Multivalent DNA Binding Complex Generated by Small Maf and Bach1 as a Possible Biochemical Basis for beta -Globin Locus Control Region Complex*

Kazuhiko IgarashiDagger §parallel , Hideto HoshinoDagger parallel , Akihiko MutoDagger parallel , Naruyoshi SuwabeDagger , Shinichi Nishikawa, Hiromitsu NakauchiDagger , and Masayuki YamamotoDagger

From the Dagger  Institute of Basic Medical Sciences and Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tenno-dai 1-1-1, Tsukuba 305, Japan and the  Kyoto University Faculty of Medicine, Sakyo-ku, Kyoto 606, Japan

    ABSTRACT
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Abstract
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Procedures
Results
Discussion
References

The human beta -globin locus control region (LCR) is required to properly regulate chromatin domain opening, replication timing, and globin gene activation. The LCR contains multiple NF-E2 sites (Maf recognition elements, MAREs) that allow the binding of various basic leucine zipper (bZip) proteins like p45 NF-E2, Nrf1, Nrf2, Bach1, and Bach2, in some cases as obligate heterodimers with a small Maf protein. In addition to the bZip domain, the Bach proteins bear a BTB/POZ domain, which has been implicated in the regulation of chromatin structure. We show here that Bach1 is highly expressed in hematopoietic cells and constitutes one of the two MARE-binding activities in murine erythroleukemic (MEL) cells. We further demonstrate that Bach1/MafK heterodimers interact with each other through the BTB domain, generating a multimeric and multivalent DNA binding complex. These results strongly implicate Bach1/MafK heterodimer as an architectural transcription factor that mediates interactions among multiple MAREs. Such a factor could then provide a model for assembly of the theoretical beta -globin LCR "holocomplex." Other BTB domain proteins have already been demonstrated to be involved in remodeling chromatin, and thus this class of proteins likely promote the formation of nucleoprotein complexes required to establish the architecture of regulatory domains.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mammalian beta -globin loci contain a group of genes whose expression is regulated by the locus control region (LCR),1 which is located far 5' to the globin genes. The beta -globin LCR is composed of five DNase I-hypersensitive sites (HS-1 to HS-5) and is able to direct copy number-dependent and position-independent expression of the globin genes in transgenic mice (1-3). This remarkable feature of the LCR is based on its potential to provide an open, accessible chromatin configuration over the entire globin gene locus (4). A variety of cis acting regulatory DNA elements within the core regions of individual HS sites act in concert in contributing to the activities attributed to the LCR (5-7). These DNA elements, which are present in redundant combinations within each of the core elements of the LCR, are bound by both ubiquitous as well as erythroid (or hematopoietic) cell-restricted transcription factors (6, 8, 9). One of these cis acting regulatory elements, present in HS-2, -3, and -4, is characterized by a consensus binding site for the transcription factor NF-E2 (10, 11). This DNA motif is closely related to the phorbol 12-O-tetradecanoate 13-acetate-responsive element (TRE) and has also been termed a Maf recognition element (MARE; Refs. 12 and 13) to avoid confusion of the cis element with the NF-E2 (p45 + small Maf) protein. Two tandemly arranged MAREs strongly contribute to the overall activity of HS-2 (5, 9, 14-17). The functional importance of the MARE within HS-3 was also demonstrated previously (7, 18). In addition, a specific combination of a MARE and a binding site for GATA factors within HS4 is neccessary to induce DNase I-hypersensitivity (19). These observations suggest an important role for MARE-binding factors in execution of beta -globin LCR function.

Hematopoietic transcription factor NF-E2 is a heterodimeric factor that consists of a large and a small basic leucine zipper (bZip) subunit and binds to the MAREs (10, 20-22). The large subunit is p45 which is expressed in hematopoietic cells (10, 22, 23). The partner molecule of p45 is a small Maf family protein, MafK (also known as p18), MafG, or MafF (20, 21, 24-28). In addition to p45, several other bZip proteins like Nrf1 (LCR-F1/TCF-11; Refs. 29-31), Nrf2 (ECH; Refs. 32-34), Bach1, and Bach2 (35) have been shown to bind to the MARE in vitro.

Several models have been postulated to account for the multiple and complex activities of the beta -globin LCR, and observations from various experiments are consistent with the hypothesis that the LCR forms a single, synergistic, and dynamic entity which has been described as the holocomplex (36-38). While such a model implicitly assumes that DNA-binding proteins interact with one another to establish the underpinning LCR architecture, little is known about protein/protein interactions between LCR-binding factors. Known examples include interactions of GATA-1 with itself, Sp-1, and EKLF (39-41). In this context, the Bach family proteins may be of particular interest as MARE-binding proteins since they bear a BTB domain (35). BTB domains are found in a variety of different DNA-binding proteins but principally those of the zinc-finger class (42). The biochemical function of this domain is to mediate the formation of homo- and hetero-oligomers (43, 44), and while some of the proteins containing a BTB domain, for example those encoded by trithorax-like (GAGA factor) and E(var)3-93D/mod(mdg4), are clearly involved in the regulation of chromatin structure (45-48), the function of the BTB domain in this process is still unknown.

