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. C α 3 Ј E and HS3 contain identical putative MAREs. ( B ) Comparison of the putative MAREs in the IgH C α 3 Ј E and HS3 with NF-E2-type MARE and TRE. 

Contexts in source publication

Context 1

... Furthermore, the two MAREs within the 3 Ј enhancer of the mouse IgH gene are bound by Bach2– small-Maf heterodimers in B-cell extracts (Figure 2). Finally, we show here that Bach2 is expressed at high levels in immature B cells but not in terminally differentiated B cells that express high levels of the IgH gene (plasmacytes; Figure 4). A similar expression profile of Bach2 was also evident in human B-lineage cells (E.Ito, T.Toki and K.Igarashi, unpublished observation). Since Bach2 does not act as a transcriptional activator and rather represses activity of the IgH 3 Ј enhancer regions (Figure 6), we interpret our findings to show that Bach2 functions by inhibiting premature initiation of robust transcription of the IgH locus in undifferentiated B cells. Consistent with this model, the IgH 3 Ј enhancer is inactive before terminal differentiation where Bach2 is expressed abundantly, but is very active in plasmacytoma or myeloma cells, in which Bach2 is not expressed (Madisen and Groudine, 1994; this study). The present results established that Bach2 represses the IgH 3 Ј enhancer by forming a heterodimer with a small Maf protein, because synergistic repression with MafK of the enhancer activity was lost upon deletion of the leucine zipper from Bach2 (Figure 6D). However, the MARE within the HS3 is not the only target cis -element. This was indicated by the results that deletion of the HS3 MARE did not completely abolish cooperative transcription repression by Bach2 and MafK (Figure 6E). In this regard, it should be noted there are several other TREs within HS1, HS2, HS3 and HS4 (Pettersson et al. , 1990; Lieberson et al. , 1991; Matthias and Baltimore, 1993; Madisen and Groudine, 1994; Grant et al. , 1995; Chauveau and Cogne, 1996). Because MARE is closely related to TRE, some of these TREs may actually be a MARE and bind Bach2–small-Maf heterodimer within cells. Indeed, Maf family members can bind to sequences that diverge considerably from the consensus MARE (Ho et al. , 1996). We are currently trying to identify other Bach2 target sites within the 3 Ј enhancer regions. We examined the effect of the HS3 MARE deletion in pro-B and pre-B cells because there was a possibility that the deletion would relieve repression and activate HS1234 enhancer activity in these early-stage cells. However, the HS3 MARE deletion did not cause activation of the reporter gene expression in these cells (data not shown). As the HS1234 is more than 6 kb in length and contains many cis -elements, its enhacer activity may not reflect function of a singular cis -element. Rather, output may be determined as a combination of activities of positive and negative cis - and trans -elements. Regarding repression, another candidate effector is BASP/Pax5 which is known to repress IgH 3 Ј enhancer activity in early B cells (Singh and Birshtein, 1993). An interesting possibility is that Bach2 could modulate the activity of the IgH locus through its BTB domain, which is one of the hallmarks of Bach proteins and distinguishes them from other p45-related factors (Oyake et al. , 1996). The BTB domain has been implicated in transcription repression by the transcription factors BCL6 and PLZF (Chen et al. , 1993; Ye et al. , 1993). These proteins interact with co-repressor molecules like SMRT through the BTB domain (Dhordain et al. , 1997; Grignani et al. , 1998; Lin et al. , 1998). Bach2 indeed binds to SMRT in a yeast two hybrid assay (Figure 7), raising the possibility that it recruits the co-repressor to the IgH locus. In the transfection assay, Bach2 repressed the IgH 3 Ј HS1234 mini-enhancer reporter gene that contains multiple binding sites for various B cell transcription factors. Such a dominant effect of Bach2 might be explained by its interaction with co-repressors like SMRT. We examined effect of histone deacetylase inhibitor trichostatin A, and found that it does not abrogate transcription repression by Bach2 (A.Muto and K.Igarashi, unpublished observation). Because trichostatin A inhibits some, but not all, of histone deacetylase (Carmen et al ., 1996), further studies are necessary to reveal the mechanism by which Bach2 represses transcription. It will also be interesting to analyze whether Bach2 participates in the function of the IgH 3 Ј enhancer in terms of altering the chromatin structure of the locus, thereby contributing to the LCR activity of this element (Madisen and Groudine, 1994). BTB-domain-containing proteins have previously been shown to be involved in regulating chromatin structure (Albagli et al. , 1995). We showed recently that Bach1 is a candidate molecule that binds to the β -globin LCR. Bach1 functions as a novel type of architectural transcription factor that mediates interactions among multiple MAREs within the β -globin LCR depending on the protein interaction through the BTB domain (Igarashi et al ., 1998). In this sense, it should be noted that IgH 3 Ј LCR also contains at least two separated MAREs (Figure 1) and other putative target sites. Hence Bach2 may orchestrate assembly of a regulatory complex on this region by mediating interactions among the cis -elements. Previous results suggest that stem cells express various genes at low levels that later become up-regulated when the stem cells differentiate into a specific hematopoietic cell lineage (Hu et al. , 1997). The commitment and differentiation of stem cells, which express these ‘differen- tiation markers’ at low levels, toward a particular cell lineage occurs by the induction and repression of specific sets of genes (Hu et al. , 1997). From this point of view, the up-regulation of Bach2 during differentiation of stem cells into B220 ϩ /IgM – bone marrow cells, as well as its high expression in B220 B31-1 cells, indicates a potential role of Bach2 in lineage commitment of B cells. Several transcription factor genes have been shown to be essential for proper development of B cells (Fitzsimmons and Hagman, 1996). Among these factors, EBF and BSAP/ Pax5 show a very similar expression pattern to that of Bach2 (Barberis et al. , 1990; Hagman et al. , 1993). Expression of these proteins is restricted to the B cell lineage; they are turned on at an early stage of B cell development and down-regulated upon maturation to plasma cells. Ikaros, another hematopoietic-specific transcription factor, is required for development of both B and T cells, and is expressed in the stem-cell fraction as well (Georgopoulos, 1997). Bach2 may act together with these B- and lymphoid-specific transcription factors, leading to the differentiation of B cells from uncommitted stem cells. In conclusion, the present study has identified Bach2 as a B cell-specific component of a small Maf heterodimer and implicated Bach2 in the regulation of B cell-specific gene expression. The results thus provide crucial inform- ation and together with further studies will enhance our ability to understand how the development of B cells is regulated by members of the bZip family of transcription ...

