Molecular Cloning and Functional Characterization of a New
Cap'n' Collar Family Transcription Factor Nrf3*
Akira
Kobayashi
§,
Etsuro
Ito¶,
Tsutomu
Toki¶,
Keiji
Kogame
,
Shinichiro
Takahashi
,
Kazuhiko
Igarashi
,
Norio
Hayashi
, and
Masayuki
Yamamoto**
From the
Department of Biochemistry, Tohoku
University School of Medicine, 2-1 Seiryomachi,
Aoba-ku, Sendai 980-8575, ¶ Department of Pediatrics and
Clinical Laboratory, Hirosaki University School of Medicine,
Zaifu-cho, Hirosaki 036, and ** Center for Tsukuba Advanced Research
Alliance and Institute of Basic Medical Sciences, University of
Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan
 |
ABSTRACT |
The NF-E2-binding sites or Maf
recognition elements (MARE) are essential
cis-acting elements in the regulatory regions of erythroid-specific genes recognized by the erythroid transcription factor NF-E2, composed of p45 and MafK. Recently, two p45-related factors Nrf1 and Nrf2 were isolated, and they are now
collectively grouped as the Cap'n' collar (CNC) family. CNC factors
bind to MARE through heterodimer formation with small Maf proteins. We report here the identification and characterization of a novel CNC
factor, Nrf3, encoding a predicted 73-kDa protein with a basic region-leucine zipper domain highly homologous to those of other CNC
proteins. In vitro and in vivo analyses showed
that Nrf3 can heterodimerize with MafK and that this complex binds to
the MARE in the chicken
-globin enhancer and can activate
transcription. Nrf3 mRNA is highly expressed in human placenta and
B cell and monocyte lineage. Chromosomal localization of human Nrf3 is
7p14-15, which lies near the hoxA gene locus. As the
genetic loci of p45, nrf1, and
nrf2 have been mapped close to those of
hoxC, hoxB, and hoxD, respectively,
the present study strongly argues for the idea that a single ancestral
gene for the CNC family members may have been localized near the
ancestral Hox cluster and have diverged to give rise to four closely
related CNC factors through chromosome duplication.
 |
INTRODUCTION |
Regulation of biological processes is accomplished through
interactions among various tissue- or developmental stage-specific transcription factors. For instance, the transcription factor NF-E2 is
critical for erythroid-specific expression of the porphobilinogen deaminase gene (1). NF-E2 is composed of two subunits; the p45
component contains the Cap' n' collar (CNC)1-type basic
region-leucine zipper (bZip) domain, which
is highly conserved with the bZip domain
of the Drosophila transcription factor CNC (2).
Recently, several mammalian transcription factors carrying the CNC/bZip
domain were identified, such as Nrf1/LCR-F1/TCF11 (3-5),
Nrf2/ECH (6, 7), Bach1, and Bach2 (8). The other subunit of the
NF-E2 complex is composed of the small Maf proteins, members of the Maf
proto-oncoprotein family (9, 10). To date, three small Mafs, MafF,
MafG, and MafK, have been identified (11-14).
A homodimeric complex of small Maf proteins recognizes a DNA sequence
motif called Maf recognition
element (MARE, TGCTGA(G/C)TCAGCA or
TGCTGACGTCAGCA), which contains either a
12-O-tetradecanoylphorbol-13-acetate-responsive element
(TGA(G/C)TCA) or cAMP-responsive element
(TGACGTCA) (15, 16). Homodimeric complexes of small Mafs
repress transcription through binding to MARE, since they lack
canonical trans-activation domains. On the other hand, CNC
proteins require a small Maf protein for their DNA binding activities.
A CNC-small Maf heterodimer complex binds to both the NF-E2 consensus
sequence (TGCTGA(G/C)TCA(T/C)) and MARE (in
particular to the
12-O-tetradecanoylphorbol-13-acetate-responsive element-type
MARE). These regulatory motifs are frequently observed in the
regulatory regions of erythroid-specific genes, such as the locus
control region (LCR) of
-globin genes, as well as in several
non-erythroid gene promoters (17).
To assess the biological roles of the CNC family proteins,
p45, nrf1 and nrf2 genes were
disrupted individually (18-20). Whereas the loss of p45 function
impaired megakaryocyte maturation and platelet formation,
erythropoiesis in p45-null mice was normal. Nrf2 is essential
for the induction of phase II-detoxifying gene expression such as
glutathione S-transferase and NADPH-quinone oxidoreductase
(NQO1) by phenolic antioxidants, but its loss (20), even in combination
with p45 (21, 22), did not cause anemia. The results of the
nrf1 gene disruption are somewhat controversial; one report
showed early embryonic lethality (19), whereas another study showed
loss of definitive hematopoiesis (23). These results indicate that
compensation among CNC family proteins may exist in erythroid-specific
gene regulation and that lineage-specific regulation of gene expression
through MAREs appears to be accomplished by multiple CNC family proteins.