In light of these facts, we explored the possibility that Bach1 exerts a unique function as a MARE-binding protein. In this report, we first show a close association of Bach1 expression and hematopoiesis and the presence of MARE-binding activity in MEL cells that is composed of Bach1/small Maf heterodimer. We further demonstrate that the presence of the BTB domain allows the Bach1/MafK heterodimer to interact with each other, generating a multimeric and multivalent DNA binding complex. These results strongly suggest that a multivalent DNA binding complex consisting of Bach1/small Maf protein multimers could function as an architecural component of regulatory domains such as a beta -globin LCR by binding to its multiple MAREs.

    EXPERIMENTAL PROCEDURES
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References

RT-PCR Analysis-- Bone marrow mononuclear cells were obtained from 6-week-old female C57BL/6 mice and fractionated as described previously (26). Total RNA was isolated from the sorted bone marrow cells (105 cells/purification) and used for cDNA synthesis. PCR was carried out using primers to amplify beta -actin, hypoxanthine phosphoribosyltransferase (HPRT), and bach1 (5'-CGGATAATTTCGCTCTCACG-3' and 5'-TAAAAAGGAAAGCGGGCAGTCGGAG-3') cDNAs, the amplicon from bach1 genomic DNA being much larger because of the presence of an intron.2 All PCR reactions were carried out for 26 and 30 cycles to ensure linearity of amplification. The products were resolved on 2% agarose gels, transferred onto ZetaProbe membranes, and hybridized with radiolabeled DNA fragments specific for each target cDNA.

ES Cell Differentiation-- In vitro differentiation of ES cells to hematopoietic cells was carried out according to Nakano's method using OP9 stromal cell line (49). Flk1+ cells were collected using the anti-Flk1 antibody (50).

Cell Extracts-- Whole cell extracts were prepared from MEL B8 cells or DS19 cells as described previously (51). Where indicated, 0.5% Nonidet P-40 was included in the extraction buffer. MEL B8 cells were treated with 1.5% Me2SO for 4 days to induce differentiation.

Plasmids-- To construct the Bach1-expression plasmid, bach1 cDNA (35) was first modified as follows. A BspHI site was introduced into the translation initiation site by site-directed mutagenesis (5'-GGATGTCT-3' to 5'-TCATGACT-3', the translation initiation codon is underlined). This treatment changed the second codon from serine to threonine. In addition, the translation stop codon was changed to a HindIII site (5'-GACGAGTAAACC-3' to 5'-GACGAGAAGCTT-3', the translation stop codon in underlined). The BspHI (blunted)/HindIII fragment was isolated and inserted into pET21b(+) (Novagen) between NdeI (blunted) and HindIII. To construct a plasmid that expresses Bach1 without the BTB domain, a EcoRI (blunted)/HindIII fragment that encodes codons 173 to 739 was isolated from the above plasmid and inserted between the BamHI (blunted) and HindIII sites of pET21b(+). The resulting plasmid, pETBach1Delta BTB, expresses a Bach1 variant (Bach1Delta BTB), lacking the amino-terminal 172 amino acids including the BTB domain, tagged with the T7 epitope at the amino terminus. The MafK-expression plasmid was constructed by inserting FLAG epitope-tagged murine mafK cDNA (31) into the NdeI (blunted) site of pET21b(+).

Expression plasmids for GAL4 fusions of Bach1 and p45 were constructed as follows using pGBT9 (CLONTECH). The Bach1 cDNA was isolated from the pA1 (35) as a SalI fragment and inserted in the BamHI site of pGBT9, after filling-in the relevant DNA ends. The entire open reading frame was fused to the GAL4 DNA binding domain by this treatment. The p45 cDNA was isolated as an NcoI (overlapping with the start methionine codon)/SalI fragment from the engineered plasmid (21) and was inserted in the EcoRI site of pGBT9 after filling in the relevant DNA ends. This treatment fused the amino-terminal 272 amino acid residues of p45 including the transactivation domain. These GAL4 fusion cDNAs were isolated from the respective plasmids as HindIII fragments and transferred to the mammalian expression plasmid pEF-BssHII (27).

Recombinant Proteins-- Recombinant Bach1 proteins were expressed in Escherichia coli BL21(DE3) and purified using nickel affinity resin (Invitrogen) in the presence of 6 M urea. MafK was partially purified by isolating inclusion bodies. Each protein was refolded as described previously (52). Purity of the proteins was over 90% for Bach1 and Bach1Delta BTB and roughly 20% for MafK. Maltose-binding protein fusions of Bach1 and p45 were described previously (21, 35).