Context 2

... 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 (TGCTGA G / C TCAGCA) containing a 12- O -tetradecanoyl- phorbol-13-acetate (TPA)-responsive element (TRE), TGA G / C TCA (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 mega- karyocyte-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 (TGCTGA G / C TCA T / 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 sug- gested (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 Ј 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 Ј 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. 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 C α 3 Ј E and HS3, are identical to the MARE consensus sequence (Figure 1). It should be noted that C α 3 Ј 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. 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, involve- ment of Bach2 in the MARE-binding complex was confirmed using an anti-Bach2 monoclonal antibody (Figure 2C): addition of the Bach2 monoclonal antibody super- shifted the MARE-binding complex. These results estab- lished that the TRE within the 3 enhancers C 3 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. 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. 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 ϩ ) B cells isolated from bone marrow showed a high level of Bach2 expression. Mac-1 ϩ cells (mono-macrophage lineage) did not express Bach2. These results clearly established that Bach2 is a B-cell-restricted transcription factor. 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 ϩ cells into two populations, depending on the cell surface expression of IgM. The B220 ϩ /IgM – fraction contains pro- and pre-B cells, whereas the B220 ϩ /IgM ϩ 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 ϩ cells from spleen . As shown in Figure 3C, the B220 ϩ /IgM – fraction, which represents an early stage of B-cell lineage development, showed the highest level of expression of bach2 mRNA, whereas the B220 ϩ /IgM ϩ bone marrow cells and B220 ϩ spleen cells expressed less bach2 mRNA. The mitogen lipopolysaccha- ride (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 ϩ fractions, both of which express the pre-pro-B-cell-surface-marker-pattern like c-Kit ϩ , CD43-, HSA (CD24) – and IgM – (N.Yanai and M.Obinata, ...

