From the Department of Virology, Institute of Medical
Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku
108-8639, Tokyo, Japan and the
Department of Molecular and
Experimental Medicine, The Scripps Research Institute, La
Jolla, California 92037
Received for publication, August 22, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Maf oncoprotein is a basic-leucine zipper (bZip)
type of transcriptional activator. Since many transcription factors are
known to form functional complexes, we searched for proteins that
interact with the DNA-binding domain of Maf using the phage display
method and identified two homeodomain-containing proteins, Hoxd12 and MHox/Prx1/Phox1/Pmx1. Studies with mutants of Hox and Maf proteins showed that they associate through their DNA-binding domains; the
homeodomain of Hox and the bZip domain of Maf, respectively. Reflecting
the high similarity of the bZip domain, all other Maf family members
tested (c-/v-Maf, MafB, MafK, MafF, and MafG) also associated with the
Hox proteins. Pax6, whose homeodomain is relatively similar to MHox,
also could interact with Maf. However, two other bZip oncoproteins, Fos
and Jun, failed to associate with the Hox proteins, while a distantly
related Hox family member, Meis1, could not interact with Maf. Through
interactions with the bZip domain, the Hox proteins inhibited the DNA
binding activity of Maf, whereas the binding of Hox proteins to their
recognition sequences was not abrogated by Maf. We further showed that
coexpression of the Hox proteins repressed transcriptional activation
and transforming activity of Maf. These results suggested that the
interaction of a set of Hox proteins with Maf family members may
interfere not only with their oncogenicity but also with their
physiological roles.
v-maf oncogene, originally identified in the genome of
acute transforming avian retrovirus AS42, induces musculoaponeurotic fibrosarcoma in chickens and causes transformation of chicken embryo
fibroblast (CEF)1 cells (1,
2). Its product, v-Maf, contains a basic-leucine zipper (bZip)
structure at its carboxyl terminus, forms a homodimer, and recognizes
relatively long palindromic sequences named Maf-recognition elements
(MARE: TGCTGACTCAGCA and TGCTGACGTCAGCA) (3-5). MARE sequences include the phorbol
12-O-tetradecanoate-13-acetate-responsive element
(TGACTCA) and cyclic AMP-responsive element (TGACGTCA), which are recognized by various homo- and heterodimers of AP-1 (Jun/Fos) and the ATF/CREB family of bZip proteins. Maf is a
transcriptional transactivator, and the DNA binding and transactivation
activities are necessary for its transforming ability (4, 6).
Structural analysis of v-maf and its cellular counterpart
c-maf revealed that the v-Maf protein had no structural
change compared with c-Maf except that it was fused to the viral Gag
protein at the initiator methionine residue of c-Maf (2). In accordance
with this finding, expression of c-maf under control of a
retroviral long terminal repeat causes efficient transformation of CEF
cells (5). Recently, c-maf overexpression was found in a
fraction of human multiple myelomas that resulted from chromosomal
translocation to the immunoglobulin heavy or light chain locus (7).
Up-regulation of c-maf was also found in melanoma cells due
to the insertion of the melanoma-associated retrovirus (MelARV)
(8).
To date, several maf-related genes have been isolated from
various vertebrates, including human, mouse, rat, chicken, quail, frog,
and zebrafish. They are divided into two subfamilies according to the
structures of the encoded proteins. The first group, which consists of
c-Maf, MafB, Nrl, and MafA/L-Maf, has an amino-terminal transactivation
domain in addition to the COOH-terminal bZip domain and are called
large Maf proteins. The second group, MafK, MafF and MafG, lack the
amino-terminal domain and thus are called small Maf proteins.
One of the most important control mechanisms of transcriptional
regulation by bZip factors is homo- and heterodimerization through
leucine zipper domains. For example, the small Maf proteins can
positively regulate a set of erythroid-specific genes as heterodimers with another b-Zip protein, NF-E2 p45, a member of the Cap'n'collar family of transcription factors (9, 10). However, the small Maf
proteins also can act as repressors for the erythroid-specific genes by
forming homodimers or heterodimers with c-Fos (11). Similarly, the
small Maf proteins can form dimers with another bZip factor Bach2 and
act as a B-cell-specific negative regulator of the immunoglobulin heavy
chain 3' enhancer (12). As for v-/c-Maf, it has been shown to form
heterodimers with both Jun and Fos (4, 13). The functional importance
of Maf/Jun or Maf/Fos heterodimers is not yet known, but these
heterodimers are different in their DNA binding specificity from Maf
homodimers or AP-1 complexes and are likely to regulate a distinct set
of cellular genes.
Jun and Fos are the most well characterized nuclear oncoproteins,
and their interaction with members of other bZip protein families such
as ATF/CREB and C/EBP has been examined (14-16). Furthermore, Jun and
Fos are known to form many kinds of functional complexes with different
classes of transcription factors. For example, direct interaction of
the Jun/Fos heterodimer with the NF-AT transcription factor is
necessary to activate expression of T-cell-specific genes (17, 18).
Interactions of Jun and Fos with the glucocorticoid receptor or some
members of the Ets family also have been reported (19-21). These
studies shed light not only on their physiological roles but also on
the mechanisms of cell transformation. In the case of c-Maf,
interaction with the proto-oncogene product c-Myb was found to regulate
transcription of the CD13/APN gene during myeloid cell
differentiation (22). Its close relative, MafB, also has been shown to
interact with and to repress transcriptional activity of c-Ets-1, which
leads to inhibition of differentiation of erythroid cells (23).
Collectively, fine tuning of cell type-specific gene expression and
lineage-specific cell differentiation seems to be achieved by
cooperative and inhibitory interactions of transcription factors.