Footprinting and EMSA-- DNA binding reactions were carried out at 37 °C for 10 min under the conditions described previously (53), with indicated amounts of proteins in figure legends. For footprinting experiments, binding reactions incorporated treatment with bovine pancreas DNase I (Amersham Pharmacia Biotech) after addition of CaCl2 and MgCl2 to 5 and 2.5 mM, respectively, and products were separated on sequencing gels. To compare dissociation of the protein-DNA complexes, a master binding reaction (equivalent to five reactions) was prepared as above, and binding was allowed for 10 min. Then excess amount of oligonucleotide competitor containing the tandem MAREs of HS-2 (21) was added to the master reaction, and incubation was continued. At various time points, aliquots were removed and processed for DNase I digestion. For EMSA, separation was on 1% agarose (Fig. 6) or 4% polyacrylamide (Figs. 3, 4, and 9) gels. Antisera were included in the binding reactions at 10-20-fold dilution with incubation for 30 min on ice before addition of the probe DNA. The 240-bp HS-2 DNA (26) was labeled with 32P at the 3'-end of the bottom strand (see Fig. 5A) by a filling-in reaction with the Klenow fragment. The 32P-labeled Cbeta E oligonucleotide probe (21) was derived from the 3' enhancer of chicken beta -globin gene.

Co-precipitation Assay-- Binding reactions were carried out as EMSA with 32P-labeled HS-2 oligonucleotide (21) (0.5 ng) in the presence of either unlabeled Cbeta E or biotinylated Cbeta E oligonucleotide (1 ng). After DNA binding reactions, streptavidine beads (Dynal) were added, and the mixtures were incubated for 20 min at room temperature. Streptavidine beads were recovered from the binding reactions using a magnet and washed two times with the binding buffer supplemented with 0.1 M NaCl. The amount of recovered radioactivity was then quantified. Radioactivity recovered with unlabeled Cbeta E oligonucleotide served as the background control. Amounts of proteins used were as follows: Bach1, 150 ng; Bach1Delta BTB, 150 ng; and MafK, 200 ng.

Chemical Cross-linking Analysis-- Equimolar amounts of Bach1 (1.5 µg) and Bach1Delta BTB (1.3 µg) were incubated with 0.005 or 0.02% of glutaraldehyde on ice for 10 min in 200 µl of phospate-buffered saline containing 1 mM dithiothreitol and 0.05% Tween 20. After adding Tris-HCl, pH 7.0, to 100 mM, the reaction mixtures were concentrated with microcon-10 (Amicon), electrophoresed on sodium dodecyl sulfate, 7.5% polyacrylamide gel, and transferred onto PVDF membranes (Pall). Proteins were detected with an antibody against hexahistidine (Invitrogen).

Transfection Assay-- Transfection of MEL DS19 cells was carried out using DOTAP reagent (Boehringer Mannheim). The luciferase reporter plasmid carried the herpes simplex virus thymidine kinase promoter and five copies of GAL4 sites.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Bach1 Expression during Hematopoiesis-- To examine Bach1 expression in normal hematopoietic cells, we isolated RNA from various hematopoietic organs as well as the brains of adult mice. RNA blotting analysis with total RNA preparations clearly indicated that bach1 is expressed in bone marrow cells and in thymus, as well as in the livers of embryos at day 13.5 post-coitus (Fig. 1A), suggesting a link with active hematopoiesis. To determine the hematopoietic lineage specificity of bach1 expression, we separated hematopoietic cells into various fractions by cell sorting (using antibodies that recognize a variety of hematopoietic lineage markers) and carried out semi-quantitative RT-PCR assays on RNA recovered from these purified cells (Fig. 1B). bach1 was found to be expressed in Ter-119 (erythroblast and later stages), Mac-1 (mono-macrophage), Gr-1 (granulocyte), B220 (B cells), and CD4/8-double positive (T cells) fractions. Interestingly, bach1 was also expressed in the pure stem cell fraction (Sca-1/c-Kit double-positive, lineage marker-negative, and CD34-negative/low), indicating that its expression in hematopoietic cells commences at an early stage of differentiation and shows little lineage-restriction thereafter.


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Fig. 1.   A, expression of bach1 mRNA in hematopoietic organs. Total RNA (20 µg) isolated from livers of day 13.5 post-coitus embryos (lane 1) or from tissues of 6-week-old Balb/c mice (lanes 2-5) were hybridized with the bach1-specific DNA probe after separation on a formaldehyde-agarose gel and transfer onto a nylon membrane. Integrity of RNA was verified by hybridization with a beta -actin probe. Positions of 28 and 18 S RNAs are indicated with bars on the left. Relative expression levels normalized to beta -actin mRNA are as follows: fetal liver, 3.0; bone marrow, 2.6; spleen, 0.4; thymus, 4.0; and brain, 1. B, expression of bach1 mRNA in normal hematopoietic cells. Hematopoietic cells from mouse bone marrow, thymus, and spleen were fractionated depending on the surface marker expression. The bach1-expression in each fraction was determined by RT-PCR analysis. Sca-1/c-Kit-double positive and lineage-negative, B220, Gr-1, Mac-1, and TER-119 fractions were from bone marrow, CD4/8-double positive fraction was from thymus, and Thy-1.2 fraction was from spleen. Linearity of amplification was ensured by comparing products from at least two different PCR cycles. Relative expression levels normalized to beta -actin mRNA are as follows: B220, 2.2; Gr-1, 2.3; Mac-1, 3.1; TER-119, 3.0; Thy-1.2, 1.0; and CD4/8, 3.2.

