The erythroid transcription factor NF-E2 is an
obligate heterodimer composed of two different subunits (p45 and p18),
each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an
enhancer-binding protein for expression of the
-globin
gene. We show here that dimethyl sulfoxide treatment of wild-type
murine erythroleukemia cells, but not a mutant clone of dimethyl
sulfoxide-resistant cells, increases NF-E2 activity significantly,
which involves both up-regulation of DNA binding and transactivation
activities. Both activities were reduced markedly by treatment of cells
with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but
this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH2-terminal
region of p45 and a suppression domain in the basic region-leucine
zipper of MafK. These findings indicate that induction of NF-E2
activity is essential for erythroid differentiation of murine
erythroleukemia cells, and serine/threonine phosphorylation may be
involved in this process. In addition, they also suggest that a MafK
homodimer can suppress transcription, not only by competition for the
DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.
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INTRODUCTION |
The erythroid transcription factor NF-E2 is present in extracts of
erythroid cell lines (1, 2) and has been shown to be a heterodimer
formed between the two basic region-leucine zipper (b-zip)1 proteins,
i.e. the p45 and the p18 subunits. p45, which together with
the Drosophila cap'n-collar (CNC) protein defines a b-zip subfamily, is expressed in hematopoietic cells of the erythroid, megakaryocytic, and mast cell lineages (1), whereas p18 is one of the
small Maf family proteins including
MafK2 (1-4), and its expression is
not restricted to hematopoietic cells (5). Involvement of NF-E2 in
erythroid cell differentiation of mouse erythroleukemia (MEL) cells has
been suggested by its role as an enhancer-binding protein for
expression of the
-globin gene, by the lack of
-globin mRNA expression in a MEL cell line (CB3) devoid of
NF-E2 protein as a result of integration of Friend viral sequences
within the p45 NF-E2 gene locus (6), and by the restoration
of
-globin mRNA by forced expression of
p45 cDNA in these cells (6, 7). It has also been found that
NF-E2·DNA binding increases during erythroid differentiation of MEL
cells (8-10). These results suggest NF-E2 to be one of the key
transcription factors that regulate erythroid differentiation of MEL
cells. Unexpectedly, targeted disruption of the p45 gene in
mice showed essentially no abnormality in erythropoiesis (11, 12). It is possible in these conditions, however, that p45-related b-zip factors, such as Nrf1 (13), or Nrf2 (14), may compensate for deficient NF-E2 activity.
Previous studies showed that p45 can be phosphorylated by a
cAMP-dependent protein kinase in vitro (15) and
that both DNA binding of NF-E2 and
-globin locus control
region enhancer activity are diminished significantly in a
cAMP-dependent protein kinase-deficient clone of MEL cells
after hexamethylenebisacetamide treatment compared with wild-type cells
(15). These findings suggest that NF-E2 function may be regulated by a
cAMP-dependent protein kinase; however, the exact mechanism
of NF-E2 regulation remains unclear. With this view in mind, we
examined the possibility that NF-E2 activity may be regulated by a
post-translational mechanism in MEL cell differentiation. Our results
in this study show that dimethyl sulfoxide (Me2SO)
increased NF-E2·DNA complex formation in the wild-type MEL cells, in
part by the induction of p45 gene expression, and that both
DNA binding and transactivation activity of NF-E2 are regulated by
serine/threonine phosphorylation such as a Ras signaling cascade. Our
findings also suggest that an MafK homodimer can suppress transcription
not only by competition for the DNA binding site, but also by directly
inhibiting transcription. Hence, MafK can be viewed as an active
transcription repressor.
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MATERIALS AND METHODS |
Cell Culture--
A clone of dimethyl sulfoxide-sensitive (DS)
MEL cells (DS-19) (16) and a clone of dimethyl sulfoxide-resistant (DR)
MEL cells (DR-1) (17, 18) were grown in suspension in modified Ham's
F-12 medium containing 10% heat-inactivated bovine calf serum (Hyclone
Laboratories, Logan, UT), as described previously (18). DS cells
undergo erythroid differentiation after treatment with
Me2SO and express erythroid markers such as
-globin (19, 20), heme pathway enzymes (18, 21), and erythropoietin receptor (22,
23); they ultimately cease to grow in culture after a few days by
completing differentiation (24). In contrast, DR cells fail to show
terminal erythroid differentiation when treated with Me2SO,
and they continue to grow as hemoglobin-free cells in culture (17).