Fluorescence in situ hybridization (FISH) analyses revealed
that the chromosomal localization of the p45,
nrf1, and nrf2 genes are mapped close to
the hoxC (12q13.1-13.3), hoxB (17q21), and hoxD (2q31) gene clusters, respectively (24), suggesting
that the p45-related factors may be derived from a single ancestral gene through chromosome duplication, as is the case for the
hox gene clusters. Since no CNC factor has been
identified near the hoxA locus (7p14-15), this also
implies the existence of an additional CNC factor. We report here the
identification and characterization of this new CNC family member, Nrf3
(NF-E2 related factor
3).
 |
EXPERIMENTAL PROCEDURES |
Isolation of Nrf3--
Expressed
sequence tag (EST) cDNA fragment for Nrf3
(THC181377) was isolated by PCR using genomic DNA of HeLa cells as a
template. 30 cycles of PCR were performed at 94 °C for
30 s; 60 °C for 30 s; 72 °C for 1 min. Primers used
were 5'-GATATTTTTAGTAGATTAAGAGATGACC-3' and
5'-GCACTTCATGAAAAAGTTGTGGC-3'.
Human and Mouse Nrf3 cDNA Cloning--
A human placenta
gt11 cDNA library (a generous gift from Dr. Shigeru Taketani,
Kansai Medical University) was plated on 150-mm Petri dishes at a
density of 5 × 104 plaque-forming units per plate,
and 1 × 106 plaques were screened with labeled EST
fragment (25). To isolate mouse Nrf3, a mouse brain cDNA library
(Stratagene) was screened using a 0.5-kbp HindIII fragment
of human Nrf3 cDNA as a probe. Hybridization was carried out at 65 or 50 °C in 50 mM Tris-HCl (pH 7.5), 1 M
NaCl, 10 mM EDTA, 1× Denhardt's solution, 0.1% SDS, and
0.1 mg/ml salmon sperm DNA. Membranes were washed twice for 30 min at
65 °C in 0.1 × SSC and 0.1% SDS solution (human cDNA library screening) or 50 °C in 2 × SSC and 0.1% SDS solution
(mouse cDNA library screening). Positive plaques were isolated and purified.
5'-Rapid Amplification of the cDNA End Analyses and DNA
Sequencing--
5'-Rapid amplification of the cDNA end analyses
were carried out with the MarathonTM cDNA amplification
kit (CLONTECH). DNA sequences were determined on
both strands by the dideoxynucleotide method using an ABI377 automated
sequencer (Perkin-Elmer).
Cell Culture and RNA Blot Analysis--
NALL-1, NALM-6, BALM-2,
NAMALWA, and RPMI8226 cells were gifts of Dr. Kenji Ohtani (Fujisaki
Cell Center of Hayashibara Biochemical Laboratories, Inc.), and
KOPT-K1, THP6, and NALM-17 cells were gifts of Dr. Yasuhide Hayashi
(University of Tokyo). MGS cells were a gift of Dr. Shinkichi Yokoyama
(Yamagata University). KG-1, HL60, U937, and THP1 were obtained from
Japanese Cancer Resources Bank. Poly (A)+ RNA samples from
various cultured cell lines were prepared by the guanidine-acidified
phenol chloroform method (26) and purified with oligotex
(dT)30 column chromatography (Roche). RNA samples were
electrophoretically separated on a 1.0% agarose gel containing 1.1 M formaldehyde and transferred onto ZetaProbe membranes
(Bio-Rad). RNA blots containing multiple human tissue RNAs were
purchased from CLONTECH (2 µg of poly
(A)+ RNA per sample). Radiolabeled probe was prepared from
the 500-bp HindIII fragment of the human clone,
SKhNrf3/1-1.
Construction of Expression Plasmids for Transient Transfection
Assays--
BosNrf1, BosNrf2, and BosNrf3 were generated by
subcloning the 2.6-kbp HindIII/XbaI fragment from
pcDNAI/Neo-Nrf1 (11), the 2.6-kbp BamHI/XbaI
fragment from pcDNAI/Neo-Nrf2 (11), and the 2.5-kbp fragment
from SKmNrf3-1, respectively, into the blunt-ended XbaI
site of pEFBos (27).
The quail fibroblast cell line QT6 (28) was maintained in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum and
plated onto 24-well dishes (5 × 104 cells per well
with 0.3 ml medium) 24 h before transfection by the calcium
phosphate precipitation method (29). 10 ng of firefly luciferase (Luc)
reporter plasmid, 50 ng of pENL (an internal control), and various
combinations of effector plasmids were used (see figure legends). Luc
reporter plasmids pRBGP2 and pRBGP4 were previously described (10). QT6
cells were washed twice with phosphate-buffered saline 12 h after
transfection, fed with fresh media, and incubated for an additional
24 h. Luc activity was measured following the supplier's protocol
(Promega) with a Biolumat luminometer (Berthold). Assays were performed
in triplicate in independent transfection experiments, and the results
were normalized with respect to pENL
-galactosidase activity.