In vitro differentiation of embryonic stem (ES) cells recapitulates in vivo hematopoiesis and hence provides an excellent system to analyze transcription factor expression during development of hematopoietic cells. One of such systems utilizes a specific stroma cell and can induce efficient hematopoietic cell differentiation in a timed manner, including erythroid lineage. Analysis of Bach1 expression in this system revealed striking induction of Bach1 upon hematopoietic differentiation (Fig. 2A). It should be noted that induction of Bach1 preceded both primitive (epsilon y) and definitive (beta -major) globin gene expression. Fetal liver kinase-1 (Flk1) is a marker for the earliest stage of hematopoiesis in the ES differentiation system as well as in vivo (50, 54), and Bach1 expression was highly induced in the Flk1+ fraction within the differentiating ES cells (Fig. 2B). Taken together, these results indicated that Bach1 expression commences from the earliest phase of hematopoietic cell development. Because p45 NF-E2 expression is confined to more differentiated, lineage marker-positive cells,3 two of the MARE effectors, Bach1 and p45 NF-E2, appear to play distinct roles during hematopoiesis.


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Fig. 2.   Expression of Bach1 during in vitro hematopoietic differentiation of ES cells. A, ES cells were induced to differentiate along hematopoietic lineage on OP9 cells, and RNA samples at indicated time point (Day) were analyzed for expression of Bach1, globin genes, and HPRT by RT-PCR analysis. B, Flk1+ cells were isolated from ES cells after 4 days of induction of differentiation. Bach1 expression in Flk1+ cells was compared with that of uninduced ES cells.

Bach1·Small Maf Complexes in MEL Cells-- Even though various recombinant bZip proteins can bind to MAREs as heterodimers with MafK or other small Maf proteins, the p45/small Maf heterodimer (NF-E2) is the only combination that has been unequivocally demonstrated to be present in hematopoietic cells (20). To examine cells of hematopoietic origin for the presence of a Bach1·small Maf complex, we carried out EMSA using protein extracts derived from MEL cells (representing definitive erythroid cells) and various antibodies. Two major MARE-binding activities were detected within whole cell extracts prepared from MEL B8 cells (Fig. 3A, lane 2). The faster mobility band increased after induction of erythroid differentiation (by Me2SO, Fig. 3A, lane 3), and addition of an anti-p45 antiserum inhibited formation of only this faster mobility band while the slower band was unaffected (lane 7). On the other hand, addition of an anti-Bach antiserum inhibited formation of only the more slowly migrating band (Fig. 3A, lane 5). The anti-p45 and anti-Bach antisera did not cross-react with the slower or faster bands, respectively, thus verifying their respective specificities. Since MEL cells express Bach1, but not Bach2 (35), Bach2 was excluded as a possible component of the slower band. Addition of an anti-small Maf antiserum inhibited the formation of both bands (lane 6), indicating the presence of MafK (or some other related protein like MafG or MafF) in both complexes. Similar MARE-binding activity, reacting with the anti-Bach antiserum, was also detected in another MEL line DS19 cells, albeit relative amounts of the lower and upper bands differed (Fig. 3B).


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Fig. 3.   A, MARE-binding activities within MEL cells. EMSA was carried out with whole cell extracts that were prepared in the absence of 0.5% Nonidet P-40 from MEL cells and with the Cbeta E probe. MEL cells were treated with (lanes 3-8) or without (lane 2) Me2SO for 4 days. Preimmune serum (PI) and antisera were included in the binding reaction as indicated. p45- and Bach1-containing complexes are indicated with arrows. Nonspecific binding complexes, as defined by competition assay with unlabeled probe DNA, are indicated with arrowheads. B, comparison of MARE-binding activities between MEL B8 and DS19 cells. EMSA was carried out as above with extracts from B8 (lane 1) or DS19 (lanes 2 and 3) cells in the absence (lanes 1 and 2) or presence (lane 3) of anti-Bach antiserum. MARE-binding complexes are indicated as above. C, effect of anti-Fos antibodies. EMSA was carried out with the uninduced MEL B8 cell extract and the Cbeta E probe. Anti-Bach antiserum (lane 3) and anti-Fos monoclonal antibodies (4-10G and 2G9C3 in lanes 4 and 5, respectively; both from Santa Cruz Biotechnology) were included in the binding reactions.

To exclude the possibility that the slower band in the EMSA experiments might also be derived from other p45-related proteins or from the binding of AP-1 complexes, we examined the effect of adding an anti-ECH antibody (33), which recognizes both p45 and Nrf2 (data not shown), as well as two anti-Fos antibodies, which react with c-Fos, FosB, Fra-1, and Fra-2. Since the anti-ECH antibody inhibited only the faster mobility band (Fig. 3A, lane 8) while the anti-Fos antibodies did not react with either (Fig. 3C), we conclude that the slower band does not include either Nrf2 or AP-1 constituents. These results taken together indicate that the faster and slower mobility bands were constituted of p45/small Maf (NF-E2) and Bach1/small Maf heterodimers, respectively, and that furthermore, somewhat to our surprise, very little, if any, other protein species exist in MEL B8 cells that bind to the NF-E2 element.

