B220+/IgM− and B220+/IgM+ cells (lanes 2 and 3, respectively). Spleen B220+ fraction was also compared as the most differentiated B cell fraction (lane 4). Relative levels of bach2 mRNA, normalized for β-actin mRNA, are shown on the right side. (D) Primary spleen B cells were stimulated with LPS and bach2 mRNA expression was determined by RT–PCR. Download figure Download PowerPoint To determine the cell-lineage specificity of Bach2 expression in vivo, we fractionated hematopoietic cells from bone marrow, spleen and thymus into various lineages using lineage-specific monoclonal antibodies (mAbs) and made comparisons by RT–PCR (Figure 3B). Only the B220-positive (B220+) B cells isolated from bone marrow showed a high level of Bach2 expression. Mac-1+ cells (mono-macrophage lineage) did not express Bach2. These results clearly established that Bach2 is a B-cell-restricted transcription factor. Expression of Bach2 during B-cell differentiation The relative expression levels in bone marrow and spleen suggests that Bach2 is expressed during the earlier stages of B-cell differentiation. This possibility has been addressed by fractionating the B220+ cells into two populations, depending on the cell surface expression of IgM. The B220+/IgM− fraction contains pro- and pre-B cells, whereas the B220+/IgM+ fraction contains immature- and mature-B cells (Hardy et al., 1991; Rolink and Melchers, 1991). As a source of mature B-cell population we also assessed B220+ cells from spleen. As shown in Figure 3C, the B220+/IgM− fraction, which represents an early stage of B-cell lineage development, showed the highest level of expression of bach2 mRNA, whereas the B220+/IgM+ bone marrow cells and B220+ spleen cells expressed less bach2 mRNA. The mitogen lipopolysaccharide (LPS) is known to induce proliferation and differentiation of resting B cells. Upon LPS treatment of spleen B cells, Bach2 expression was further down-regulated (to 50%, Figure 3D). Taken together, these results indicated that Bach2 expression decreases during the maturation of B cells. The relationship between Bach2 expression and B-cell development was further examined using various B cell lines at different stages of B-cell differentiation (Figure 4A). Among the cell lines examined, B31-1 is a stroma-dependent B-cell line and is at the earliest stage (N.Yanai and M.Obinata, unpublished observations). When cultured with the stromal cell line TBR31-1, B31-1 cells are a mixture of B220− and B220+ fractions, both of which express the pre-pro-B-cell-surface-marker-pattern like c-Kit+, CD43-, HSA (CD24)− and IgM− (N.Yanai and M.Obinata, unpublished observations). RT–PCR analysis revealed that bach2 was expressed abundantly at the earliest stage of B-cell development (i.e. B31-1 on TBR31-1; Figure 4A) and, in addition, in the stroma-independent pre-B, pro-B, immature-B and mature-B cell lines. In contrast, it was not detected in two different plasmacytoma cell lines, X63/0 (X63-Ag8.653) and J558L. Two stromal cell lines (TBR31-1 and ST-2) expressed the bach2 gene at a low level, raising the possibility that Bach2 may play some roles in stromal cells as well. Figure 4.Expression of Bach2 in B cell lines. (A) Expression of bach2 mRNA in various B cell lines was examined by RT–PCR. Cell lines were stromal cell lines TBR31-1 and ST-2, pre-pro-B cell line B31-1, pro-B cell lines 38B9 and 63–12, pre-B cell lines 18–81 and NFS5.3, immature-B cell lines WEHI 231 and WEHI 279, mature-B-cell lines CH1 and BAL17, and plasmacytoma cell lines X63/0 and J558L. The PCR products of β-actin mRNA are shown at the bottom. (B) Immunoblotting analysis of the expression of Bach2 protein in the B–cell lines. Twenty-five micrograms of whole-cell extracts prepared from each B-cell line were separated with 7.5% of SDS–polyacrylamide gel, transferred onto membranes, and reacted with anti-Bach2 antiserum. Positions of mol. wt markers are shown at the left-hand side. (C) Immunoblotting analysis of the expression of small Maf proteins in B cell lines. Whole-cell extracts of indicated cell lines as well as MEL cells were examined for the presence of small Maf proteins with anti-small-Maf antiserum. The ∼20 kDa antigen is indicated with an arrow. Positions of mol. wt markers are shown on the left-hand side. Download