Disregulated expression of a nuclear oncoprotein thus may affect the
cell differentiation program, which in turn may lead to cell transformation.
To understand the mechanism of transcriptional regulation and cell
transformation by Maf, it is important to clarify the regulatory cross-talk that occurs with other transcription factors. To this end,
we utilized the phage display method to screen a cDNA expression library for genes whose products interact with Maf and isolated two
genes that encode homeodomain-containing transcription factors, Hoxd12
and MHox/Prx1/Phox1/Pmx1. These two proteins repressed DNA binding and
transforming activities of Maf by interacting with its bZip domain. The
possible biological significance of this interaction network also will
be discussed.
Phage Display--
cDNA was made from poly(A)+
RNA isolated from whole chicken at embryonic day 8 and was inserted
into the EcoRI and HindIII digested
pT7Select1-1b vector (Novagen), which should display products of the
inserted cDNA on the surface of T7 phage particles as a fusion
product with its capsid protein. Phage were packaged in
vitro and used to infect the host bacterial strain, BLT5403 (Novagen). Enrichment of recombinant phage that expressed candidate Maf-binding proteins by biopanning was performed as recommended by the manufacturer with some modifications. Briefly, wells of microtiter plates were coated with bacterially expressed and purified maltose binding protein (MBP) or MBP-Maf fusion protein in
Tris-buffered saline (TBS) and blocked with 10% Blockace
(Dainihon Seiyaku, Osaka, Japan) in TBS. A 10-µl aliquot of fresh
phage lysate (1 × 108 pfu/µl) containing 2.2 × 106 independent clones was incubated in a MBP-coated
well at room temperature for 30 min to absorb phage particles that have
affinity for MBP or blocking reagent. Then the lysate was transferred
into a MBP-Maf-coated well, incubated for 30 min, removed, and the well
washed five times with 200 µl of TBS containing 0.1% of Tween 20. The phages that remained in the well were eluted with 20 µl of TBS
containing 0.1% Tween 20 and 1% sodium dodecyl sulfate (SDS) and
amplified in BLT5403 cells. After seven rounds of this two-step
biopanning procedure, the titer of MBP-Maf bound phage was five
times higher than background. The eluted phages were combined with host
cells to form plaques on agar plates. The cDNA insert of individual
phages was amplified by the polymerase chain reaction (PCR) using
T7Select UP and DOWN primers (Novagen), subcloned into the
EcoRI-HindIII site of pUC19, and subjected to
nucleotide sequence analysis.
Coprecipitation Analysis by MBP or GST Fusion
Proteins--
Construction of the prokaryotic expression vector for
the MBP-Maf fusion protein has been described (4, 11, 24). The procedures for expression of MBP and MBP-Maf proteins in
Escherichia coli and for purification by amylose resin
chromatograhy (New England Biolabs) also have been described
previously (4).
To produce a GST-fusion protein of Hoxd12 in E. coli, a
hoxd12 cDNA isolated by the phage display method was digested with BamHI at the 3'-noncoding region, treated with T4 DNA
polymerase, ligated with EcoRI linkers, and digested with
EcoRI. The resultant EcoRI fragment encoding the
103 COOH-terminal amino acids of Hoxd12 was inserted into the
EcoRI site of the pGEX-3X plasmid (Amersham Pharmacia
Biotech). GST and GST-Hoxd12 proteins were purified by
glutathione-Sepharose 4B column chromatograpy (Amersham Pharmacia Biotech).
cDNA fragments encompassing the entire open reading frames of
hoxd12, mhox, and pax6 of chicken and
meis1 of mouse were amplified by reverse transcriptase-PCR
from total RNA from day 8 whole chicken embryo and from the Balb/c/3T3
cell line, respectively, using specific primers (hoxd12,
5'-AGAGCGCGCAGTCCTTTGTTGGAAATGTG-3' and
5'-AGAGCGCGCCGCGCTAGTACATAGAGAG-3'; mhox,
5'-AGAACGCGTACGAGGAAAAGCCCCCGCTG-3' and
5'-GAGACGCGTGCGAAGCAGCTGCCCCCAG-3'; pax6,
5'-GAGACGCGTGCCTGCCCCGGCCCACCATGC-3' and
5'-GAGACGCGTGAATTAACACATGTTTTACTG-3'; meis1,
5'-AGAACGCGTAGGAAGGGAGCCAGAGAGG-3' and
5'-AGAACGCGTAGATGAAGGTTACATGTAGTG-3'). Products were
digested with BssHII or MluI and inserted into
the MluI site of pGEM3-MluI or
pGEM4-MluI vectors (25). To construct
hoxd12
Structures of plasmids used for in vitro translation of the
maf-related genes, mafB, mafK,
mafF, and mafG of chicken (11, 24, 25) and
v-maf mutants (5) have been described.
For in vitro translation of human c-Jun and c-Fos, a
PmaCI-StyI fragment of c-jun cDNA
and a HaeII-BanI fragment of c-fos cDNA, both of which contain entire open reading frames, were
blunt-ended by T4 DNA polymerase, ligated with MluI linkers
(pGACACGCGTGTC), and inserted into pGEM4-MluI.
These plasmids were linearized with appropriate restriction enzymes and
were transcribed and translated in vitro using the TNT
Coupled Wheat Germ Extract System (Promega) in the presence of
[35S]methionine. 10 µl of the programmed extracts were
added to 800 µl of phosphate-buffered saline containing 1% bovine
serum albumin and 0.05% Tween 20 together with 20 µl of amylose
resin or glutathione-Sepharose 4B immobilized with MBP or GST fusion
proteins, respectively. After overnight incubation at 4 °C, samples
were washed three times with 800 µl of phosphate-buffered saline
containing 0.05% Tween 20 and analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE).