Both DS and DR cells were split every 3-4 days to maintain a
logarithmic growth.
cDNA Probes--
cDNAs for mouse p45
and mafK were generated by polymerase chain reaction using
cDNAs reverse transcribed from MEL cell RNA and a set of primers as
described previously (3). Human ribosomal DNA was obtained from
the Japanese Cancer Research Resources Bank, Tokyo, and used as
an internal control.
Northern Blot Analysis--
Total RNA from untreated and treated
MEL cells was isolated using the acid guanidium thiocyanate- phenol
chloroform method (25). 20 µg of each RNA was loaded onto a 1.2%
agarose-formaldehyde gel, electrophoresed, and transferred to a sheet
of Zeta-Probe filter (Bio-Rad). The filters were hybridized overnight
with appropriate RNA probes at 50 °C in a solution containing 50%
formamide, 1.5 × SSPE (270 mmol/liter NaCl, 15 mmol/liter
Na2HPO4, and 1.5 mmol/liter EDTA), 1% SDS,
0.5% bovine lactotransfer optimizer (BLOTTO), 0.2 mg/ml yeast transfer
RNA, and 0.5 mg/ml sonicated salmon sperm DNA. Hybridized filters were
washed and exposed to Kodak X-Omat XAR-5 films (Eastman Kodak).
mRNA concentrations were quantified by LKB Ultrascan XL enhanced
laser densitometry (Pharmacia Biotech Inc.). Experiments were performed
two or three times using separate preparations of RNA, and
representative results are shown in the figures.
Western Blot Analysis--
Nuclear extracts were prepared from
1 × 107 cells according to the method described
previously (26). 10 µg of nuclear extracts was separated
electrophoretically using 10% polyacrylamide gel. Immunoblotting and
detection by enhanced chemiluminescence were performed as described
previously (18).
DNA Gel Mobility Shift Assays--
To evaluate the DNA binding
activities of NF-E2, DNA gel mobility shift assays were performed using
the oligomer 5'-GTGGTGCTGAGTCATAGGAGAAG-3', which contains the 11-base
pair NF-E2 consensus and its flanking sequences. 5 µg of nuclear
extract was incubated with 5'-end-labeled oligomers in a binding buffer
containing 20 mM HEPES buffer (pH 7.8), 60 mM
KCl, 0.2 mM EDTA, 6 mM MgCl2, 0.5 mM dithiothreitol, 10% v/v glycerol, and 1.5 µg of an
equimolar mixture of poly(dI-dC) and poly(dA-dT). The reaction mixture
was incubated at room temperature for 15 min. In antibody-mediated
competition assays, 1 µl of rabbit anti-mouse p45 serum was first
incubated with nuclear extracts on ice for 10 min and then incubated
with the probes for an additional 10 min. For dephosphorylation, 5 µg
of nuclear extracts from Me2SO-treated DS cells was
incubated with 0.001-0.01 unit of calf intestine phosphatase for 15 min before the addition of the 32P-labeled probe. The
mixture was then loaded onto a 4% polyacrylamide gel, and
electrophoresis was carried out at room temperature.
