From the Department of Pediatrics, School of Medicine
and § Department of Biology, Faculty of Sciences, Hirosaki
University, Hirosaki 036-8563, the ¶ Department of Science for
Laboratory Animal Experimentation, Research Institute for Microbial
Diseases, Osaka University, 3-1 Yamadaoka, Suita City, Osaka 565-0871, the
Center for Tsukuba Advanced Research Alliance and Institute
of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, and
the ** Department of Biochemistry, Hiroshima University School of
Medicine, Kasumi 1-2-3, Minami-Ku, Hiroshima 734-8551, Japan
Received for publication, May 17, 2000, and in revised form, November 6, 2000
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ABSTRACT |
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The transcription factor Bach1 is a member of a
novel family of broad complex, tramtrack,
bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region
leucine zipper factors. Bach1 forms a heterodimer with MafK, a member
of the small Maf protein family (MafF, MafG, and MafK), which
recognizes the NF-E2/Maf recognition element, a cis-regulatory motif
containing a
12-O-tetradecanoylphorbol-13-acetate-responsive element.
Here we describe the gene structure of human BACH1,
including a newly identified promoter and an alternatively RNA-spliced
truncated form of BACH1, designated BACH1t,
abundantly transcribed in human testis. The alternate splicing
originated from the usage of a novel exon located 5.6 kilobase pairs
downstream of the exon encoding the leucine zipper domain, and produced
a protein that contained the conserved BTB/POZ, Cap'n collar, and
basic region domains, but lacked the leucine zipper domain essential
for NF-E2/Maf recognition element binding. Subcellular localization
studies using green fluorescent protein as a reporter showed that
full-length BACH1 localized to the cytoplasm, whereas BACH1t
accumulated in the nucleus. Interestingly, coexpression of BACH1 and
BACH1t demonstrated interaction between the molecules and the induction
of nuclear import of BACH1. These results suggested that BACH1t
recruits BACH1 to the nucleus through BTB domain-mediated interaction.
The transcription factor Bach1 belongs to the Cap'n collar
(CNC)1-related basic region
leucine zipper (bZip) factor family (1), which includes p45 NF-E2,
Nrf1/LCR-F1/TCF11, Nrf2/ECH, and Nrf3 (2-9). Mammalian CNC
members heterodimerize with small Maf proteins (MafK, MafG, and MafF)
(10, 11) which then recognize NF-E2/Maf-recognition elements (MARE)
(TGCTGAG/CTCAT/C) that contain
12-O-tetradecanoylphorbol-13-acetate-responsive elements
(TRE) (TGAG/CTCA) (9, 12-19). Gene targeting experiments revealed that
several CNC family members had distinct roles in mammalian gene
regulation. Homozygous disruption of the murine p45 NF-E2
gene results in defective megakaryopoiesis and profound thrombocytopenia, leading to postnatal death (20); nrf1-null mutant mice are anemic due to a noncell autonomous defect in definitive erythropoiesis and die in utero (21), and
nrf2-null mutant mice are viable but have an impaired
xenobiotic inductive response involving phase II detoxifying enzymes
(22).
Unlike other CNC members, Bach1 and Bach2 posses BTB/POZ (broad
complex, tramtrack, bric-a-brac/poxvirus and
zinc finger) domains (23), which have been implicated in transcription
repression by transcription factors BCL6 and PLZF, involved in cases of
non-Hodgkin lymphoma and acute promyelocytic leukemia, respectively
(24, 25). These factors interact with the corepressors N-CoR and SMRT
via the BTB/POZ domain (26). Transcription factors that contain BTB/POZ
domains are of particular interest as they are thought to play a
variety of structural and organizational roles (27). For example,
Drosophila Mod (mdg4) and GAGA factors are involved in
chromatin organization and transcription regulation through the
establishment of higher order domains (28-30). Bach1-MafK heterodimers
generate higher order complexes through the Bach1 BTB domains (31), and
the resultant complex binds target DNA sequences consisting of multiple
MAREs, generating DNA loops (32).
The BTB/POZ domain acts as a specific protein-protein interaction
domain, with most factors forming homo- and/or hetero-oligomers via
BTB/POZ domain binding (24, 33). The Drosophila pipsqueak (psq) gene encodes a BTB/POZ-containing protein required for early oogenesis (34). This gene is large and complex, encoding multiple transcripts and protein isoforms. The structure of the gene product (PsqA) and the nature of the truncated form (psq PZ fusion
protein) suggest that these proteins interact directly with other
proteins, including Psq isoforms, through BTB domains. However, no
BTB/POZ gene family members that encode multiple protein isoforms have been reported in mammals.