Expression of CNC Proteins in Yeast Cells and Yeast Two-hybrid
Analysis--
Expression plasmids of GADNrf1, GADNrf2, and
GADNrf3 were generated by inserting the 2.6-kbp
DraI/XbaI fragment from pcDNAI/Neo-Nrf1 (11),
the 2.6-kbp BglII/XbaI fragment from
pcDNAI/Neo-Nrf2 (11), and the 1.8-kbp EcoRI
fragment from SKhNrf3/1-1, respectively, into the appropriate sites of
GAD424. GBD-MafK was generated by subcloning the 1.0-kbp
BamHI/HindIII fragment from pQE30-MafK (11) into
the blunt-ended ClaI site of GBD-pCla which was
created by inserting a ClaI linker (8-mer, 5'-catcgatg-3')
into the EcoRI site of GBT9. Saccharomyces
cerevisiae SFY526 were transformed with several combinations of
plasmids by the lithium acetate method. Measurement of
-galactosidase activity of transformants was performed as described
(30).
Expression of MBP-CNC-bZip and MBP-MafK in Escherichia
coli--
MBPNrf1, MBPNrf2, MBPNrf3, and MBPMafK were
constructed by inserting 500-bp PCR-generated
BamHI/XbaI fragments from pcDNAI/Neo-Nrf1, pcDNAI/Neo-Nrf2, and SKhNrf3/1-1, and the 1.0-kbp
BamHI/HindIII fragment from pQE30-MafK (11) into
appropriate sites of pMAL-C2 (New England Biolabs). Primers used were
5'-CGGGATCCAAAGGCAGCAAGGAGAAGCA-3'/SP6 primer (Nrf1),
5'-CGGGATCCCCATTCACAAAAGACAA-3'/SP6 primer (Nrf2), and
5'-CGGGATCCCAGAAGATAAGGAGTAGATA-3'/T3 primer (Nrf3). All
constructs were confirmed by sequencing.
BL21(DE3) pLysS strain of E. coli was transformed with each
expression vector and incubated overnight. Each culture was diluted 10-fold with fresh LB medium containing 100 µg/ml ampicillin and incubated for additional 2 h at 37 °C. The cells were harvested 3 h after the addition of
isopropyl-1-thio-
-D-galactopyranoside at a final
concentration 0.2 mM and mildly sonicated in extraction buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol). Lysates were prepared by centrifugation at 5,000 × g
for 20 min at 4 °C and loaded on columns of amylose resin (New
England Biolabs). The columns were washed with extraction buffer and
subsequently with buffer B (20 mM Hepes-NaOH (pH 7.9), 20 mM NaCl, 4 mM MgCl2, 1 mM EDTA, 20% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 1 mM dithiothreitol).
Recombinant proteins were eluted with buffer B containing 10 mM maltose and analyzed by SDS-polyacrylamide gel
electrophoresis and Coomassie Brilliant Blue staining (31).
Electrophoretic Gel Mobility Shift Analyses (EMSA) and Off-rate
Kinetic Experiments Using MBP-CNC Proteins--
Oligonucleotides
encoding the chicken
-globin enhancer MARE sequence
(5'-TCGCCCGAAAGGAGCTGACTCATGCTAGCCC-3') were
labeled with
[32P]ATP by T4 polynucleotide kinase. The
reaction mixture contained 20 mM Hepes-NaOH (pH 7.9), 1 mM EDTA, 60 mM NaCl, 1 mM
dithiothreitol, 4 mM MgCl2, 25 µg of
poly(dI-dC) and poly(dA-dT)/ml, 100 µg of bovine serum albumin/ml,
and 50 ng of purified proteins. The reactions were electrophoresed at
4 °C, 150 V on a 4.5% polyacrylamide gel in 0.5 × TBE (45 mM Tris-HCl (pH 8.2), 45 mM borate, and 1.25 mM EDTA). Competition assays using various cold probes were
performed as described previously (8). In off-rate kinetic experiments, EMSA reaction mixture was first incubated with radiolabeled probe at
37 °C for 5 min. After the addition of cold double-stranded oligonucleotides (560-fold molar excess), an aliquot of the mixture was
taken and analyzed by polyacrylamide gel electrophoresis at several
time points (described in figure legend). Amounts of binding complexes
were quantified by an imaging analyzer (Fuji BAS-2000).