Competition between Bach1 and p45 NF-E2 for Small Maf Proteins and MARE-- The expression profiles of Bach1 and p45 NF-E2 during in vitro differentiation of ES cells and the presence of their MARE-binding activities in MEL cells raised a further provocative question. Do these two proteins compete with one another for small Maf heterodimerization, and if so, do the two heterodimers efficiently compete for NF-E2 binding sites in DNA? To address these questions and to examine the relative affinities of the heterodimeric species for DNA, we titrated recombinant Bach1 and p45 proteins in the presence of a limiting amount of MafK with an excess of MARE probe DNA. For this competition assay, we employed a Bach1 fusion protein that lacks the BTB domain in order to avoid complications that could occur because of Bach1-mediated oligomer formation (see below).

As shown in Fig. 4, addition of recombinant p45 to the mixture of proteins and DNA sites efficiently inhibited Bach1 heterodimer formation. A roughly equal amount of p45 protein was required to reduce the relative abundance of the Bach1·MafK complex by approximately 50%. Hence, Bach1 and p45 appear to have similar affinities for MafK. In the presence of 40 ng of both proteins, the amount of p45·MafK complex was roughly 2-fold greater than that of the Bach1·MafK complex. Hence, each heterodimer possesses comparable binding affinity for the MARE, with perhaps slightly higher affinity of p45·MafK. These results imply that inside cells, Bach1 and p45 NF-E2 probably compete both for small Maf partner molecules as well as for NF-E2 binding sites within a similar, narrow concentration range.


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Fig. 4.   Competitive DNA binding by Bach1 and p45. EMSA was carried out in the presence or absence of partially purified MafK, 40 ng of maltose binding protein (MBP)/Bach1 fusion, and increasing amounts of MBP/p45 fusion, as indicated at the top. Bach1 and p45 fusion proteins possess similar molecular mass (91 and 87 kDa, respectively). Under the conditions, MafK is relatively limiting, whereas probe MARE DNA is in excess. Relative levels of Bach1·MafK complex are shown below the lanes.

BTB Domain Modulates DNA Binding-- Establishing that Bach1 could be one of the effectors of MAREs within the beta -globin LCR, we liked to address possible unique Bach1 function due to its BTB domain. We expressed full-length Bach1 as well as a Bach1 derivative lacking the BTB domain (Bach1Delta BTB; Fig. 5A) and MafK in E. coli. The probe DNA was a 240-bp fragment from the HS2 of the beta -globin LCR that contains tandem MAREs (Fig. 5A). In DNase I protection assays, purified Bach1/MafK or Bach1Delta BTB/MafK heterodimers showed virtually identical binding to the sites (Fig. 5B). No other region of the probe DNA was protected from DNase I digestion (Fig. 5B and data not shown). To compare stability, DNA-protein complexes were challenged with excess unlabeled competitor DNA. As shown in Fig. 5, C and D, Bach1Delta BTB dissociated more rapidly from the tandem MARE than Bach1 did. Hence, binding of Bach1 heterodimer to the tandem MAREs could be stabilized because of its BTB domain.


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Fig. 5.   BTB domain modulates Bach1 DNA binding. A, schematic representation of the Bach1 proteins and the 240-bp HS-2 DNA. B, DNase I protection assay with the HS-2 DNA. Increasing amounts of Bach1 or Bach1Delta BTB (10, 40, and 150 ng) were incubated with the probe DNA in the presence of crude recombinant MafK (200 ng). The position of the tandem MAREs is indicated with bar and arrows, respectively. C, dissociation of the DNA-protein complexes was compared by incubating for the indicated duration with an excess amount of competitor DNA. Proteins were Bach1 plus MafK (lanes 2, 4, and 6) and Bach1Delta BTB plus MafK (lanes 3, 5, and 7). Position of the MAREs is indicated with a bar. D, gel scan of cleavage pattern. Protection patterns at each time point in panel C were scanned using BioImage Analyzer BAS1500 and compared. Patterns obtained with Bach1/MafK and Bach1Delta BTB/MafK are shown in thick and thin lines, respectively.

We next examined the same 240-bp DNA in an EMSA in order to compare the nature of the nucleoprotein complexes formed. As shown in Fig. 6, MafK bound to the probe as a homodimer (lane 2) while neither Bach1 nor Bach1Delta BTB alone exhibited binding (lanes 11 and 12). In the presence of MafK, Bach1Delta BTB formed two distinct retarded bands (Fig. 6, bands c and d). Band d became more intense with increasing amounts of protein, suggesting that it represented a complex in which both of the MAREs in the probe were occupied. Bach1 also generated two distinct bands under the same conditions (bands a and b). The mobility of band a was slightly retarded relative to that of the d band of Bach1Delta BTB, suggesting that in this case each of the MAREs was occupied by a Bach1/MafK heterodimer. Hence, Bach1/MafK appears to bind more stably to the two sites than Bach1Delta BTB/MafK. The band b migrated extremely slowly compared with other bands and moved diffusely, indicating the presence of heterogeneous complexes containing multiple Bach1 and MafK species. These results support the contention that Bach1/MafK heterodimers interact with each other through the BTB domain, generating oligo-heterodimers.