In the present study, we identified an alternatively spliced isoform of
human BACH1 (designated BACH1t). The
BACH1t isoform encoded a truncated form of BACH1 protein
that lacked the leucine zipper domain, but retained the BTB domain, CNC
domain, and basic region. Although both BACH1 and
BACH1t transcripts were expressed in all tissues examined,
both were most abundant in testis. In transfected cells, full-length
BACH1 was localized to the cytoplasm, whereas BACH1t accumulated in the
nucleus. BACH1t formed hetero-oligomers with BACH1 through the BTB/POZ
domains, and induced nuclear accumulation of BACH1. Thus, the
subcellular localization of BACH1 is regulated by its non-DNA-binding
isoform BACH1t.
Isolation of Human BACH1t cDNA and BACH1 Gene--
A human
testis cDNA library (CLONTECH) was plated on
150 mm Petri dishes at a density of 5 × 104
plaque-forming units/plate, and 1 × 106
plaque-forming units screened with the 1.5-kb PstI fragment
of human BACH1 cDNA. Duplicate filters were made from
each plate and hybridized to the probe in 6× SSC and 0.25% skim milk
at 55 °C overnight. Following washing under high stringency
conditions, positive clones were identified and purified by two
additional rounds of plaque hybridization. All isolated clones were
directly sequenced using primers Plasmid Construction--
Eukaryotic expression plasmids, GFP
fusion protein expression plasmids, and FLAG-tagged expression plasmids
were generated by ligating BACH1 or BACH1t
cDNA into BamHI and XhoI sites of pcDNAI/Neo (Invitrogene), EcoRI and BamHI
sites of pEGFP-NI (CLONTECH), and EcoRI
and SalI sites of pCMV2 (Sigma), respectively. To construct prokaryotic expression plasmids, BACH1 and BACH1t
cDNA fragments encoding the CNC-basic region were inserted into
pMAL-c2 (New England Biolabs). The resulting plasmids encoded the
C-terminal 187 and 74 amino acids of BACH1 and BACH1t, respectively.
RT-PCR Analysis--
PCR was carried out using a multiple tissue
cDNA panel (CLONTECH) as template, and primers
to amplify glyceraldehyde-3-phosphate dehydrogenase
(5'-TGAAGGTCGGAGTCAACGGATTT-3' and 5'-CATGTGGGCCATGAGGTCCACCAC-3'), BACH1 (B1-E4, 5'-TTCATGG CACAACGGATAATTTCACTG-3'; and
F-GSP-A, 5'-GTAACGCCAGTTCACCATCAGGAGTACT-3'), and BACH1t
(B1-E4 and T-GSP-A, 5'-TGAGACTCCAGCCTTATCTTAGCAGCTA-3'). The
BACH1 genomic DNA-derived amplicon was relatively large due
to the presence of an intron. PCR reactions were carried out for 24-32
cycles to ensure linearity of and products resolved on 1.5%
agarose/Tris borate electrophoresis buffer gels.
5'-Rapid Amplification of cDNA Ends (5'-RACE)--
5'-RACE
was carried out using human testis marathon-ready cDNA
(CLONTECH) as template and primers designed to
amplify the 5'-end of BACH1t (adapter primer 1, 5'-CCATCCTAATACGACTCACT ATAGGGC-3'; adapter primer 2, 5'-ACTCACTATAGGGCTCGGCGGC-3'; T-GSP-A; and T-GSP-A2, 5'-TGCAACACTACTATCTTCCCTGGTGCCC-3'). PCR was carried out according to
the supplier's recommendations. The first reaction was performed using
adapter primer 1 and T-GSP-A, and incubated at 94 °C for 30 s,
followed by 5 cycles at 94 °C for 5 s and 72 °C for 4 min; 5 cycles at 94 °C for 5 s and 70 °C for 4 min; 20 cycles at
94 °C for 5 s and 72 °C for 4 min. Nested PCR was then
performed using adapter primer 2 and T-GSP-A2 and incubated at 94 °C
for 30 s, followed by 5 cycles at 94 °C for 5 s and
72 °C for 4 min, and 20 cycles at 94 °C for 5 s and 70 °C
for 4 min. Nested PCR products were subcloned to pCRII (Invitrogene)
plasmids and DNA sequenced.