Chromosome Mapping by Fluorescence in Situ
Hybridization--
Metaphase spreads for FISH were prepared from
phytohemagglutinin-stimulated normal human lymphocytes using a
thymidine synchronization/deoxybromouridine release technique. Nrf3
cDNA was labeled by nick translation with biotin-16-dUTP
(Boehringer Mannheim). In situ hybridization was performed
with the protocol of Inazawa et al. (32) with minor modifications. Briefly, for hybridization, 200 ng of probe was added to
10 ml of a hybridization mixture containing 50% formamide, 2× SSC,
10% dextran sulfate, and 20 µg/ml bovine serum albumin at 37 °C
for 90 min. Slides were denatured in 70% formamide, 2× SSC at
72 °C for 3 min, dehydrated, and hybridized overnight in a moist
chamber at 37 °C with the probe. The slides were washed in 50%
formamide, 2× SSC and in 1× SSC at 37 °C for 10 min each, and then
in 4× SSC at room temperature for 10 min. After washing, the slides
were incubated with avidin-fluorescein isothiocyanate at 37 °C for
50 min and then counterstained with DAPI in anti-fade solution.
Analysis was carried out using an Olympus BX50-FLA fluorescence microscope and photographs were taken with Kodak Ektachrome ASA 400 film.
Nucleotide Sequence Data--
The nucleotide sequence data
reported in this paper will appear in the DDBJ, EMBL, and
GenBankTM nucleotide sequence data bases with the following
accession numbers, AB010812 (human) and AB013852 (mouse).
 |
RESULTS |
Isolation of a cDNA Clone Encoding a Novel CNC Family
Member--
Loci for the p45, nrf1, and
nrf2 genes have been mapped near the hoxC,
hoxB, and hoxD gene clusters, respectively (24), but no CNC factor mapping close to the hoxA locus had yet
been identified, suggesting that a novel CNC factor may exist. We
searched for CNC factors in the TIGR human EST data base and found a
668-bp EST clone (GenBankTM accession number THC181377)
encoding an amino acid sequence homologous to that of the C-terminal
end of Nrf1. As this EST clone may encode a novel CNC factor, we set
out to isolate the corresponding or related DNA fragments.
We first attempted to isolate a genomic DNA fragment corresponding to
this EST clone, since nrf1 and nrf2 genes
are known to contain a large C-terminal exon (5, 6). The C-terminal exon sequence was amplified by PCR using genomic DNA of HeLa cells as a
template, and a fragment with sequence matching exactly to the EST
clone was successfully obtained (data not shown). To obtain a full
length cDNA clone, a human placenta cDNA library was screened using the PCR clone, and four positive overlapping phage clones were
isolated (data not shown; see below).
To isolate the mouse homologue, we screened a mouse brain cDNA
library using one of the human partial cDNA clones and obtained eight positive phage clones. As shown in Fig.
1A, the longest mouse cDNA
clone encodes a 1983-bp open reading frame flanked by 237 and 339 bp of
5'- and 3'-untranslated regions (UTRs), respectively. Although no
preceding in-frame stop codon was noted in the 5'-UTR of this cDNA,
we identified a termination codon 15 nucleotides upstream from
5'-terminal of this cDNA in the mouse genomic
clone,2 supporting our
assigned translation initiation codon. The nucleotide sequence
GAGATGA surrounding the ATG codon (underlined) matches the
translation initiation site consensus sequence reported by Kozak (33).
The open reading frame encodes a predicted protein of 660 amino acid
residues with a calculated molecular mass of 72,706 Da (Fig.
1A). Two copies of RNA destabilizing signal (ATTTA) (underlined; Ref. 34) reside in the 3'-UTR.

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Fig. 1.
Cloning and structure of Nrf3.
A, nucleotide sequence and deduced amino acid sequence of
mouse Nrf3 cDNA. The CNC and basic domains and leucine residues in
zipper domains are indicated by the dotted line,
box, and circles, respectively. A polyadenylation
consensus sequence is indicated by the thick
underline. Two mRNA destabilizing sequences in 3'-UTR
and one CNC factor-conserved amino acid sequence
(DSGLSL) are underlined. B, comparison
of amino acid sequences of mouse and human Nrf3. Sequence information
from human Nrf3 genomic sequence data of bacteria artificial chromosome
clone (AC004520) is underlined.
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Sequence analysis of the longest human clone (number 1-1) and
comparison of the predicted protein with that of the mouse revealed that the human clone is likely to be partial (Fig. 1B),
since the N-terminal coding region was shorter than that of the mouse protein. Despite extensive 5'-rapid amplification of the cDNA end
analyses using human placental mRNA, we were unable to isolate any
additional upstream sequence (data not shown). However, the genomic
sequence data of a bacteria artificial chromosome clone containing this
locus was recently entered in the GenBankTM data base from
the Genome Sequencing Center of Washington University (accession number
AC004520) and that sequence information has been incorporated into the
human cDNA sequence (Fig. 1B). Comparison of the
N-terminal structure of the human protein with that of the mouse
revealed that their deduced coding sequences are less conserved (68%
homology) than those of other CNC family members (89, 97, and 80%
overall identities, respectively, for p45, Nrf1, and Nrf2)
between mouse and human.