Plasmids--
p45 and MafK expression vectors were described
previously (3, 5). FLAG epitope tags were introduced into
p45 and MafK cDNA by polymerase chain reaction such
that p45 and MafK are tagged at the NH2 termini. Resulting
cDNAs were cloned in pEFBssHII, generating p45 FLAG and MafK FLAG
expression vectors. GAL4 DNA binding domain fusions were constructed
using pGBT9 (CLONTECH). The
NcoI/HindIII fragment of pIKIII (3) was cloned in
the EcoRI site of pGBT9 after blunting of the end. Portions
of p45 cDNAs were also inserted into pGBT9 using
conventional restriction enzyme sites, resulting in G4-p45 fusion
plasmids. Various portions of murine mafK cDNA (5) were
isolated by polymerase chain reaction and cloned also in pGBT9,
resulting in G4-MafK fusion plasmids. These fusion cDNAs
were transfected into the BssHII site of pEFBssHII by
isolating the HindIII fragment, resulting in G4-p45(1-272), -(1-83), -(1-36), -(39-83), -(86-272), G4-MF, G4MF
N, and
G4MF
C. An expression plasmid for H-RasV12 was kindly
provided by Dr. M. Nakafuku. A plasmid expressing dominant positive
MAPKK (SESE-MAPKK) (27) was kindly provided by Drs. Y. Gotoh and E. Nishida. pRBGP10 reporter plasmid was generated by inserting a single
copy of the NF-E2 site derived from the chicken
-globin
3'-enhancer into pRBGP3 (3).
Transfection and Luciferase Assays--
pRBGP2 that contains
three copies of NF-E2 site (3), pRBGP10, or GAL4 × 5 luciferase
plasmid was used as a reporter. Transfection of DNA into DS or DR cells
was carried out using the transfection reagent DOTAP (Boehringer
Mannheim). Briefly, a total of 5 × 105 cells was
incubated with 2 µg of reporter and effector plasmids and 3 µg of
pSV-
-galactosidase (as an internal control) in 1 ml of culture
medium without serum for 7 h at 37 °C. Then 2 ml of fresh
medium containing 10% bovine calf serum was added, and the cells were
incubated in the absence or presence of 1.5% Me2SO or
kinase inhibitors for another 8-72 h. Cell lysates were prepared using
reporter lysis buffer (Promega, Madison, WI). Luciferase and
-galactosidase activities were determined using the luciferase assay
system and
-galactosidase enzyme assay system (Promega), respectively. Luciferase activity was normalized on the basis of
-galactosidase activity. NIH3T3 and QT6 cells were transfected by
the calcium-phosphate precipitation procedure as described previously.
To examine the effect of H-RasV12 and MAPKK, cells were
incubated in the presence of 0.25% fetal bovine serum for 36 h
after transfection.
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RESULTS |
Effect of Me2SO on NF-E2·DNA Complex
Formation--
To define the role of NF-E2 in erythroid
differentiation, we first examined the effect of Me2SO on
NF-E2·DNA complex formation in DS and DR cells using a gel mobility
shift assay. In DS cells a band was detected which increased its
intensity significantly after treatment of cells with Me2SO
for 72 h (Fig. 1A). This
band was suppressed completely by the addition of anti-p45 antiserum, but not by preimmune serum, indicating that it is an NF-E2·DNA complex (Fig. 1B). In contrast to DS cells, the
corresponding band in DR cells did not increase after Me2SO
treatment (Fig. 1A). A band corresponding to an AP-1·DNA
complex that migrated slightly slower than the NF-E2·DNA complex was
not influenced by Me2SO treatment in either DS or DR cells
(Fig. 1A).

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Fig. 1.
Effect of Me2SO on NF-E2·DNA
complex formation in DS and DR cells. Cells were incubated with
1.5% Me2SO (DMSO) for 72 h. NF-E2·DNA
complex formation was determined by DNA gel mobility shift assays as
described under "Materials and Methods." Panel A, effect
of Me2SO on NF-E2·DNA complex formation in DS and DR cells. Panel B, effect of anti-p45 antiserum (Ab)
on the formation of NF-E2·DNA complex. 1 µl of rabbit anti-mouse
p45 serum was first incubated with nuclear extracts on ice for 10 min
and then incubated with the probes for additional 10 min.
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To clarify whether the induction of NF-E2·DNA complex formation by
Me2SO is caused by increased gene expression, we examined the mRNA levels for p45 and mafK by RNA blot
analysis and their protein levels by immunoblot analysis. In DS cells,
Me2SO treatment increased p45 mRNA levels
significantly at 72 h (Fig.