Northern Hybridization--
Northern blots containing multiple
human tissue RNA samples were purchased from
CLONTECH. Each tissue sample contained 2 µg of
poly (A)+ RNA. Blots were hybridized with
32P-labeled human BACH1 or rat
glyceraldehyde-3-phosphate dehydrogenase cDNA probes, and
subsequent washings carried out as described previously (17).
Electrophoretic Gel Mobility Shift Assays--
Nuclear extracts
were prepared from a quail fibroblast cell line, QT6 transfected with
BACH1, or other expression vectors as described previously
(16, 35). An oligonucleotide containing the chicken Transient Transfection--
QT6 cells (36) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and seeded in six-well dishes 24 h before
transfection. Following replacement of the cell culture medium with
Opti-MEM (Life Technologies, Inc.), cells were transfected with
GFP fusion, Flag-tagged protein, or nontagged protein expression
vectors by lipofection using LipofectAMINE (Life Technologies, Inc.)
according to the supplier's recommendations.
Determination of the DNA-binding Consensus Sequence--
Binding
site selection and PCR amplification were performed as described
previously (37) with some modifications. Briefly, 69-base pair
single-stranded synthetic oligonucleotides were prepared with the
sequences
5'-GCGGATCCTGCAGCTCGAC(n30)GTCGACAAGCTTCTAGAGCA-3', where n30 represents a stretch of 30 random base pairs.
Double-stranded oligonucleotides were prepared with specific forward
(5'-GCGGATCCTGCAGCTCGAG-3') and reverse primers
(5'-TGCTCTAGAAGCTTGTCGAC-3') and the double-stranded oligonucleotide pool then incubated with recombinant maltose-binding protein (MBP)-BACH1, MBP-BACH1t, or MBP-LacZ fusion proteins bound to
amylose resin in binding reaction buffer (25 mM HEPES, 100 mM KCl, 1 mM EDTA, 10 mM
MgCl2, 5% glycerol, 1 mM dithiothreitol, 0.2 mg/ml poly(dI-dC), 0.2 mg/ml bovine serum albumin) on ice for 30 min.
The resins were washed three times in binding reaction buffer and
boiled to release bound oligonucleotides into the supernatant. The
recovered oligonucleotides were amplified by PCR with the same forward
and reverse primers. After this procedure had been repeated five times,
the resultant PCR products were cloned into the pCRII (Invitrogen)
plasmid vector, and nucleotide sequences of the inserts determined.
Immunoprecipitation--
Cell extracts were prepared from
transfected QT6 cells by lysis with ice-cold lysis buffer (10 mM Tris, pH 7.6, 5 mM EDTA, 50 mM
NaCl, 50 mM NaF, 30 mM
Na2P2O7, 1% Triton X-100, 100 µM NaVO4, 100 µM
phneylmethylsulfonyl fluoride). Lysates were clarified by
centrifugation at 12,000 rpm for 5 min and incubated with anti-FLAG antibody M2-agarose (Sigma) for 2 h at 4 °C. Precipitated
complexes were pelleted by brief centrifugation and washed five times
with lysis buffer. Washed complexes were directly applied to SDS-PAGE and transferred to polyvinylidene difluoride membranes for Western blotting analysis using anti-Bach antibody (F69-1) (23).
Immunofluorescence and GFP Fusion Protein Analysis--
Cells
transfected with GFP-tagged protein or other expression vectors were
incubated for 16 h on coverslips. Cells were then fixed in PBS
containing 4% formaldehyde for 30 min, followed by 0.1% Triton X-100
treatment for 15 min at room temperature. Following treatment with
blocking reagent (Dako) for 5 min, cells were stained with 10 µg/ml
biotin-labeled M2 anti-FLAG monoclonal antibody (Sigma) for 30 min at
room temperature, washed five times in Tris-buffered saline containing
1 mM CaCl2 (TBS/Ca), and stained with
streptavidin-conjugated R-phycoerythrin (Dako) diluted 1:20 for 10 min
at room temperature. After five washes with TBS/Ca,
fluorochrome-labeled cells expressing GFP fusion proteins were
visualized using a microscope equipped with fluorescence optics (Nikon)
and images recorded using SensiaII Fujichrome film.