Nrf3 Is a New Member of CNC Family--
Inspection of the domain
structure of the new mouse clone revealed the presence of a bZip domain
which is homologous to that of other CNC transcription factors: 53, 56, and 50% identity, respectively, for p45, Nrf1, and Nrf2 (Fig.
2A), indicating that this
protein is a member of the CNC family. We therefore named this factor
Nrf3 (NF-E2 related
factor-3).

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Fig. 2.
Comparison of Nrf3 with other CNC
factors. A, schematic alignment of the structures of
murine p45-related factors, Nrf3, p45, Nrf1, and Nrf2. The CNC
domain and basic region are depicted by stippled boxes. An
Nrf1 and Nrf2/ECH conserved region, Domain 2 (7), is
shown by hatched boxes. B, comparison of the
CNC-bZip domains of CNC family proteins. Conserved amino acids are
indicated by dots. The basic region and leucine residues in
the zipper domain are indicated by the thick line and
filled diamonds, respectively.
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In the bZip region, the basic region of Nrf3 was especially highly
conserved with that of other CNC factors (see Fig. 2B), suggesting that Nrf3 may share their DNA binding specificity. A short
region "DSGLSL" that is absolutely conserved among the CNC family
members (24) is also present in Nrf3 (see Fig. 1A, underlined). The leucine-zipper sequence of Nrf3, however,
was much more variable than those of other CNC factors.
DNA Binding Activity of Nrf3--
The three CNC factors currently
analyzed (i.e. p45, Nrf1, and Nrf2) have all been
shown to bind MAREs as heterodimers with one of the small Maf proteins,
but not as homodimers. Since one of these small Mafs, MafK (9, 11, 12),
is highly coexpressed with Nrf3 (see below) in the placenta, these two
factors are likely to be partners, at least in this tissue. We
therefore examined whether a complex of Nrf3 and MafK could bind to the
MARE of the chicken
-globin enhancer by EMSA.
Bacterially expressed MBP-fused bZip domains of the CNC proteins were
tested for their ability to bind MARE. In the absence of MafK, no DNA
binding was observed (Fig. 3A,
lanes 2-4). Although a homodimer of MafK could
bind the probe (lane 5), the affinity appeared to
be much weaker than the CNC-MafK heterodimers (lanes 6-8). The binding of Nrf3-MafK was efficiently competed out
with addition of cold double-stranded oligonucleotides encoding the MAREs of the chicken
-globin enhancer and the hypersensitive site 2 (HS-2) of human
-globin LCR (Fig. 3B, lanes
3 and 5), but not by mutated MAREs (lane
4). Similarly, the MARE sequence of the erythroid
5-aminolevulinate synthase gene promoter, to which NF-E2 cannot
efficiently bind,3 did not compete with the
binding of the labeled probe (lane 6). These
results demonstrate the specificity of the recognition for MAREs by the
Nrf3-MafK heterodimer complex.

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Fig. 3.
DNA binding activity of Nrf1-, Nrf2-,
and Nrf3-MafK in EMSA. A, MBP, MBP-Nrf1,
MBP-Nrf2, and MBP-Nrf3 proteins (50 ng) were incubated with
chicken -globin enhancer MARE in the presence (lanes
5-8) or absence (lanes 1-4) of
MBP-MafK protein (50 ng). B, DNA recognition specificity of
Nrf3-MafK complex was examined by competition assay. 100-fold excess
competitors were added to the reaction. Competitors were
double-stranded cold oligonucleotides containing wild type
( E, lane 3) or mutated -globin
3'-enhancer MARE ( E/M, lane 4), LCR
HS-2 MARE (HS2, lane 5) and
erythroid-specific 5-aminolevulinate synthase MARE
(ALAS, lane 6). C,
DNA binding affinities of CNC factor-MafK complexes were determined by
off-rate kinetic experiments. After addition of 560-fold excess
competitors, a portion of the binding mixture was taken at 0, 3, 6, 9, 15, 20, and 25 min time points, respectively, and separated by
polyacrylamide gel electrophoresis. D, the dissociation
curve of CNC factors-MafK complex from DNA. Intensity of the time 0 band was arbitrary assigned to 1, and other data were normalized on the
basis of this. The values represent average of three independent
experiments.
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To assess binding affinities of the three CNC factors to MARE, a series
of off-rate kinetic experiments was carried out (Fig. 3C).