Gel Mobility Shift Analysis--
XhoI restriction
sites were introduced at the first leucine residue of the zipper
structure of Maf and Jun by the method of Kunkel (26) using
oligonucleotide primers (5'-CTCCGACTCgAGGACGTGCCGC-3' for
v-maf and 5'-CACTTTTTCcTCgAgCCTGGCAATCC-3' for
v-jun) without changing amino acids, and the resultant
plasmids were used to construct a chimera of maf and
jun (maf(jun-zip)). Maf, Jun, Fos, and Hox
proteins were transcribed and translated in vitro using the
TNT Coupled Wheat Germ Extract System (Promega), and were analyzed by a gel mobility shift assay as described previously (4).
Oligonucleotide probes containing MARE (probe 7) or AP-1 (probe 11)
also have been described (4). A double-stranded oligonucleotide probe
containing the Hoxd12 recognition sequence (27) was made from the
oligonucleotides: 5'-GATCCTTCACTCCGTTTTACGACAGGAGTA-3' and
5'-GATCTACTCCTGTCGTAAAACGGAGTGAAG-3'.
Luciferase Assay--
For eukaryotic expression,
BssHII fragments of hoxd12 or Hoxd12
3 × 105 CEF cells, grown on a 35-mm dish, were
transfected with a total of 1 µg of plasmid (125 ng of luciferase
reporter plasmid, 750 ng of expression plasmids, and 125 ng of
pEF-Rluc plasmid) using 1 µl of Superfect Transfection
Reagent (Qiagen) as recommended by the manufacturer. The cells
were harvested at 18 h post-transfection, and luciferase activity
was measured using the Dual-Luciferase Reporter Assay System (Promega).
Focus Formation Assay--
To construct a NotI
cassette plasmid with the internal ribosome entry site (IRES), an
EcoRI-NotI-EcoRI adaptor
(5'-AATTCGGCGGCCGCGGATCCCCTCGAGTTCG-3' and
5'-pAATTCGAACTCGAGGGGATCCGCGGCCGCCG-3')
and an NcoI-SmaI-NotI-EcoRI adaptor (5'-AATTCGGCGGCCGCGGATCCGACCCGGGATC-3'
and
5'-pCATGGATCCCGGGTCGGATCCGCGGCCGCCG-3') were added to the 5' and 3' ends, respectively, of the
EcoRI-NcoI fragment of pCITE-1 (Novagen)
containing the IRES of encephalomyocarditis virus and inserted into the
EcoRI site of a modified pUC vector. An NcoI
restriction site was introduced into the 75th codon of hoxd12 and Hoxd12
Preparation and maintenance of CEF cells have been described
previously (24). For the focus formation assay, 2.5 µg of the recombinant retrovirus vector plasmid DNA was transfected into 1.2 × 106 CEF cells grown on a 60-mm dish using 12.5 µl of
Effectene Transfection Reagent (Qiagen) as recommended by the
manufacturer. On the day following transfection, the cells were
trypsinized and appropriately diluted with fresh CEF cells to give a
total of 1.2 × 106 cells/60-mm dish. The cells were
then overlaid with medium containing 0.4% agar and tested for focus formation.
Isolation of Hoxd12 and MHox as Maf-associated Proteins--
We
selected cDNAs encoding proteins that interact with the carboxyl
terminus DNA-binding domain of Maf by the phage display method (for
datails see "Experimental Procedures"). In brief, a cDNA
library constructed from chicken whole embryo RNA was inserted into the
T7Select vector, which expresses protein encoded by inserted cDNA
on the surface of the phage particle as a fusion with phage capsid
protein. Recombinant phage particles that express putative Maf-binding
proteins were enriched by multiple rounds of biopanning on
microtiter plates coated with purified Maf protein fused to maltose-binding protein (MBP-Maf). After seven rounds of selection, cDNA inserts of individual phages were amplified by PCR, subcloned, and sequenced. As a result, of 16 clones analyzed, 11 clones were derived from at least three independent cDNA molecules of the chicken mhox/prx1/phox1/pmx1 gene. Four other clones were
derived from at least two independent cDNA species of chicken
hoxd12.
As shown schematically in Fig.
1A, both MHox and Hoxd12
proteins contain a homeodomain, which is a DNA-binding motif composed of 60 amino acids. Although all cDNA species isolated were partial, they all fused to the capsid protein of the T7 phage in frame and
retained their homeodomains. Furthermore, they had no sequence similarity with each other except for their homeodomains (Fig. 1B), suggesting that Maf binds to these conserved domains of
MHox and Hoxd12. hoxd12 is a member of the Abdominal
B (Abd-B) group of hoxD cluster genes and
has been shown to be involved in limb pattern formation (29). MHox was
originally isolated as a member of the paired class homeobox proteins
from mice that are expressed only in cells of mesodermal origin and
bind to A/T-rich elements of the muscle creatin kinase enhancer
(30). Its chicken and human counterparts, prx1 and
phox1, were isolated as homeobox genes predominantly
expressed in the developing limb (31) and as an interacting partner of
the serum response factor (32), respectively. mhox is also
identical to a recently reported gene named pmx1, which was
found at a chromosome translocation break region in human acute
myelogenous leukemia cells (33). The fact that two homeodomain-encoding
cDNAs were isolated independently and that homeobox proteins are
transcriptional regulators prompted us to further analyze these
cDNAs.
The Homeodomain Is Necessary for Hox Association with Maf--
To
confirm the interaction of these homeobox proteins with Maf, we cloned
the entire open reading frames of hoxd12 and mhox cDNA by reverse transcriptase-PCR, subjected them to in
vitro transcription and translation in the presence of
[35S]methionine, and tested for coprecipitation with MBP
or MBP-Maf protein immobilized onto amylose resin. As shown in Fig.