Molecular Cloning of the BACH1t and BACH1 Genes--
In an attempt
to clone the human homologue of murine Bach1, we screened a
human endothelial cell cDNA library under nonstringent hybridization conditions with a 1.4-kb NcoI fragment of
murine Bach1 cDNA as a probe, and isolated seven
positive clones. Upon Southern blotting and sequence analysis, three
clones (63, 67, and 71) were found to encode human BACH1, based on
sequence similarity to murine BACH1. Clone 63 contained a 2.3-kb insert
that covered the entire BACH1 open reading frame (ORF).
During our characterization of human BACH1, Ohira et
al. (38) and Blouin et al. (39) reported the cloning of
human BACH1 cDNA and gene localization at 21q22.1. Although the cDNA clone reported by Ohira et al.
contained longer 3'- and 5'-noncoding regions than clone 63, coding
region sequences were completely identical. Comparison with cDNA
sequences isolated by Blouin et al. showed eight coding
region mismatches, resulting in two amino acid differences at positions
158 and 171 (Thr
Northern blot analysis using a multitissue blot
(CLONTECH) and a 1.5-kb PstI fragment of
clone 63, including CNC-bZip region sequence, as a probe revealed the
existence of a smaller sized transcript in testis, as reported by the
previous studies (data not shown) (38, 39). To clone this molecule, we
screened a human testis cDNA library using the PstI
fragment of clone 63 as a probe. Southern blot and DNA sequence
analysis revealed that the eight isolated clones lacked sequences
encoding the C-terminal region that contained the leucine zipper domain
and instead possessed a novel sequence (Fig.
1, A and B). We
designated the truncated sequence as BACH1t. Eleven other
isolated clones obtained by the same screening method included the
leucine zipper domain. As none of the isolated clones were full-length,
we performed 5'-RACE using the Marathon cDNA amplification kit
(CLONTECH) and primer T-GSP-A, derived from
BACH1t-specific sequence, to obtain the complete BACH1t ORF.
As shown in Fig. 1C, the sequence of the 5'-noncoding region
from the isolated BACH1t clones was different to previously
reported sequences (38, 39).
To eliminate the possibility that the smaller transcript was an
artifact of DNA manipulation or PCR amplification, we isolated a
genomic clone encoding BACH1 by PCR using the primers B1geno+ and
B1geno BACH1t Is Ubiquitously Transcribed--
To examine the expression
patterns of BACH1 and BACH1t in various tissues,
we performed RT-PCR analysis using combinations of a primer located in
exon IV (primer B1-E4) with primers in exons V (primer F-GSP-A) or VI
(primer T-GSP-A), using a multitissue cDNA panel
(CLONTECH) as template. Although both
BACH1 and BACH1t were transcribed in all tissues
examined, expression profiles varied depended on tissue type (Fig.
2A). Both BACH1 and
BACH1t mRNAs were most abundantly expressed in the
testes.
Because BACH1 cDNAs have three different 5'-noncoding
sequences, we analyzed the tissue specificity of these putative first exons by RT-PCR with each isomer-specific primer and a primer common to
all isomers located in exon V (Fig. 2B). Results showed that, although the alternate first exons were all transcribed, tissue-specific usage remained unclear.
Northern analysis showed that the short mRNA form was highly
abundant in testis, but undetectable in other tissues (38, 39). In
contrast, the long form was of similar abundance in several tissues,
but higher in spleen and leukocytes than in testis. Thus, these results
appeared to be disconcordant with the PCR results (see Fig.
2A). To clarify whether BACH1 and
BACH1t accounted for the long and short forms, respectively,
we performed Northern blot hybridization on multitissue blots
(CLONTECH) probed with an ~300-base pair
PstI/EcoRI fragment of BACH1 cDNA
(clone 63), corresponding to exon V, as a BACH1-specific
probe. Unexpectedly, the results showed that the short form as well as
the long form was detectable in testis (Fig. 2C). Northern
blot hybridization was performed using BACH1t-specific
probes, but specific bands were not detected due to high background.
Although BACH1t could not be distinguished from
BACH1 by Northern blot analysis, results suggested that the
long RNA form contained both BACH1 and BACH1t sequence.
BACH1t Does Not Recognize NF-E2/MARE--
To examine the DNA
binding properties of BACH1t, we performed gel retardation analysis
using labeled double-stranded oligonucleotides containing NF-E2 motifs
and proteins extracted from QT6 fibroblasts transfected by various
combinations of BACH1, BACH1t, MAFK,
and MAFG expression constructs. As shown in Fig.