These CNC factors were mixed with MafK and radiolabeled MARE probe, and
the mixtures were subjected to the addition of cold double-stranded
oligonucleotides. The mixtures were then separated at several time
points by polyacrylamide gel electrophoresis. Fig. 3D shows
half-times of Nrf1-, Nrf2- and Nrf3-MafK-DNA complexes. The
half-time of dissociation of Nrf1- and Nrf3-MafK complex from MARE was
approximately 5 min, while that of Nrf2-MafK complex was within
3 min. These results suggest that Nrf1- and Nrf3-MafK complexes could
bind the MARE probe more tightly than Nrf2-MafK complex in this
experimental condition.
Nrf3-MafK Dimer Formation in Yeast Cells--
We then examined
dimerization affinities between Nrf3 and MafK using the yeast
two-hybrid system. For this purpose the S. cerevisiae strain
SFY526 was utilized which carries a lacZ reporter gene with
binding sites for GAL4 (upstream activating sequence). Hybrid proteins
of human Nrf1, Nrf2 (both full length), and Nrf3 (lacking the
N-terminal) fused to the GAL4 activation domain (GAD) were used for
"prey," whereas a fusion of human MafK to the GAL4 DNA-binding
domain (GBD) was used as "bait" (Fig.
4). Dimerization activities between these
chimeric proteins were determined by measuring the LacZ activity.

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Fig. 4.
Schematic presentation of GBD-MafK and
GAD-CNC factors. cDNAs for human Nrf1 and Nrf2
(full-length) and Nrf3 (lacking the N-terminal) were fused to the GAL4
activation domain (GAD), and cDNA for human MafK was
fused to the GAL4 DNA binding domain (GBD). One of the
Nrf2/ECH conserved domain, Domain 2 (7), is shown as
hatched boxes.
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As shown in Table I, control experiments
using GBT9 plasmid (GBD alone) and GAD-Nrf1, GAD-Nrf2, or
GAD-Nrf3 did not exhibit LacZ reporter activity. Similarly, GBD-MafK
and GAD424 plasmid (GAD alone) showed no LacZ activity. In contrast,
considerable enhancement of LacZ activity was observed when the yeast
cells were transformed with GBD-MafK and GAD-Nrf1, GAD-Nrf2, or
GAD-Nrf3, indicating the presence of interaction between MafK and these CNC family members. GAD-Nrf2 and -Nrf3 showed much stronger
enhancement of LacZ activity than GAD-Nrf1 did in this yeast
interaction assay. These results thus show that MafK-Nrf2 and
-Nrf3 may have stronger heterodimerizing activities than MafK-Nrf1
does, although there is a possibility that a part of the enhancement of
LacZ activity is derived from the activation domain of CNC factors in
the GAD constructs.
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Table I
Interaction of p45-related CNC factors with MafK in a yeast two-hybrid
system
Yeast strain SFY526 containing lacZ gene under the control
of the GAL1 promoter was transformed with the plasmids indicated, and
-galactosidase activity was determined.
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Nrf3 Is a Transcriptional Activator--
The
trans-activation activity of Nrf3 was examined by transient
transfection assay using two reporter plasmids, pRBGP2, encoding three
copies of chicken
-globin MARE, and pRBGP4, containing mutated
binding sites (10). The reporter plasmids were transfected with various
amounts of Nrf3, Nrf1, and Nrf2 expression plasmids into QT6
quail fibroblasts. Co-transfection of the Nrf3 expression plasmid with
the pRBGP2 reporter resulted in activation of the reporter by
approximately 4-fold (Fig. 5A,
compare lanes 1 and 4) in a
dose-dependent manner. This activation was not observed with the pRBGP4 reporter (Fig. 5B), demonstrating that the
activation by Nrf3-MafK is dependent on binding to the MAREs.
Furthermore, while the expression of high doses of MafK efficiently
repressed the pRBGP2 reporter plasmid (Fig. 5C,
lane 2), co-expression of Nrf3 overcame this
MafK-mediated repression (see lanes 3-5),
indicating that Nrf3 is a transcriptional activator.

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Fig. 5.
Transcriptional activity of Nrf3.
A, mouse Nrf3, Nrf1, and Nrf2 expression plasmids (1, 10, and 100 ng) were transfected into QT6 cells together with pRGBP2
reporter. B, mouse Nrf3, expression plasmid (1, 10, and 100 ng) was transfected into QT6 cells with pRGBP4 reporter. C,
co-transfection of Nrf3 and MafK expression plasmids with pRBGP2
reporter. Increasing amounts of the Nrf3 expression plasmid (1, 10, and
100 ng) were transfected in the presence of mouse MafK expression
plasmid (10 ng) and pRBGP2 reporter.
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Nrf2 has been shown to be the strongest
trans-activator among the CNC factors (11), and this
observation was reproducible in the present analysis (see Fig.
5A). We speculate that this may be either due to the
stronger trans-activation activity of Nrf2 or the
high affinity of Nrf2 for endogenous MafK than Nrf1 or Nrf3.
Nrf3 showed strong enhancement of the LacZ reporter expression in the
yeast two-hybrid assay (see Table I), suggesting that Nrf3 may activate
transcription less efficiently than Nrf2 does in QT6 cells.
