From the 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
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ABSTRACT |
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The human -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
-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.
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INTRODUCTION |
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The mammalian -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
-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
-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 -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 -globin
LCR by binding to its multiple MAREs.
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EXPERIMENTAL PROCEDURES |
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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 -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,
pETBach1BTB, expresses a Bach1 variant (Bach1
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(+).
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 Bach1BTB 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 CE oligonucleotide probe (21) was derived
from the 3' enhancer of chicken
-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 CE or biotinylated C
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 C
E oligonucleotide served as the background control.
Amounts of proteins used were as follows: Bach1, 150 ng; Bach1
BTB,
150 ng; and MafK, 200 ng.
Chemical Cross-linking Analysis--
Equimolar amounts of Bach1
(1.5 µg) and Bach1BTB (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.
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RESULTS |
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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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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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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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BTB Domain Modulates DNA Binding--
Establishing that Bach1
could be one of the effectors of MAREs within the -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 (Bach1
BTB; Fig.
5A) and MafK in E. coli. The probe DNA was a 240-bp fragment from the HS2 of the
-globin LCR that contains tandem MAREs (Fig. 5A). In
DNase I protection assays, purified Bach1/MafK or Bach1
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,
Bach1
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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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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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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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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DISCUSSION |
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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 -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
-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 -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
-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
-globin LCR.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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