3, the BACH1 and small MAF heterodimer
recognized the NF-E2 motif (Fig. 3, lanes 7 and 8). In
contrast to BACH1, no interaction between BACH1t and NF-E2 sites was
observed, irrespective of the presence of small MAFs (Fig. 3,
lanes 9 and 10).
To investigate the possibility that BACH1t recognized a unique motif,
we then attempted to determine the optimal binding site for BACH1t by
PCR-assisted selection of binding sites. Fusion proteins of the
MBP-C-terminal fragment containing the basic region of BACH1t, or the
MBP-C-terminal region containing the bZip domain of BACH1 were used for
five rounds of selection. Although BACH1 exhibited binding preferences
toward oligonucleotides containing the TRE consensus sequence, BACH1t
showed no obvious binding activity to specific DNA sequences (Fig.
4). These results suggest that BACH1t is
a non-DNA-binding isoform.
BACH1t Associates with BACH1-MafK Heterodimer through the BTB
Domain--
Previous studies have shown that Bach1 BTB domains mediate
protein-protein interactions (31). To clarify interactions between BACH1t and the BACH1/small MAF heterodimer, we performed
coimmunoprecipitation analysis using fibroblasts transfected with
FLAG-labeled BACH1t expression construct. Cotransfections were
performed using various combinations of FLAG-labeled BACH1t, murine
Bach1-MafK fusion protein (B1K), and Bach1 lacking BTB domain-MafK
fusion protein ( BACH1t Mediates Nuclear Import of BACH1--
To examine the
interaction between BACH1t and BACH1 at the subcellular level,
GFP-labeled BACH1 or GFP-BACH1t, BACH1t expression vectors were
transfected into fibroblasts and immunofluorescence analysis performed.
Unexpectedly, BACH1 and BACH1t were distributed separately in the
transfected QT6 cells, with the former cytoplasmically localized and
excluded from the nucleus, and the latter accumulated in the nuclei
(Fig. 6A). However, when the
FLAG-MAFK expression vector was transfected to QT6, MAFK was
accumulated in the nucleus as expected.
To further examine the interaction between BACH1t and BACH1, we
cotransfected both expression constructs into QT6 cells. As shown in
Fig. 6B (a and b), although BACH1t
localization was not affected, significant accumulation of GFP-labeled
BACH1 was observed in the nucleus. To exclude the possibility that this
was due to the presence of the GFP label, we performed the same
transfection assay using FLAG-tagged BACH1 and GFP-labeled BACH1t, and
GFP-labeled BACH1 and nontagged BACH1t, with the same changes in BACH1
localization observed in each experiment (data not shown). We then
performed cotransfection of expression vectors for GFP-labeled BACH1
and FLAG-labeled BACH1t lacking BTB domain (FLAG-
Because the DNA binding activity of BACH1 requires the presence of
small MAF proteins, we examined the subcellular localization of BACH1
in the presence of MAFK. When GFP-labeled BACH1 and FLAG-tagged MAFK
were coexpressed in QT-6 fibroblasts, a small amount of BACH1 accumulated in the nucleus (Fig. 6B, e and
f). However, MAFK protein, localized in the nucleus in the
absence of BACH1, showed very similar distribution to BACH1 when
coexpressed, such that MAFK was also more abundant in the cytoplasm.
These results suggest that interactions between small MAF and BACH1
influence subcellular localization. These observations were consistent
with the results obtained by electrophoretic mobility shift assays,
which showed that significant NF-E2 binding activities existed in
nuclear extracts when small MAF and BACH1 expression vectors were
cotransfected (Fig. 3). However, we cannot exclude the possibility that
more NF-E2 binding activity existed in the cytoplasm of QT-6 cells.
In the present study, we described the isolation and
characterization of an alternatively spliced BACH1
isoform, BACH1t, that originated from the usage of a novel
exon located downstream of the exon encoding the leucine zipper domain.
The BACH1t isoform encoded a truncated form of BACH1 protein
that lacked a leucine zipper domain. Although both BACH1 and
BACH1t transcripts were abundantly expressed in human
testis, expression was observed in all tissues tested. In transfected
cells, BACH1 was localized to the cytoplasm, whereas BACH1t accumulated
in the nucleus. BACH1t formed hetero-oligomers with BACH1 through the
BTB/POZ domains and induced the nuclear accumulation of BACH1. These
results suggested that BACH1t may play an important role in the control
of the subcellular localization of BACH1.