Context 3

... the B220 ϩ /IgM ϩ bone marrow cells and B220 ϩ spleen cells expressed less bach2 mRNA. The mitogen lipopolysaccha- ride (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 ϩ fractions, both of which express the pre-pro-B-cell-surface-marker-pattern like c-Kit ϩ , 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. 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. To determine at which point Bach2 expression commences during the differentiation of B cells, we examined hematopoietic stem cells (c-Kit ϩ /Sca-1 ϩ /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 ϩ /Lin – cells, which include progenitor cells of various lineages as well as in lineage- markers-positive (Lin ϩ ) 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 ϩ /Lin – cells expressed Bach2 at low levels. Considering the fact that the Lin ϩ fraction contained differentiated cells of various lineages and that Bach2 expression in this fraction is restricted to the B220 ϩ cells, the expression level in the stem-cell fraction was estimated to be relatively low compared with expression in B220 cells in the Lin ϩ fraction. 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 ϩ cells, and that B220 – cells give rise to B220 ϩ 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 ϩ 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 . 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 C α 3 Ј 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). 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 μ g of reporter plasmid (1 μ g 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 (Bach2 ∆ zip), and hence could not form a heterodimer with a small Maf protein (Figure 6D). In this experiment, we used 0.1 μ g of Bach2-expression plasmid per transfection (0.9 μ g was used in Figure 6C). Both wild-type Bach2 and Bach2 ∆ zip repressed the reporter gene activity to 60% in the absence of MafK. However, Bach2 ∆ zip 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 Bach2 ∆ zip that was independent of dimer formation may be due to its interaction with other proteins through remaining regions such as the BTB domain. Such an interaction could inhibit the function of other transcription factors that are involved in enhancer activity of the HS1234. In order to examine roles of the MARE within HS3 in the context of the large cis -regulatory region, we deleted the MARE from the 6.6 kb HS1234 mini-enhancer ( ∆ MARE). As shown in Figure 6E, the deletion caused a modest decrease in the reporter gene expression (compare lanes 1 and 5). Co-expression of Bach2 and MafK still showed some synergistic repression of such a reporter gene activity, even though its extent was reduced. Based on these observations, we could draw two conclusions. First, in plasmacytoma cells, the enhancer function of the HS1234 is partly dependent on the MARE within the HS3. Secondly, the HS3 MARE is not the sole target of the Bach2–small-Maf heterodimer. Other functional MAREs appear to be present within the IgH 3 Ј enhancer regions (see Discussion). The results described in Figure 6D and E, taken together, strongly implicated Bach2 as a negative regulator of IgH 3 Ј enhancer that functions by forming a heterodimer with small Maf proteins. Since several BTB-domain-containing proteins have been shown to bind to co-repressors like SMRT, and because we had shown that Bach2 functions as a transcription repressor, we examined whether Bach2 interacts with SMRT in a yeast two-hybrid system. Bach2 was fused to the GAL4 DNA binding domain, and the fusion protein was then expressed in yeast cells. The Bach2 fusion protein did not activate the GAL4-dependent HIS3 reporter gene (Figure 7). However, when it was co-expressed together with SMRT–GAL4 activation domain fusion protein, the Bach2–GAL4 fusion activated the reporter gene, resulting in histidine autotrophy. In contrast to the Bach2 fusion, the Bach1 fusion did not show any interaction with SMRT, as evidenced by its failure to support yeast growth in the presence of the SMRT fusion. These results thus indicate that Bach2 but not Bach1 binds specifically to SMRT. The results of the RT–PCR assay and EMSA, shown in this study, have established that Bach2 functions in B cells as a major MARE-binding factor. In terms of cell differentiation, B cell is the second hematopoietic cell lineage in which partners of MARE-effectors have been identified, with a precedent being NF-E2 p45 in erythroid cells (Andrews et al. , 1993a,b; Igarashi et al. , 1994). This is somewhat surprising in view of our previous observation that, among various hematopoietic cell lines tested, Bach2 ...