2A, both Hoxd12 and MHox
proteins were specifically coprecipitated with MBP-Maf but not with
MBP. Deletion of the homeodomain of the Hoxd12 protein (Hoxd12 Maf Also Interacts with Pax6--
MHox and Hoxd12 are not the
closest members among the homeoprotein family (Fig. 1B). We
thus suspected that other homeodomain-containing proteins also could
associate with Maf. To test this possibility, we chose Pax6, which
contains the paired class of homeodomain with high homology to that of
Mhox. We also chose Meis1, whose homeodomain is distantly related to
both MHox and Hoxd12 (Fig. 1B). Pax6 and Meis1 were
translated in vitro and subjected to the coprecipitation
assay with the MBP-Maf fusion protein. As shown in Fig. 2B,
Pax6 protein specifically associated with MBP-Maf, whereas Meis1 did
not. These results showed that Maf could interact with a set of, but
not all, homeodomain-containing proteins.
bZip Domain of Maf Is Necessary for Interaction with Hox
Proteins--
We next tried to identify domains in Maf required for
interaction with the homeobox proteins. Hoxd12 and MHox proteins fused to GST were expressed in E. coli, purified by glutathione
beads, and used to test for association with a set of in
vitro translated mutant Maf proteins. Schematic structures of the
Maf mutants used in this assay are shown in Fig.
3A, together with their DNA
binding abilities and transforming activities (4, 5).
Fig. 3B shows the coprecipitation results for a set of
mutant Maf proteins with GST or GST-Hoxd12. Essentially the same
results were obtained using GST-MHox (data not shown). The full-length v-Maf protein was efficiently coprecipitated with GST-Hoxd12 but not
with GST, confirming the specific association of Maf and Hoxd12 proteins. Mutants with a deletion of the COOH-terminal, but which retain most of the zipper structure (CD1 and CD2), could associate with
Hox, but further deletion of three out of six leucine repeats (CD3)
resulted in a large reduction in the amount of coprecipitated protein.
Deletion of the entire bZip domain (CD4) lead to complete loss of
association. Disruption of the Maf Family Proteins, but Not Jun and Fos, Interact with Hoxd12 and
Mhox--
We also tested the interaction between other bZip proteins
and Hox proteins with the GST-pull-down assay (Fig.
4). In vitro translated MafB
protein, a close relative of v-/c-Maf, was efficiently coprecipitated
with the GST-Hoxd12 protein. All three small Maf subfamily proteins,
MafK, MafF and MafG, also interacted with Hoxd12. However, c-Jun could
only marginally bind to the GST-Hoxd12 protein, and c-Fos could not
bind at all. The efficiency of coprecipitation of Jun (3% of input)
was similar to those of the Maf mutants CD3, L2PL4P, MD26.22, and MD45
(2-4% of input, see Fig. 3B) and was much lower than those
of Maf and the Maf family members (more than 20% of input). Again,
essentially the same results were obtained using GST-MHox (data not
shown). Thus, the interaction of the Hox proteins seemed to be specific
for Maf family proteins.
DNA Binding Activity of Maf Is Inhibited by Interaction with
Hox--
The fact that Maf and Hox proteins interact with each other
through their DNA-binding domains led us to examine the effect of the
association on their DNA binding activities. For this purpose, a
constant amount of linearized template plasmid containing
maf (Nd5) was cotranscribed and translated
in vitro with increasing amounts of hox template
(1:1 to 1:5) and subjected to an electrophoretic gel mobility shift
assay using the MARE probe (Fig. 5). The
intensity of the retarded band of the Maf-DNA complex was decreased by
the presence of increasing amounts of Hoxd12 and MHox proteins, while the homeodomain deletion mutant (Hoxd12
On the other hand, cotranslation of Hoxd12, MHox, Pax6, or Meis1 had no
effect on binding of the Jun homodimer to the AP-1 site (Fig.
6A). DNA binding of the
Jun/Fos heterodimer also was not abrogated by Hoxd12 or MHox (Fig.
6B). These results again indicated specific interaction of
Hox proteins with Maf.
We next constructed a chimeric Maf molecule whose leucine zipper was
substituted by one from Jun. As expected, this molecule, Maf(Jun-zip),
could efficiently form a homodimer and bind to MARE (Fig.
6C), because the DNA binding specificity of bZip proteins depends on the basic region. Importantly, the DNA binding activity of
Maf(Jun-zip) was abrogated as efficiently as that of the Maf homodimer
by Hoxd12. Based on these results, together with the fact that Hox
proteins do not interact with Jun, and that the basic and leucine
zipper regions of Maf are necessary for interaction with Hox (see Fig.
3), we believe that Hox proteins interact with the basic region of Maf
only when it forms a dimer, and the leucine zipper can be substituted
by those from other bZip proteins.
We next asked if Hox proteins could interact with heterodimers of Maf
and other bZip proteins. For this purpose, we translated constant
amounts of Maf and Fos together with increasing amounts of Hoxd12 and
subjected the samples to gel mobility shift analysis using an
oligonucleotide probe that can be efficiently bound by the Maf
homodimer and Maf/Fos heterodimer (4). As shown in Fig. 6D,
the intensity of the retarded band of the Maf/Fos heterodimer was
decreased in the presence of Hoxd12 similarly to that of the Maf
homodimer. One possible explanation for this inhibition of DNA binding
is that Hoxd12 formed a ternary complex with Maf and Fos by interacting
with the basic region of Maf. Although more careful examination is
necessary to determine the mechanism of inhibition, these results
suggest that Hox proteins interfere with the DNA binding activities of
not only the Maf homodimer but also heterodimers of Maf and other bZip proteins.