Characterization of human BACH1 demonstrated a complex gene
structure, with multiple differentially spliced transcripts and protein
isoforms. The present results and data base information indicated that
the BACH1 gene has three alternate exon I or noncoding exon
sequences (Fig. 1D), and RT-PCR analysis showed that all three alternate exons were transcribed (Fig. 2B). Such
complex structures of genes encoding BTB/POZ proteins have not been
described previously in mammals.
Recently, Hoshino et al. found that Bach2 was mainly
localized in the cytoplasm through its evolutionarily-conserved
C-terminal cytoplasmic localization signal (CLS) (41). The CLS directs leptomycin B-sensitive nuclear export of reporter proteins through the
binding of leptomycin B to the nuclear exporter carrier protein Crm1/exportin 1. This inhibits a nuclear export signal, leading to
Crm1/exportin 1-dependent nuclear export. As oxidative
stressors diminished CLS activity and induced nuclear accumulation of
Bach2, the resulting rapid nuclear accumulation may allow Bach2 to
regulate gene expression in response to oxidative stress. The
functional significance of the CLS is reinforced by the fact that this
region is highly conserved between Bach1 and Bach2, which suggests that Bach1 activity may also be regulated by a similar mechanism. In the
present study, we found that BACH1 was localized cytoplasmically in
transfected cells, while the naturally occurring truncated form of
BACH1, which lacks the CLS, was found to be localized in the nucleus.
Furthermore, our results demonstrated that BACH1t recruited BACH1 to
the nucleus. It is possible that the subcellular localization of BACH1
is determined by the net balance of nuclear localization signal (NLS)
and CLS activities. Thus, if one molecule of BACH1 associated with one
molecule of BACH1t through their BTB domains, the resulting heterodimer
would have two NLS and one CLS, such that the NLS activity would be
stronger than that of the CLS. The present data also showed that small
MAF protein promoted the accumulation of BACH1 in nuclei, but to a far
lesser extent than compared with BACH1t (Fig. 6). Interestingly, BACH1 influenced the subcellular localization of MAFK but not of BACH1t, suggesting that there are differences in NLS activity between BACH1t
and MAFK. These results indicated that there may be at least two
different pathways in the control of BACH1 subcellular localization: an
oxidative stress pathway and a BACH1t pathway. To our knowledge, the
control of subcellular localization by heterodimer formation with an
alternate isoform has not been described for other CNC family members.
Recently, it was reported that the BACH1 gene localized to
an ~400-kb region on 21q22.1 (38, 39), within the potential Down's
syndrome-associated gene region proposed by Korenberg et al.
(42). Furthermore, the BACH1 gene is part of the
APP-SOD1 region involved in some of the features associated
with monosomy 21. Down's syndrome, the most common birth defect
causing mental retardation, is characterized by specific phenotypes
including subfertility or sterility and hypogonadism in males. In
contrast, several women with Down's syndrome have given birth to
offspring. It has been proposed that the effect of trisomy 21 on
spermatogenesis and fertility is a consequence of the behavior of the
extra chromosome during the meiotic prophase (43). However, an abundant
expression of BACH1 and BACH1t in testis suggests
that these molecules may have an important role in this tissue and
raises the possibility that overexpression of BACH1 and
BACH1t caused by the presence of three gene copies may lead
to some aspects of the Down's phenotype, including hypogonadism or
sterility. Analysis of transgenic mice with an extra copy of
BACH1 or a targeted BACH1 gene knockout may
provide further information to elucidate its function and involvement
in Down's syndrome and monosomy 21.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
gt11+
(5'-CAGCGAATTCGGTGGCGACGACTCCTGGA GC-3') and
gt11
(5'-CAGCGAATTCTTGACACCAGACCAACTGGTA-3'), the longest positive clone
insert (clone 97) subcloned into the EcoRI site of
pBluescript KS (+) (Stratagene) and both strands sequenced using an ABI
PRISM cycle sequencing kit (Applied Biosystems). Genomic clones
encoding BACH1 were isolated by screening a commercial P1 phage
human genomic library (Genome inc.) by polymerase chain reaction (PCR)
analysis using primers B1geno+ (5'-AGGATGCTGCTCTGGCCTT GC-3') and
B1geno
(5'-CTACTATCTTCCCTGGTGCCC-3'). Following mapping of the
restriction enzyme sites of the positive clone, exon-intron boundaries
were determined by Southern blot analysis and DNA sequencing.