Precise functions of Nrf3 activation, dimerization, and DNA binding
domains, however, remain to be elucidated.
Expression Pattern of Nrf3--
To investigate the functional role
of Nrf3, the expression profile of human Nrf3 was determined by RNA
blot analysis. High levels of Nrf3 mRNAs were detected in placenta
and also in various other tissues, but at lower levels (Fig.
6A). In all cases, two distinct species of Nrf3 transcripts were observed.

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Fig. 6.
Expression profile of Nrf3.
A, RNA blot analysis with poly(A)+ RNAs derived
from human adult tissues. Positions of molecular markers are shown on
both sides. B and C, RNA blot analysis using
mRNA from various hematopoietic lineage cell lines. Positions of
28 S and 18 S rRNA are indicated by lines.
|
|
We also examined the expression of Nrf3 in various hematopoietic cell
lines. High levels of Nrf3 mRNA were detected only in a B cell
line, Raji, and monocytic cell lines THP-1 and U937, whereas low levels
of Nrf3 transcripts were widely present in all cases except CCRF-CEM
and KG-1 (Fig. 6B). The expression of Nrf3 in the B cell
lineage was intriguing and further examined using additional B and T
cell lines. We found that Nrf3 is expressed in RPMI8226, a myeloma cell
line and three Burkitt's lymphoma cell lines, Raji, BALM-2, and
NAMALWA, but not in pre-B cell lines, NALM-6, NALM-17, and NALL-1 (Fig.
6C). Nrf3 mRNA was also not expressed in two T cell
lines, KOPT-K1 (Fig. 6C) and CCRF-CEM (Fig. 6B).
These results suggest that Nrf3 may play an important role in B cell
and monocyte lineages.
nrf3 Gene Is Mapped Close to HoxA Gene Cluster--
We determined
the chromosomal localization of nrf3 gene by FISH analysis
of a total of 142 metaphase and prometaphase
phytohemagglutinin-stimulated lymphocytes. Fluorescent spots on one or
both chromatids of chromosome 7 were observed in 94 metaphases
(66.2%), and 25 plates (14.8%) displayed double signals on both
homologues of chromosome 7. No significant signals were observed at any
other chromosomal location. DAPI Q-banding was used to locate the probe
on the short arm of chromosome 7. Localization of the hybridization
signals was determined by comparing 10 selected FISH photos with the
corresponding Q-band photos and assigned to 7p14-15 (Fig.
7, A and B, shown
by arrowhead), where the hoxA locus is also
mapped. In addition, a human bacteria artificial chromosome clone
containing the nrf3 gene sequence was isolated from 7p15
(data not shown).

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Fig. 7.
Chromosome assignment of the human
nrf3 gene by FISH. A, metaphase
chromosome spread hybridized with Nrf3 probe, showing the presence of
fluorescent signals at chromosome region 7p14-15
(arrowhead). B, the same chromosomes
demonstrating Q bands with DAPI.
|
|
An evolutionary tree comparing bZip domain sequences among CNC family
revealed that Nrf3 might have been the first derivative from an
ancestral gene (Fig. 8) and that the CNC
family may consist of two subfamilies, p45-related factors and the Bach
family.

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Fig. 8.
A phylogenetic tree of murine CNC family
proteins. Phylogenetic tree was constructed by analyzing the amino
acid sequences of CNC-bZip domains using the Genetyx-Mac UPGMA method
(Software Development).
|
|
 |
DISCUSSION |
In this study, we isolated and characterized a new CNC family
transcription factor Nrf3, which binds to MARE as a heterodimer with
small Maf proteins and functions as a transcriptional activator. The
nrf3 gene maps near the hoxA gene cluster. Since
genes for p45, nrf1, and nrf2
are closely linked to the hoxC, hoxB, and hoxD clusters, the present result implies that CNC factors
may be derived from a single ancestor, as is the case for the
hox gene clusters. The evolutionary tree analysis and other
results clearly indicate that the CNC family is composed of two
distantly related subfamilies, the p45-related and Bach subfamilies. It is interesting to note that members of the former subfamily all contain
a conserved "DSGLSL" region, referred to as domain A (24), in the N termini.
The basic region of the Nrf3 bZip domain shows high structural
similarity to those of other p45-related CNC factors (see Fig. 2B), suggesting that they recognize similar or overlapping
DNA sequences. Indeed, EMSA showed that Nrf3-MafK binds to the MARE in
the chicken
-globin enhancer, which is known to be a common target
sequence of p45-, Nrf1-, and Nrf2-small Maf heterodimers. In
yeast two-hybrid assays, Nrf3 (and Nrf2) enhanced the LacZ reporter expression to a much greater extent than Nrf1, suggesting that
CNC factors differ in their ability to dimerize with small Maf proteins
and hence in their DNA binding affinities (shown in Fig. 3,
C and D). However, trans-activation
activity of Nrf3 was weaker than that of Nrf1 and much weaker than that
of Nrf2, indicating that the activation potentials also differ
between factors. These divergences in heterodimer formation and
trans-activation abilities of the CNC factors were most
likely acquired during their evolution.