DNA Binding of Hox Is Not Abrogated by Maf--
Since the
DNA-binding domains of Hox proteins can associate with Maf, we tested
whether DNA binding of Hox proteins was also inhibited by Maf.
Unexpectedly, binding of Hoxd12 protein to its recognition sequence was
not affected by Maf (Fig. 7A).
Maf also did not have any effect on binding of MHox protein to the
muscle creatin kinase enhancer sequence (data not shown). We then
examined the relative affinity of Hox proteins to DNA and to Maf.
In vitro translated, 35S-labeled Hoxd12 protein
was mixed with increasing concentrations of oligonucleotide containing
the Hoxd12-binding site or an unrelated sequence and then incubated
with the MBP-Maf fusion protein immobilized on amylose resin. As shown
in Fig. 7B, the amount of Hoxd12 protein coprecipitated with
MBP-Maf was significantly decreased by addition of the Hoxd12-binding
site oligonucleotide but not by the unrelated oligonucleotide, clearly
demonstrating that the affinity of Hoxd12 protein to its binding site
was higher than its affinity to the Maf protein. It thus seems
reasonable that Maf is unable to compete with the Hox recognition
sequence for the binding of Hox proteins.
Hoxd12 Inhibits Transactivation and Cell Transformation by
Maf--
The negative effect of Hox proteins on the DNA binding
activity of Maf prompted us to investigate the effect of Hox proteins on the transactivation and cell transforming potential of Maf. The
transactivation assay was performed on CEF cells using a luciferase reporter plasmid containing three tandem repeats of MAREs upstream of
the TATA sequence of the rabbit
To measure the antagonistic effect of Hox proteins on transformation by
Maf, we developed a replication competent avian retrovirus vector
system (pRV-9), which can express two exogenous genes in a single cell
(Fig.
9A).2
Upon transfection into CEF cells, we expect one gene to be expressed from fully spliced subgenomic RNA and the other to be expressed from
any RNA species by utilizing the IRES. We constructed
pRV-9/c-maf-IRES-hoxd12 and
pRV-9/c-maf-IRES-hoxd12 In this study, we isolated two homeodomain-containing proteins,
Hoxd12 and MHox, that interact with v-/c-Maf, using the phage display
method. The Hox proteins also could associate with the other Maf
protein family members, MafB, MafK, MafF, and MafG, but not with Jun
and Fos. The Hox proteins negatively regulated the DNA binding,
transactivation and cell-transforming abilities of Maf.
To date, several classes of transcriptional regulators are known to
interact with Maf and/or Maf family proteins. For instance, Jun, Fos,
(4, 13) and Bach13 have been
identified as heterodimeric partners of v-/c-Maf. In addition to these
bZip proteins, several members of other transcription factor
superfamilies have been reported to interact with Maf proteins. For
instance, USF2, a member of the basic helix-loop-helix zipper transcription factor family, has been shown to interact with c-Maf and
inhibit its DNA binding activity (34). c-Maf was also shown to form a
transcriptionally inert complex with c-Myb in a developmentally regulated manner in cells of the myeloid lineage (22). Similarly, MafB
has been reported to repress transcriptional activity of c-Ets-1
through direct interaction, which results in inhibition of erythroid
cell differentiation (23). These transcription factors recognize the
bZip domain of Maf proteins, which is highly conserved among the family members.
As shown in this study, Maf and Hox proteins can form complexes through
interaction between the conserved bZip domain of Maf and the
homeodomain of the Hox proteins. To date, more than 150 homeodomain-encoding genes have been identified in the vertebrate genome. Among them, Hoxd12 and MHox, identified in this study, are
members of different subclasses and display higher amino acid sequence
homology to other Hox proteins than to each other. These facts lead us
to investigate whether other Hox proteins interact with Maf. We found
that Pax6, whose homeodomain was more similar to MHox than to Hoxd12,
could associate with Maf. In contrast, Maf could not bind to Meis1,
whose homeodomain was distantly related to both Hoxd12 and MHox. These
observations indicated that a set of, but not all,
homeodomain-containing proteins could interact with Maf family proteins.
The Hox family transcription factors are known to play pivotal roles in
the establishment of cell identity and regional information as well as
for cell differentiation and morphogenesis. Maf also act as a
differentiation factor in specific cell types, although it was
originally identified as an oncogene product. For example, c-Maf is a
key regulator for the specific expression of the interleukin-4 gene and
differentiation of the Th2 subset of helper T-cells (35). The
mafB gene has also been shown to establish specific
rhombomeres in the developing hindbrain and is responsible for the
kreisler and valentino mutations of mouse and
zebrafish, respectively (36, 37). As both Maf and Hox are
transcriptional regulators, we examined the effect of their physical
interaction on their functions and found that Hox proteins inhibited
the DNA binding ability of Maf. In contrast, Maf could neither
interfere with the DNA binding activities of Hox proteins or form a
ternary complex with Hox bound to DNA, indicating that Maf was inert
with regard to the biological activities of Hox proteins. However,
interaction of Maf and Pax6 is of interest because Pax6 contains
another DNA-binding motif paired domain in addition to the homeodomain.
It therefore might be possible that Maf interacts with Pax6 at the
homeodomain when Pax6 is bound to DNA by its paired domain. It
previously has been shown that Pax6 and large Maf family members were
expressed in overlapping regions of the eye and that both Pax6-binding
sites and MAREs were identified in transcriptional regulatory regions of crystallin genes in a variety of species. Accordingly, large Maf
proteins have been shown to activate lens specific expression of
crystallin genes through such MAREs (38-40). Pax6 also has been reported to activate the Recently, it has been reported that a large Maf family protein, Nrl,
and a paired-type homeodomain protein, Crx, synergistically regulate
the photoreceptor cell-specific expression of the rhodopsin gene (46).