-globin enhancer
NF-E2 site (5'-CCCGAAAGGAGCTGACTCATGCTAGCCC-3') was labeled with
[
-32P]ATP using T4 polynucleotide kinase.
Binding reactions and electrophoresis on 4% nondenaturing
polyacrylamide gels (PAGE) were carried out as described previously
(16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Ser and Gly
Glu, respectively). These
differences may be due to allelic variation.
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Fig. 1.
Structure of human BACH1
gene and nucleotide sequences of the novel human BACH1
isoform, BACH1t. A, schematic
illustration of human BACH1 and its naturally occurring truncated form
BACH1t. BACH1t lacks the leucine zipper domain. B, the 3'
region of the nucleotide and deduced amino acid sequences of
BACH1t. The arrow and the asterisk
indicate the boundary of newly identified sequence and translation stop
codon, respectively. The CNC domain and basic regions are
underlined with a solid line and a
broken line, respectively. The remaining amino
acid residues of the leucine zipper domain are circled.
Amino acid residues are shown using the single-letter
code and are numbered from the initiation
methionine. C, comparison of the 5' region nucleotide
sequences of BACH1t and reported BACH1 (38, 39).
Conserved nucleotide sequences are indicated by asterisks,
and the translation initiation codon is boxed. Nucleotide
sequences of BACH1t are numbered on the
right. The arrow indicates the position for the
intervening sequence (IVS). D, schematic
illustration of the isolated human BACH1 genomic clone.
BACH1 (upper) and BACH1t
(lower) transcripts are shown below the map. The sequences
of Ia, Ib, and Ic correspond to 5'-untranslated region sequences of
BACH1 cDNA described by Ohira et al. (38),
Blouin et al. (39), and us in this paper. White
boxes indicate 5'- and 3'-untranslated region sequences.
Black boxes indicate coding sequence.
E, intron-exon junctions of the human BACH1 gene.
Exon sizes and estimated intron sizes are indicated. All spliced sites
conform to the GT/AG rule. The size of exon V is quoted from reference
38.
(Genome inc.). The gene structure and exon-intron boundaries
of BACH1 were determined by Southern blot hybridization and
DNA sequence analysis (Fig. 1, D and E) and
revealed that all spliced sites conformed to the GT/AG rule (40).
Analysis of the structure of the 3'-region BACH1t-specific
exons revealed four exons (exons VI, VII, VIII, and IX) located
downstream of exon V, which encoded the leucine zipper domain (Fig.
1D). The 5'-noncoding region of the BACH1t clones
was located 16 kb upstream of the exon that contained the translation
initiation codon. A data base search for related sequences identified a
human genomic sequence (GenBankTM accession no. AF124731) that covered
a 108-kb region. We identified three segments within this sequence that were identical to previously reported 5'-noncoding sequences of BACH1 (38, 39) and to BACH1t, indicating that the
segments were alternative exon I or noncoding exon sequences for the
BACH1 gene. We designated these exons described by Ohira
et al. (38), Blouin et al. (39), and by this
present study, as Ia, Ib, and Ic, respectively (Fig.
1D).
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Fig. 2.
BACH1t expression in human tissues.
A, BACH1 and BACH1t expression was
assessed by RT-PCR using a multitissue cDNA panel as template.
Linearity of amplification was ensured by comparing amplicons from
different PCR cycles. B, RT-PCR analysis of usage of the
three alternate 5'-noncoding sequences of the BACH1 gene.
The putative first exons, Ia, Ib, and Ic, were ubiquitously
transcribed. C, Northern blot analysis using multitissue
blots (CLONTECH) and a BACH1-specific
probe. Both the short and long forms of BACH1 transcripts
were detected in testis.
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Fig. 3.
Loss of binding of BACH1t to NF-E2
sites. Nuclear protein extracted from QT6 fibroblasts transfected
by various combinations of BACH1, BACH1t, or
small MAF expression vectors was incubated with a NF-E2 site
probe from the chicken -globin 3' enhancer. Arrow
indicates shifted signals derived from BACH1 and small MAF protein
complexes.
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Fig. 4.