figure Download PowerPoint Immunoblotting analysis with an anti-Bach2 antiserum confirmed that Bach2 is expressed in the pro-B, pre-B, immature-B and mature-B cell lines, and that it is absent in the plasmacytoma cell lines (Figure 4B). Furthermore, the expression profile of Bach2 in the different B-cell lines correlates well with the presence or absence of the MARE-binding complex, as shown in the EMSA studies (Figure 2). On the other hand, the anti-small-Maf antibody reacted with an ∼20 kDa antigen which was present in all of the B-cell lines including plasmacytoma, as well as in the murine erythroleukemic (MEL) cells, in which mafK is known to be expressed (Andrews et al., 1993b; Igarashi et al., 1995a, b; Figure 4C; data not shown). Hence, Bach2 determines the B-cell lineage and stage-specificity of MARE effectors by interacting with more broadly expressed small Maf proteins. Such a B-cell- and stage-specific Bach2 heterodimer will most probably play an important role during B-cell development and differentiation. Expression of Bach2 during B-cell commitment To determine at which point Bach2 expression commences during the differentiation of B cells, we examined hematopoietic stem cells (c-Kit+/Sca-1+/CD34 low or negative/lineage markers negative). A single cell of the stem cell fraction was shown previously to be able to reconstitute bone marrow cells in lethally irradiated mice (Osawi et al., 1996). Expression of Bach2 was compared with those in more differentiated c-Kit+/Lin− cells, which include progenitor cells of various lineages as well as in lineage-markers-positive (Lin+) differentiated cells. As shown in Figure 5A, a significant level of bach2 mRNA could be detected in the stem cell fraction, whereas the c-Kit+/Lin− cells expressed Bach2 at low levels. Considering the fact that the Lin+ fraction contained differentiated cells of various lineages and that Bach2 expression in this fraction is restricted to the B220+ cells, the expression level in the stem-cell fraction was estimated to be relatively low compared with expression in B220+ cells in the Lin+ fraction. Figure 5.Expression of Bach2 during B cell commitment. (A) Lin−/ Sca-1+/c-Kit+/CD34- (lane 1), Lin−/c-Kit+ (lane 2) and Lin+ (lane 3) fractions were sorted from bone marrow cells of adult mice. bach2 expression in each fraction was examined by RT–PCR. (B) Pre-pro B cell line B31-1 cells cultured on stromal ST-2 cells were fractionated into B220− and B220+ fractions. Twenty-five micrograms of whole-cell extracts were separated with SDS–7.5% polyacrylamide gel and examined for Bach2 expression with anti-Bach2 antiserum. Download figure Download PowerPoint B220 is known as one of the earliest markers expressed in the B-cell lineage (Li et al., 1996). To determine the timing of Bach2 induction during B-cell development, we took advantage of the fact that the B31-1 cells, grown on the TBR31-1 stromal cells, are a mixture of B220− and B220+ cells, and that B220− cells give rise to B220+ cells in vitro (N.Yanai and M.Obinata, unpublished observations). Each population was purified and examined for the presence of Bach2 protein by immunoblot analysis. As shown in Figure 5B, expression of Bach2 in the less-differentiated B220− fraction is more abundant than it is in the B220+ fraction, suggesting that B220− B31-1 cells, which are already committed to the B-cell lineage, express Bach2 at a high level. Taken together, these results indicate that Bach2 is expressed at low levels in uncommitted hematopoietic stem cells, and that its expression is up-regulated during, or soon after, commitment of stem cells to the B-cell lineage. The commitment to other hematopoietic cell lineages might then lead to the down-regulation of bach2. Bach2 negatively regulates IgH 3′ enhancer activity To examine an effect of Bach2 on the IgH 3′ enhancer activity, the HS1, −2, −3 and −4 were cloned in combination as described previously, downstream of the luciferase gene under the control of IgH promoter (Figure 6A). HS3 contains an inverted repeat of the Cα3′E and carries an identical MARE that binds Bach2–small-Maf heterodimer (Figure 1). Hence, the HS1234 reporter plasmid contains at least one functional MARE. The HS1234 mini-enhancer was shown previously to be active in plasmacytoma cells