Nrl is expressed not only in photoreceptor cells but also in other
retinal cells (47). In contrast, Crx is exclusively expressed in
photoreceptor cells, and synergistic activation of rhodopsin gene
transcription is achieved only by the copresence of Nrl, which binds to
a MARE-like cis-regulatory element, and Crx, which binds to
another cis-element, Ret4, located next to the MARE on its
promoter (46, 48). Although direct physical interaction of Nrl and Crx
has not been reported, such functional synergism between Maf family
members and Hox proteins may occur depending on the context of the promoter.
As we have reported previously, the v-maf-carrying
retrovirus, AS42, induces a specific type of tumor, musculoaponeurotic fibrosarcoma, in chicken (2), whereas v-Fos and v-Jun induce nephroblastoma and osteosarcoma (NK24 virus) (28) and fibrosarcoma (ASV17 virus) (49), respectively. Although the mechanism of tissue
specificity in tumor induction is still not known, similar but clearly
distinct DNA binding specificities of Maf and Fos/Jun may explain these
differences. These oncogene products are likely to activate overlapping
but different sets of downstream target genes. In addition, the finding
that coexpression of Hoxd12 in CEF cells specifically inhibited the
transcriptional activation and transforming ability of Maf, but not of
Fos, suggests that the expression levels of a group of tissue-specific,
Maf inhibitory factors such as USF2 or Hox proteins also may be
important for oncogene-specific tumor induction. These two
possibilities are not mutually exclusive and require further study.
Disregulated expression of some homeodomain-containing proteins also
has been implicated in malignancies in humans and rodents. For example,
Pax6 has been shown to be oncogenic when overexpressed (50), and
chromosome translocations of pax3 and pax7 genes, close relatives of pax6, to the fkhr gene have
been found in human rhabdomyosarcomas (51, 52). It is especially
noteworthy that not only MHox/Pmx1 itself but also HoxA9 and HoxD13,
whose homeodomains are closely related to that of Hoxd12, have been
found to form a fusion product with the nucleoporin, Nup98, by
chromosome translocation in human acute myelogenous leukemia cells (33,
53-55). All these potentially oncogenic Hox proteins might interact
with Maf family members, considering the similarity of the amino acids
in their homeodomains. If these interactions do occur, together with
recent findings that both c-Maf and MafB induce monocytic
differentiation (56, 57), then overexpression of these Hox proteins in
cells of the myeloid lineage should inhibit differentiation-inducing functions of Maf proteins, which may result in inhibition of the differentiation program and may cause myelogenous leukemia. This idea
of a common protein target for the action of oncogenic Hox proteins is
worthy of further study, because DNA sequences recognized by these Hox
proteins are different from each other, and it does not seem plausible
that they share common downstream target gene(s) for cell transformation.
Recently, it was reported that MHox and a set of related
homeodomain-containing proteins, Chx10, B4, and Pax3, interacted with
the pRB family proteins, pRB, p107 and p130, through their homeodomains
(58). Thus, Maf may compete with pRB family members for binding to
these Hox proteins. Conversely, Hox proteins may compete for binding to
Maf with other Maf-interacting molecules, such as USF2, c-Ets1, and
c-Myb, because the COOH-terminal bZip domain of Maf is necessary for
interaction with these three proteins (22, 23, 34). Moreover, in this
study, we demonstrated that Hox proteins abrogated DNA binding of not
only the homodimer of Maf family members but also the heterodimer of
Maf and Fos, suggesting the possibility that Hox interferes with
heterodimers of Maf and other bZip partners such as Jun and
Cap'n'collar family members. Although the functional and
physiological relevance of interactions between these transcription
factors must be examined more carefully, we propose that these
different classes of transcription factors form a regulatory network
through protein-protein and protein-DNA interaction, which may allow
the fine tuning of the regulation of gene expression. Disorder of the
network by dysfunction or abnormal expression of a factor by mutation,
viral transduction, or chromosome translocation may lead to a
developmental abnormality, genetic disease, or cell transformation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HD, three methionine codons for
in vitro labeling by 35S and a termination codon
were introduced instead of the 207th lysine codon of hoxd12
cDNA by PCR using the primer,
5'-GAGCGCGCTACATCATCATTGTGTACGGTTTCCGTTTC-3', and the
hoxd12 forward primer, and inserted into
pGEM3-MluI after BssHII digestion.
HD
were inserted into a unique BssHII site of the pEF-BssHII expression vector (24), a derivative of pEF-BOS. The
pEF/v-maf expression plasmid and the luciferase reporter
plasmid 3×MARE/RBGP-luc, containing three copies of MARE
(oligonucleotide 7) and rabbit
-globin minimal promoter, have been
described (6, 24). pEF-Rluc, used to normalize transfection
efficiencies, was constructed by inserting a
XbaI-NheI fragment containing the open reading
frame of the Renilla luciferase gene (pRL-TK, Promega) into
the XbaI site of pEF-BOS, followed by deletion of the
replication origin of SV40 by HindIII digestion and
self-ligation.