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Fig. 6.   Formation of DNA-binding oligomers by Bach1. EMSA was carried out using 1.0% agarose gel. The probe DNA, Bach1, Bach1Delta BTB, and MafK are 155, 80, 61, and 18 kDa, respectively, and the mobilities of Bach1 and Bach1Delta BTB complexes were expected to be similar if the stoichiometry was the same. Nucleoprotein complexes are indicated with arrows as referred to in the text. Amounts of proteins were as follows: MafK, 50 ng (lanes 2-10); Bach1 (lanes 3-6) and Bach1Delta BTB (lanes 7-10), 5, 10, 20, or 40 ng. 40 ng of Bach1 or Bach1Delta BTB were incubated with the probe in the absence of MafK in lanes 11 and 12, respectively.

To further examine the nature of possible multimeric interactions that might be mediated through the BTB domain of Bach1, we employed a co-precipitation assay with two distinct oligonucleotide DNA probes (Fig. 7A) derived from either the 3' enhancer of the chicken beta -globin gene (containing a single MARE, labeled with biotin) or the mouse beta -globin HS2 (containing double MAREs, labeled with 32P). The probes were mixed with various combinations of proteins, and the biotinylated probe was then purified from the mixtures using streptavidin beads. As shown in Fig. 7B, a substantial proportion of the 32P-labeled probe was also recovered in the presence of both Bach1 and MafK. Recovery of the 32P-labeled probe reflected specific DNA binding by the Bach1/MafK heterodimer because it was not observed with either of the two individual proteins alone. We failed to co-precipitate the 32P-labeled probe using a mixture of Bach1Delta BTB plus MafK even though the Bach1Delta BTB/MafK heterodimer binds to the MAREs with the same apparent specificity and affinity as Bach1/MafK (see above, and data not shown). The low recovery (6%) of the 32P-labeled probe in the presence of saturating amount of proteins may be due to loss during the extensive washing procedures employed, to low efficiency of biotinylation of the other probe DNA, or to steric hindrance of biotin/streptavidine interaction by the hetero-oligomers. Taken together with the results of the EMSA, these findings clearly established that oligomeric heterodimers of Bach1/MafK are generated through the BTB domain and furthermore, that these oligomeric complexes can mediate the interaction of MAREs present on different DNA molecules.


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Fig. 7.   BTB domain tethering of the bZip heterodimer. A, schematic representation of the co-precipitation experiment. B, results of the co-precipitation experiment. Values are the means of two assays. Amounts of proteins used were: Bach1 and Bach1Delta BTB, 150 ng; MafK, 200 ng. Radioactivity recovered in the presence of both Bach1 and MafK corresponded to 6% of the input 32P-labeled HS-2 oligo DNA.

BTB Domain and Oligomer Formation-- We next carried out a chemical cross-linking assay to test whether interactions through the BTB domain depend on DNA binding and to attempt to reveal the stoichiometry of the BTB-mediated oligomer formation. As shown in Fig. 8, cross-linking of Bach1 resulted in at least three bands in addition to the monomer band (lanes 1-3). Comparison of the relative mobilities of these bands indicated that the second fastest band migrated as a dimer. The more slowly migrating bands were thus concluded to be due to formation of trimer or higher multimeric Bach1 species. The results shown underestimate the amounts of cross-linked products since the larger molecular mass complexes transfer less well onto membranes. Formation of oligomers was dependent on the BTB domain, because its deletion virtually abolished formation of cross-linked products (lanes 4-6), but it was independent of DNA binding.


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Fig. 8.   BTB domain-dependent oligomer formation by Bach1. Bach1 or Bach1Delta BTB were incubated without (lanes 1 and 4) or with 0.005% (lanes 2 and 5) or 0.02% (lanes 3 and 6) glutaraldehyde (GA) and analyzed by immunoblotting. Positions of markers (220 and 97 kDa) are indicated with bars on the left, and cross-linked products are indicated with dots. The apparent mobilities of Bach1 and Bach1Delta BTB in the SDS-polyacrylamide gel are slower than expected from their calculated molecular masses. Such aberrant mobilities are often observed for various bZip proteins (e.g. Ref. 30).

Multimeric DNA Binding Complexes in MEL Cells-- The above results indicated that Bach1 and small Maf generate multimeric DNA binding complex which migrated more slowly in EMSA. Accordingly, extraction of proteins from MEL cells in the presence of 0.5% Nonidet P-40 yielded another MARE-binding complex that showed much slower mobility upon EMSA analysis (Fig. 9, upper panel, large arrow). Because it was also reactive with the anti-Bach and anti-MafK antisera, this band may represent oligo-heterodimers of Bach1·small Maf. The abundance of this very low mobility complex was reduced in extracts prepared from Me2SO-treated MEL cells (compare lanes 6 and 7).