We recently demonstrated that phosphorylation is involved in the
p45-dependent trans-activation process (35). The
DNA binding and trans-activation activities of the p45-MafK
complex appeared to be positively regulated by serine/threonine
phosphorylation in mouse erythroleukemia cells. Since Nrf3 contains
several phosphorylation consensus sites for protein kinase A, protein
kinase C, and casein kinase II (data not shown), one plausible
explanation of the above observation (i.e. Nrf3 exhibited
strong activity in yeast assays but weak activity in transfection
assays) is that the trans-activation activity of Nrf3 may be
under the regulation of specific protein kinases in the cytokine signal
cascade, and this regulation may be absent in the fibroblast
transfection assays.
Recently, MafG was reported to interfere with
trans-activation by Nrf1 in transfection studies using COS1
cells. Based on that observation, the authors (36) claimed that Nrf1
does not cooperate with MafG for trans-activation but may
utilize a hypothetical partner molecule for this purpose in
vivo. Although the discrepancy between their observation and ours
(Fig. 5C and see Ref. 7) requires further testing, the
proposition of new bZip partner molecule(s) for the CNC factors has not
been examined yet. In this regard, we have extensively screened for
"partner molecule(s) of CNC factors" using Nrf2/ECH
molecules in yeast two-hybrid assays, but to date have not isolated any
suitable candidates with the expected function.3 Since we
observed significant differences among the CNC factors in both yeast
two-hybrid assay and EMSA, an alternative explanation for the
difference in results may be the variation in the affinity of the CNC
factors for the small Mafs.
We observed that Nrf3 and MafK were both abundantly expressed in
placenta (this study and see Refs. 11 and 37). The placental giant
cells produce a number of hormones, including four members of the
prolactin/growth hormone family: placental lactogen I and II (PL-I and
-II), proliferin, and proliferin-related protein. A regulatory element
recognized by AP-1 has been shown to be essential for the
trophoblast-specific transcription of the mouse PL-I gene (38). Since the AP-1 binding sequence is completely integrated in MARE,
the possibility exists that Nrf3 acts as a regulator of the
PL-I gene through this site. To shed more light on the biological role of Nrf3, it is necessary to investigate the mRNA distribution by in situ hybridization and elucidate the
Nrf3-positive cell types in placenta.
Nrf3 is also expressed abundantly in B cell and monocyte
lineages. It is interesting to note that, in B cell development, Nrf3
mRNA is expressed after the pre-B cell stage, especially in
Burkitt's lymphoma cell lines. This expression profile is in clear
contrast to that of Bach2; Bach2 is expressed in pro-B cell stage, and
its expression is extinguished in the plasma cell stage.3
Thus, whereas Nrf3 and Bach2 share similar binding sequence specificity (i.e. both bind to MARE), they show reciprocally exclusive
expression profiles during B cell development. Whether Nrf3 induces the
expression of regulators of the class switch and B cell maturation
remains to be clarified.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Shigeru Taketani for providing
the human placenta cDNA library. We also thank Drs. Taka-aki Abe,
Ken Itoh, Tadashi Nagai, Kazuhiro Sogawa, Ken-ichi Yasumoto, and Ruth
Yu for advice and discussion.
 |
FOOTNOTES |
*
This work was supported by Grants-in-Aid from the Ministry
of Education, Science, Sports, and Culture, Core Research for
Evolutional Science and Technology, and the Japanese Society for the
Promotion of Sciences.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) AB010812 (human) and AB013852 (mouse).
§
To whom correspondence should be addressed: Dept. of Biochemistry,
Tohoku University School of Medicine, 2-1 Seiryomachi, Aoba-ku, Sendai
980-8575, Japan. Tel.: 81-22-717-8088; Fax: 81-22-717-8090; E-mail:
akira-k{at}mail.cc.tohoku.ac.jp.
2
A. Kobayashi, unpublished observations.
3
K. Igarashi and M. Yamamoto, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
bZip, basic
region-leucine zipper;
CNC, Cap'n' collar;
EMSA, electrophoretic gel
mobility shift analysis;
EST, expressed sequence tag;
FISH, fluorescence in situ hybridization;
MARE, Maf recognition
element;
MBP, maltose-binding protein;
Nrf3, NF-E2-related factor 3;
LCR, locus control region;
UTR, untranslated region;
bp, base pair(s);
kbp, kilobase pair(s);
PCR, polymerase chain reaction;
GAD, GAL4
activation domain;
GBD, GAL4 DNA-binding domain.
 |
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