HD in the pGEM3-MluI
plasmid by PCR using the primers,
5'-GAGCCATGGGCTCCGTTCCAATC-3' and SP6 primer. The PCR fragment was digested with HindIII at the 3' polylinker
site, treated with T4 DNA polymerase, digested with NcoI,
and inserted into the NcoI and SmaI-digested IRES
cassette plasmid to make pUC/IRES-hoxd12 and
pUC/IRES-Hoxd12
HD. The NotI fragments containing IRES-hoxd12 and IRES-Hoxd12
HD were then inserted
into the unique NotI site of the replication-competent avian
retroviral vector, pRV-9. Subsequently, the MluI fragment
containing chicken c-maf or chicken v-fos was
introduced into a unique MluI site in the plasmid. To
construct the chicken v-fos MluI fragment, the initiator methionine codon and an NcoI restriction site were created
at the fusion point of gag and fos of the NK24
provirus (28), and the MluI site in the coding region was
deleted by site-directed mutagenesis using the oligonucleotides,
5'-CCCCAGGAGccATggTCAACTCGCAGG-3' and
5'-CCTTCTAtGCaTCGGACTGGGAG-3', respectively. The modified v-fos fragment was introduced into the
NcoI-MluI site of the pRAM plasmid (5) by
addition of an MluI linker to the ApaI site at
the 3'-noncoding region followed by digestion with NcoI and MluI.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
Structures of Hoxd12 and MHox.
A, schematic structures of Hoxd12 and MHox/Prx1/Phox1/Pmx1
proteins. B, comparison of amino acid sequences of
homeodomains of Hoxd12, MHox, Pax6, and Meis1. Three -helical
domains are shown at the top. The numbers of identical amino
acids with Hoxd12 and MHox are also shown on the
right.
HD)
resulted in loss of specific association with Maf, indicating that the
homeodomain is necessary for this interaction. We also could detect
recombinant GST-Hoxd12 and GST-MHox proteins (see below) immobilized on
nitrocellulose membranes by West-Western method using the MBP-Maf
fusion protein as a probe (data not shown), indicating direct
interaction of Maf and Hox proteins.
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Fig. 2.
Interaction of Hox proteins with Maf.
A, specific interaction of Hoxd12 and MHox with MBP-Maf.
35S-Labeled Hoxd12, Hoxd12 HD, and MHox
translated in vitro (indicated by asterisks) were
tested for coprecipitation with MBP or MBP-Maf and analyzed by
SDS-PAGE. B, pull-down assay of 35S-labeled Pax6
and Meis1 (indicated by asterisks) using MBP or
MBP-Maf.
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Fig. 3.
Analysis of domains of Maf necessary for
interaction with Hox. A, schematic structures of mutant
Maf proteins are shown with their DNA binding and transforming
activities. The amounts of Maf proteins coprecipitated with GST-Hoxd12
were quantitated and indicated by + (more than 20% of input),
triangle (2~4%), and (less than 2%).
B, SDS-PAGE analysis of in vitro translated
(top panel), GST-bound (middle panel), or
GST-Hoxd12-bound (bottom panel) mutant Maf proteins.
-helical structure of the leucine
zipper by amino acid substitutions of two leucine residues with
prolines (L2PL4P) significantly affected the binding with GST-Hoxd12,
suggesting that the intact leucine zipper structure is required for the
interaction. A mutant, ND5, with a deletion of the amino-terminal
region, contains the same region present in the MBP-Maf fusion protein
and thus can associate with GST-Hoxd12. The mutant, ND6, which contains
further deletions and cannot bind to DNA, could still efficiently be
coprecipitated. On the other hand, deletion of either the basic domain
(MD56) or the preceding region (MD45), which is also necessary for DNA
binding, significantly affected the interaction. A deletion of only
five amino acids in the basic region (MD26.22) effectively abrogated
the association. The Q5H mutant, which contains a glutamine to
histidine substitution in the hinge region of the basic domain and the
leucine zipper that results in enhanced transforming ability by an
unknown mechanism (5, 6), was coprecipitated with GST-Hox at a
comparable level as the wild type Maf protein. These results together
suggested that the domain spanning the basic region and the leucine
zipper structure of Maf was necessary for association with Hox proteins.
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Fig. 4.
Interaction of Hox and bZip proteins.
In vitro translated bZip proteins shown at the
top were assayed for binding with GST (middle
panel) or GST-Hoxd12 (bottom panel).
HD) had little effect (Fig.
5A). Coexpression of Pax6 also inhibited the DNA binding activity of Maf (Fig. 5B), whereas Meis1, which cannot
associate with Maf, did not affect DNA binding. These results clearly
indicated that the association of Hox proteins with Maf specifically
inhibited its DNA binding activity.
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Fig. 5.
Inhibition of DNA binding of Maf by Hox
proteins. A, a constant amount of template plasmid for
Maf (ND5) (lanes 2-12) was cotranslated in
vitro with increasing amounts of template for Hoxd12 (lanes
3-5), Hoxd12 HD (lanes 6-8), and MHox
(lanes 10-12) at a ratio of 1:1 (lanes 3,
6, and 10), 1:3 (lanes 4,
7, and 11), and 1:5 (lanes 5,
8, and 12) and then tested for binding to labeled
MARE probe by electrophoretic gel mobility shift analysis.
B, template plasmid for Maf (ND5) (lanes 2-4)
and for Pax6 (lane 3) or Meis1 (lane 4) were
cotranslated at a ratio of 1:5 and subjected to gel mobility shift
analysis using MARE probe.
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Fig. 6.