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Fig. 9.   Bach1 oligomer in MEL cells. Whole cell extracts were prepared in the presence of 0.5% Nonidet P-40 from uninduced (lanes 2-6) or Me2SO-induced MEL cells (lane 7) and analyzed as in Fig. 3. Antisera were included in the binding reactions as indicated. The slowly moving Bach1 complex is indicated with a large arrow. Upon shorter exposure, shown as the lower panel, Bach1- and p45-containing complexes showed a similar pattern to that in Fig. 3A. Nonspecific complexes are indicated with arrow heads.

Bach1 Is Not a Conventional Transactivator-- We showed previously that Bach1 functions as a transcriptional repressor through MARE in transient reporter assays in QT6 fibroblasts (35). To reveal intrinsic regulatory functions of Bach1 within erythroid environment, we examined activity of Bach1/GAL4 DNA binding domain fusion in transient transfection asssays using the MEL cells. As shown in Fig. 10, Bach1 did not activate the reporter gene activity. Rather the fusion protein repressed the GAL4-dependent promoter activity to 50%. NF-E2 p45 is a transcriptional activator, and as expected, a p45/GAL4 fusion protein activated the reporter gene activity about 20-fold. These results led us to conclude that Bach1 lacks any transactivation domain that can be transferable onto the GAL4 DNA binding domain, and they suggested a role for Bach1 other than conventional transcription activators.


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Fig. 10.   Bach1 lacks bone fide transcription activation activity. MEL cells were transfected with the plasmids that express GAL4 DNA binding domain (lane 1), GAL4/Bach1 fusion (lanes 2 and 3), or GAL4/p45 fusion (lane 4), and expression of the luciferase reporter gene activity was determined. Amounts of the expression plasmids were either 0.5 µg (lanes 1, 2, and 4) or 1.0 µg (lane 3).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The expression profile of Bach1 is in clear contrast to that of p45 NF-E2. We reported previously that Bach1 is expressed at a low level (detectable using poly(A)+ RNA in Northern blot) in various tissues in adult mice. Upon detailed analysis using tissues that were not examined previously, we now noticed that tissues like bone marrow and 13.5-days post-coitus fetal livers express Bach1 very abundantly, readily detectable in Northern blot with total RNA (Fig. 1). In addition to its abundant expression in hematopoietic organs, Bach1 expression is induced in the Flk1+ cells during hematopoietic cell differentiation of ES cells in vitro (Fig. 2). The Flk1+ cells are the most early hematopoietic precursors identified thus far (50, 54). Taken together, these observations suggest that Bach1 is one of the transcription factors that function during early phase of hematopoiesis. The results, however, do not rule out functions of Bach1 in non-hematopoietic cells.

The band that migrates more slowly than NF-E2 in EMSA with MEL cell extracts has been assumed in the literature to be AP-1 (55), but unfortunately, there is no solid experimental evidence for such an assignment. Insofar as we can determine, no one (with the exception of this study) has developed an antibody that specifically recognizes this presumed "AP-1" band in MEL cell extracts. In contrast, we provide rather compelling evidence that the presumed AP-1 band is actually a Bach1·small Maf complex since it reacts with both anti-MafK and anti-Bach antibodies, but not with anti-Fos or anti-p45 antibodies (Fig. 3). Furthermore, no one has demonstrated the presence of DNA binding complexes containing either Nrf1 or Nrf2 in either MEL or K562 cell extracts. All of the binding experiments reported for those two proteins were carried out using either recombinant or in vitro translated proteins (29, 30, 33, 34). Hence, it may also not be surprising that we were unable to detect binding complexes containing either of these other bZip proteins. Taken together, the present data indicated that, in addition to NF-E2 (p45 + small Maf) which has been shown previously to be present in MEL cells as a MARE-binding activity (10), Bach1 is a second partner for small Maf proteins in MEL B8 cells. On the other hand, MEL DS19 cells contained less Bach1 complex as compared with B8 cells (Fig. 3B). Hence amounts of Bach1 complex appear to differ significantly among various MEL cell lines. These observations might be explained by different differentiation stages at which the two MEL cell lines are stalled. Considering the fact that Bach1 expression commences at an earlier stage than p45 NF-E2, B8 cells might represent more immature erythroid cells. In any case, available data suggest strongly that Bach1 is one of the MARE effector molecules within erythroid cells and their progenitors.

The presence of Bach1 as a MARE-binding complex is intriguing in light of previous observations regarding other LCR/MARE-binding proteins. Among the MARE-binding proteins, p45 is known to disrupt nucleosomal structures upon binding to a reconstituted chromatin template in vitro (56). Such a unique activity of p45 may reflect its role in LCR function. However, neither p45, Nrf1, or Nrf2 are essential for activation of beta -globin gene expression nor are they required for erythroid cell differentiation (57-59). These observations raise an interesting question of whether Bach1 plays some unique and/or redundant role in LCR functions. One feature of Bach1 function is its site-specific DNA binding activity. The present results of footprinting analysis, EMSA, co-precipitation assays, and cross-linking experiments (Figs. 5-8) are all consistent with the hypothesis that two distinct protein/protein interactions of Bach1, separately mediated by the BTB and leucine zipper domains, lead to the formation of a multimeric and multivalent DNA binding complex with the ability to bind multiple MAREs simultaneously. Such a complex may thus facilitate the formation of the postulated beta -globin LCR holocomplex (36-38) by structurally connecting the MAREs present in three core LCR hypersensitive sites, HS2, -3, and -4. It should be noted that we failed to identify a transcription activation domain within Bach1 when fused to the GAL4 DNA binding domain (Fig. 10), further corroborating that Bach1 does not primarily function as a bona fide transcriptional activator protein, but might rather serve as an architectural component of regulatory domains.