but relatively inactive in pre-B cells (Madisen and Groudine, 1994). Accordingly, the HS1234 mini-enhancer failed to activate IgH promoter-driven reporter gene expression in pro-B- and pre-B-cell lines (Figure 6B). In contrast, the ability of the HS1234 to enhance transcription from the IgH promoter in plasmacytoma cells was evident, as shown in Figure 6B (compare lanes 9 and 10). Its stimulating activity was lower in mature B cells than in plasmacytoma cells (compare lanes 8 and 10). Figure 6.Repression of IgH 3′ enhancer activity by Bach2. (A) Schematic representation of reporter plasmid (line 3) that has IgH promoter (line 1) and a set of IgH 3′ HS1/2, −3 and −4 (Madisen and Groudine, 1994; lines 1 and 2). HS1234 fragments were linked in both orientations relative to the promoter, and both reporter genes gave essentially similar results in experiments described below. HS1234 (−) reporter plasmid carries only IgH promoter. (B) Enhancer activity of HS1234 in different cell background. IgH promoter reporter plasmids with or without the HS1234 mini-enhancer (1 μg) were transfected into the indicated B cell lines. Relative expression was determined by comparing normalized reporter gene levels induced by HS-containing plasmid with that by an enhancerless control (promoter only). Values represent the averages and standard errors of four transfections. (C) Cooperative repression of HS1234 activity by Bach2 and MafK. Bach2- and MafK-expression plasmids were transfected into X63/0 cells in various combinations, as shown below, together with reporter plasmids (0.1 μg) that carried (lanes 5–9) or lacked (lanes 1–4) the HS1234. Amounts of MafK expression plasmid were titrated (0, 0.45 or 0.9 μg) in the presence or absence of Bach2-expression plasmid (0.9 μg). The results are means of four independent experiments, each carried out in duplicate, and standard errors are indicated with thin lines. (D) Cooperative repression by Bach2 and MafK requires their interaction. Effects of co-transfection with MafK (0.9 μg) and wild-type Bach2 (0.1 μg, lanes 3–4) or a Bach2 derivative that lacked the leucine zipper (0.1 μg, lanes 5–6) were compared. (E) HS3 MARE is not the sole target of Bach2/MafK. The MARE within HS3 was deleted from the HS1234 reporter gene, and its response to Bach2 (0.1 μg) and MafK (0.9 μg) expression (lanes 5–8) was compared with the wild-type reporter plasmid (lanes 1–4). Download figure Download PowerPoint To examine the regulatory role of Bach2 in B-cell differentiation, we carried out co-transfection experiments (Figure 6C). In this experiment, we used 0.1 μg of reporter plasmid (1 μg was used in Figure 6B). Co-transfection of Bach2-expression plasmid into the plasmacytoma cells repressed reporter gene activity driven by the HS1234 (Figure 6C, lane 6). Expression of MafK alone resulted in only weak inhibition of the reporter gene activity (Figure 6C, lane 9). Interestingly, co-transfection of both Bach2- and MafK-expression plasmids resulted in more efficient repression that virtually abolished the effect of the HS1234 enhancer (Figure 6C, lanes 7 and 8), indicating that Bach2 and MafK cooperatively repressed gene expression. Neither of them showed significant repression of reporter gene in the absence of the HS1234, indicating that effects of Bach2 and MafK were mediated by HS1234. To verify the interaction of Bach2 and MafK in the observed cooperative transcription repression, we examined a Bach2 derivative that lacked the leucine zipper (Bach2Δzip), and hence could not form a heterodimer with a small Maf protein (Figure 6D). In this experiment, we used 0.1 μg of Bach2-expression plasmid per transfection (0.9 μg was used in Figure 6C). Both wild-type Bach2 and Bach2Δzip repressed the reporter gene activity to 60% in the absence of MafK. However, Bach2Δzip did not exert synergistic transcription repression with MafK, whereas Bach2 did exert such an effect (compare lanes 4 and 6). The results indicated that a heterodimer of Bach2 and MafK was responsible for the observed cooperative repression of transcription. The residual repression activity of Bach2Δzip that was independent of dimer formation may be due to its interaction with other proteins through remaining regi
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