Effect of Hox proteins on DNA binding by
other bZip proteins. A, inability of Hox proteins to
inhibit Jun binding to the AP-1 site. The template plasmid for Jun
(lanes 2-6) was cotranslated with template for Hoxd12
(lane 3), MHox (lane 4), Pax6 (lane
5), or Meis1 (lane 6), and tested for binding to
oligonucleotide probe 11 containing an AP-1 site. Endogenous
nonspecific binding activity to the probe is indicated by an
asterisk. B, effect of Hox on the Jun/Fos
heterodimer. Binding ability of the heterodimer of Jun and Fos to probe
11 (AP-1) (lanes 2-4) was tested by gel mobility shift
analysis after cotranslation with Hoxd12 (lane 3) or MHox
(lane 4). C, inhibition of DNA binding of the
Maf-Jun chimera by Hoxd12. Maf (lanes 2 and 3) or
a mutant Maf construct (Maf(Jun-zip)) whose leucine zipper was
substituted by one from Jun (lanes 4 and
5), was cotranslated with (lanes 3 and
5) or without (lanes 2 and
4) Hoxd12, and tested for binding to MARE. D,
effect of Hoxd12 on the Maf/Fos heterodimer. Constant amounts of
template plasmids for Maf (ND5) and Fos (lanes 2-5) were
cotranslated with increasing amounts of Hoxd12 template (lanes
3-5) and were subjected to gel mobility shift analysis using
probe 11, which can be bound by both Maf/Fos heterodimer and Maf
homodimer (indicated by arrows). The endogenous binding
activity is indicated by an asterisk.
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Fig. 7.
Inability of Maf to affect Hoxd12 binding to
DNA. A, effect of Maf on DNA binding by Hoxd12. A
constant amount of Hoxd12 protein (lanes 2-5) was
cotranslated in vitro with 1-fold (lane
3), 3-fold (lane 4), and 5-fold (lane
5) excess of Maf and subjected to gel mobility shift analysis
using a labeled Hoxd12-binding site oligonucleotide. B,
higher affinity of Hoxd12 to its recognition DNA sequence than to Maf.
In vitro translated, 35S-labeled Hoxd12 protein
(lane 1) was coprecipitated with MBP
(lane 2) or MBP-Maf (lanes
3-9) in the absence (lane 3) or
presence of 10 ng (lanes 4 and 7), 100 ng (lanes 5 and 8), or 1 µg
(lanes 6 and 9) of double-stranded
oligonucleotide containing the Hoxd12 recognition sequence
(lanes 4-6) or the unrelated, serum-responsive
element of human c-fos promoter (lanes
7-9), and analyzed by SDS-PAGE.
-globin promoter (3×MARE/RBGP-luc) (6, 24). As shown in Fig. 8,
cotransfection of the expression plasmid for Maf resulted in
significant induction of luciferase activity, and the magnitude of
transactivation was reduced by cotransfection of a Hoxd12 expression
plasmid in dose-dependent manner. On the other hand,
cotransfection of the Hoxd12
HD expression plasmid had little effect
on the transactivation by Maf. We confirmed the nuclear localization of
both Hoxd12 and Hoxd12
HD proteins in the transfected cells by
producing GST fusion proteins and performing immunofluorescent staining
of cells using anti-GST antiserum (data not shown). These results
indicated that the Hoxd12 protein inhibits the transactivation activity
of Maf by abrogating its DNA binding ability.
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Fig. 8.
Transcriptional regulation of MARE by Maf and
Hoxd12. A reporter plasmid, 3×MARE/RBGP-luc, was cotransfected
into CEF cells with a constant amount of expression plasmid for Maf and
an increasing amount of expression plasmid for Hoxd12 or
Hoxd12 HD and assayed for luciferase activity.
HD plasmids,
transfected them into CEF cells, and assayed for focus formation. As
shown in Fig. 9B, pRV-9/c-maf efficiently induced
foci, but pRV-9/c-maf-IRES-hoxd12 did not. On the
other hand,
pRV-9/c-maf-IRES-hoxd12
HD induced foci with a similar efficiency to that of pRV-9/c-maf.
Furthermore, coexpression of Hoxd12 did not interfere with the
transforming ability of v-Fos (pRV-9/v-fos and
pRV-9/v-fos-IRES-hoxd12). These results indicated
that the Hoxd12 protein specifically suppressed the transforming
ability of Maf.
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Fig. 9.
Supression of transforming activity of Maf by
Hoxd12. A, schematic representation of the retroviral
vector system that express two exogenously introduced genes. Upon
transfection into CEF cells, three mRNA species were generated and
produced viral proteins Gag-Pol, Env, and exogenous proteins X and Y. LTR, long terminal repeat, IRES; internal ribosome entry
site. B, culture dishes of CEF cells transfected with the
indicated recombinant retrovirus vectors.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A-crystallin gene of chicken (41) and mouse
(42) and
-crystallin in chicken (43), and to activate synergistically with Maf, the
-crystallin gene of guinea pig (44).
On the other hand, Pax6 negatively regulates
-crystallin genes of
chicken in lens fiber cells, while Maf activates these genes (38-40,
45). The fine tuning of the spacio-temporal expression of a set of
crystallin genes during lens development may require such synergistic
and antagonistic regulation by Maf and Pax6, which might in part be
accomplished by direct association of these two transcription factors.
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ACKNOWLEDGEMENTS |
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We thank Shinya Watanabe and Sumio Sugano for helpful discussions and encouragement. We are also grateful to Shigekazu Nagata for pEF-BOS vector, Nobuo Nomura for cDNA clones for human c-jun and c-fos, and Peter K. Vogt for the recombinant clone of v-jun.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture in Japan (to K. K.).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: Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan. Tel.: 81-45-924-5799; Fax: 81-45-924-5834; E-mail: kkataoka@bio.titech.ac.jp.
¶ Present address: Present address: Frontier Collaborative Research Center, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007643200
2 M. Nishizawa and K. Kataoka, unpublished observations.
3 M. Nishizawa, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: CEF, chicken embryo fibroblast; bZip, basic-leucine zipper; MARE, Maf recognition element; MBP, maltose-binding protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; IRES, internal ribosome entry site; TBS, Tris-buffered saline.
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REFERENCES |
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