One of the most striking features of the LCR is that the DNase I hypersensitive sites are formed before induction of globin gene expression. HS-2 region of the beta -globin LCR exhibits DNase I hypersensitivity in multilineage progenitors, suggesting that the altered structure of the LCR is initiated before erythroid lineage commitment (60). Two observations in this study suggest that Bach1 may be involved in LCR function prior to overt beta -globin gene expression. First, its expression commences at an early stage of differentiation of hematopoietic cells and is not restricted to erythroid cells (Figs. 1 and 2). Second, two Bach1 DNA binding complexes are either down-regulated or unchanged during Me2SO-induced MEL cell differentiation (Figs. 3 and 9); p45 NF-E2 on the other hand is induced by Me2SO treatment. Hence, it appears that Bach1 has no direct role in globin gene activation per se. It is possible that Bach1 and p45 NF-E2 play distinct roles during erythroid differentiation: Bach1 may participate in the assembly of the LCR complex during early stages of hematopoietic cell differentiation, whereas p45 and/or other p45-related factors might contribute to globin gene activation per se at later stages. In other words, Bach1 may be involved in preparing the LCR for being activated as an enhancer at later stages. Because Bach1 and NF-E2 heterodimers effectively compete for MAREs (Fig. 4), induction of NF-E2 p45 DNA binding activity during erythroid cell differentiation may cause a shift in the equilibrium of effector molecules of MAREs.

One of the interesting aspects of BTB domain-containing proteins is that at least some of these proteins have been shown to be involved in the regulation of chromatin structure (42). It was demonstrated previously that BTB domains in DNA-binding proteins inhibit DNA binding in cis (43). Hence, it has been assumed that the formation of a multimeric complex by BTB domains and DNA binding by BTB domain proteins are mutually exclusive (42, 43), thus questioning the roles BTB domains might play in regulating chromatin structure. The results presented here are apparently inconsistent with these previous observations since we observed formation of multimeric complexes bound to DNA. The differences may reflect properties of individual BTB domains and DNA binding domains. Alternatively, complexes formed through BTB domains may recognize specific DNA sequences, as suggested previously (43). In view of our results, however, it is likely that one of the functions of BTB domains in DNA-binding proteins is to mediate the interaction of widely separated cis-regulatory domains without inhibiting DNA binding. Furthermore, some BTB domains show heterotypic interaction, forming various hetero-oligomers (43). Hence, it is likely that BTB domain proteins, with or without DNA binding domains, interact with one another through the BTB domain, generating diverse multimeric and multivalent DNA binding complexes. Generation of such complexes may be responsible for regulating chromatin structure, as might be the case for BTB containing proteins like the GAGA factor, a product of trithorax-like gene, and products of Enhancer of variegation gene E(var)3-93D/(mod)mdg4.

In conclusion, this study demonstrates that the formation of a multivalent MARE-binding complex is mediated by the BTB and bZip domains of Bach1 in conjunction with MafK. Such multivalent complexes may promote the formation of nucleoprotein complexes required to establish the architecture of regulatory domains like the human beta -globin LCR.

    ACKNOWLEDGEMENTS

We thank S. Hirose, A. Ishihama, H. Ueda, T. Ohta, S. Miyoshi, Y. Matsuzaki, K. Nagata, and S. Mizuno for various suggestions, discussions, and reagents. Further thanks go to N. Minegishi for providing cDNA of ES cells and C. Yoshida, M. Nishizawa, J. Bungert, and J. D. Engel for stimulating discussions and suggestions.

    FOOTNOTES

* This work was supported by the International Scientific Research Program and grants-in-aid from the Ministry of Education, Science, Sport, and Culture of Japan, and grants from the Japanese Society for the Promotion of Science (RFTF), the Mochida Memorial Foundation, the Ciba-Geigy Foundation Japan, and the Naito Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 81-22-717-8086; Fax: 81-22-717-8090.

parallel Present address: Dept. of Biochemistry, Tohoku University School of Medicine, Seiryo-machi 2-1, Sendai 980-8575, Japan.

1 The abbreviations used are: LCR, locus control region; BTB domain, bric-a-brac, tramtrack, and broad complex domain; MARE, Maf recognition element; EMSA, electrophoretic mobility shift assay; RT-PCR, reverse transcriptase-polymerase chain reaction; HPRT, hypoxanthine phosphoribosyltransferase; ES, embryonic stem; Flk-1, fetal liver kinase-1; bp, base pair(s).

2 H. Hoshino, A. Muto, and K. Igarachi, unpublished data.

3 N. Suwabe and M. Yamamoto, manuscript in preparation.

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Abstract
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Results
Discussion
References

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