Determination of the DNA-binding consensus
sequence of BACH1t by PCR-assisted selection of binding sites. The
maltose binding protein (MBP)-C-terminal fragment containing the basic
region of BACH1t or MBP-C-terminal region containing the bZip domain of
BACH1 fusion proteins were used for this experiment. A,
BACH1t did not show any obvious binding preference to specific DNA
sequences after five rounds of selection, although BACH1 exhibited
binding preferences toward oligonucleotides containing TRE consensus.
B, sequence alignment of 27 independent clones such that the
centers of the palindromic sequences are occupied by a purine. The
consensus site, below, was derived by selecting nucleotides at each
position that were present in more than 70% (uppercase) or
30% (lowercase) of clones.
BTB) expression vectors into fibroblasts by
lipofection. When cell lysates derived from fibroblasts expressing
FLAG-labeled BACH1t and B1K were immunoprecipitated by anti-FLAG
antibody, anti-Bach antibody (F69-1) recognized the immunoprecipitated
B1K (Fig. 5, lane
3). However, after cotransfection with
BTB, the fusion
protein was not detected (Fig. 5, lane 5). These
results suggested that interaction occurred between BACH1t and the
BACH1-MafK heterodimer through the BTB domains.
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Fig. 5.
Association of BACH1t and Bach1-MafK through
the BTB domains. Following the transfection of various
combinations of FLAG-labeled BACH1, FLAG-labeled BACH1t,
Bach1-MafK, and BTB BACH1 expression vectors into QT6 fibroblasts,
immunoprecipitation was performed with transfected cell lysates and the
anti-FLAG antibody, M2. Immunoprecipitates were analyzed by Western
blotting using anti-Bach antibody (F69-1).
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Fig. 6.
Subcellular localization of BACH1
and BACH1t. A, QT6 fibroblasts were transiently
transfected with the expression vectors for GFP, GFP-labeled BACH1,
GFP-labeled BACH1t, and FLAG-tagged MAFK. Localization of GFP or
FLAG fusion protein and phase contrast images of transfected cells were
observed by inverted microscopy with or without fluorescence optics.
B, import of BACH1 into the nucleus is mediated by
cotransfection of BACH1t. QT6 fibroblasts were transiently
cotransfected with expression vectors for GFP-labeled BACH1 and
FLAG-tagged BACHt (a and b, top),
BACH1t lacking the BTB domain ( BTB BACH1t) (c and
d, middle), or MAFK (e and
f, bottom). Following the fixation of transfected
cells, FLAG fusion proteins were stained with streptavidin conjugated
to R-phycoerythrin, and the cells observed with a microscope equipped
with fluorescence optics.
BTB BACH1t) to
examine whether nuclear accumulation of BACH1 depended on the
interaction through the BACH1 and BACH1t BTB domains. As expected, the
nuclear accumulation of BACH1 alone was markedly reduced compared with cotransfection of GFP-labeled BACH1 and FLAG-labeled BACH1t expression vectors (Fig. 6B (c and d)).
Nevertheless, a small amount of BACH1 still accumulated in nuclei and
therefore we could not exclude the possibility that BACH1 and
BTB
BACH1t interacted with each other outside the BTB domains. Indeed, we
have previously found a weak BACH1 interaction mediated through a
region between the BTB and bZip
domains.2
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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Part of this work was carried out using the facilities of the Department of Molecular Genetics at Hirosaki University School of Medicine.
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FOOTNOTES |
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* This work was supported by grants-in-aid for scientific research, grants-in-aid for scientific research on priority areas (to E. I. and K. I.) and a grant-in-aid for encouragement of young scientists (to T. T.) from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the Karouji Memorial Foundation (to E. I.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF317902 and AF317903.
To whom correspondence should be addressed. Tel.:
81-172-39-5070; Fax: 81-172-39-5071; E-mail:
eturou@cc.hirosaki-u.ac.jp.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M004227200
2 K. Igarashi, data not shown.
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ABBREVIATIONS |
---|
The abbreviations used are: CNC, Cap'n collar; bZip, basic region leucine zipper; MARE, Maf recognition element; TRE, 12-O-tetradecanoylphorbol-13-acetate-responsive elements; BTB/POZ, broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger; PCR, polymerase chain reaction; GFP, green fluorescent protein; 5'-RACE, 5'-rapid amplification of cDNA ends; PAGE, polyacrylamide gel electrophoresis; CLS, cytoplasmic localization signal; NLS, nuclear localization signal; RT, reverse transcription; kb, kilobase pair(s); MBP, maltose-binding protein.
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