2A), consistent with the
observed increase in NF-E2·DNA complex formation (Fig. 1A). In contrast, DR cells did not show any increase in
p45 mRNA after Me2SO treatment.
mafK mRNA levels were not influenced by Me2SO
treatment in either DS or DR cells (Fig. 2A). Consistent with the induction of p45 mRNA in DS cells, p45 protein
levels were also increased significantly after Me2SO
treatment in DS cells but not in DR cells (Fig. 2B). In
contrast to p45 protein levels, which were readily detectable, MafK
protein levels were below the detection level in both DS and DR cells
(data not shown). These findings indicate that increased NF-E2·DNA
complex formation by Me2SO treatment occurs only in DS
cells, which subsequently undergo erythroid differentiation, and that
this process involves p45 gene up-regulation.

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Fig. 2.
Effect of Me2SO on
p45 and mafK mRNA and p45 protein levels.
Cells were incubated with 1.5% Me2SO (DMSO) for
72 h. Panel A, p45 and mafK
mRNA levels were determined by RNA blot analysis, as described
under "Materials and Methods." Data are the mean ± S.D. of
three determinations. Panel B, nuclear extracts were
prepared, separated by SDS-polyacrylamide gel electrophoresis, transferred onto a membrane, and reacted with anti-p45 antiserum.
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Me2SO Treatment of DS Cells Increases the Enhancer
Activity of the NF-E2 Site--
To examine whether the
Me2SO-induced NF-E2 complex is active in transcription, we
determined the activity of an NF-E2 binding site-luciferase fusion
construct that was transfected into DS or DR cells. The results of
these experiments are summarized in Fig.
3. As shown in the figure, luciferase
activity was increased by ~4-fold in transfected DS cells when they
were treated with Me2SO for 72 h compared with the
level in untreated transfected cells (Fig. 3A). In contrast
to DS cells, Me2SO treatment did not increase luciferase
activity in DR cells (Fig. 3A). These results indicate that
NF-E2·DNA complex formation led to the up-regulation of NF-E2
site-dependent promoter activity during erythroid
differentiation in DS cells by Me2SO treatment. They also
suggest that the failure of DR cells in erythroid differentiation may
be related to the lack of an increase in NF-E2·DNA complex formation
and therefore to the lack of NF-E2-mediated enhancer activity. It
should also be noted that induction of luciferase activity in DS cells
was already observed at 12 h (~7-fold) after Me2SO
treatment (Fig. 3B). It was earlier than an increase in the
amount of NF-E2·DNA complex which was not yet detectable at 12 h
(data not shown). Thus it is possible that Me2SO treatment
may induce not only an increase in the amount of NF-E2·DNA complex
but also transformation of the NF-E2 molecules to become functionally
active.

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Fig. 3.
Me2SO increased an enhancer
activity of the NF-E2 site in DS cells. A luciferase reporter
plasmid with three NF-E2 binding sites and the pSV- -galactosidase
was cotransfected into DS and DR cells as described under "Materials
and Methods." Cells were then incubated with 1.5% Me2SO
(DMSO) for 72 h. Luciferase (LUC) activity
was normalized to the -galactosidase value and expressed as the mean
of three separate transfection experiments. Panel A, 72-h
experiment; panel B, DS cells, 12-h experiment.
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The Role of Phosphorylation on NF-E2 Function--
Because
phosphorylation plays an important role in various signal cascades (28)
and because NF-E2 has been shown to be phosphorylated in
vitro (15), we examined the effect of protein kinase inhibitors on
NF-E2 DNA binding activity and cis-regulatory activity of
the NF-E2 site. Combined treatment of DS cells with Me2SO
and 2-aminopurine (2-AP), a broad specificity serine/threonine kinase
inhibitor, resulted in a marked decrease in Me2SO-mediated
induction of DNA binding activity of NF-E2 (Fig.
4A). In contrast to 2-AP
treatment, treatment of DS cells with genistein, a tyrosine kinase
inhibitor, had no effect on Me2SO-mediated induction of
NF-E2 binding activity (Fig. 4A), suggesting that tyrosine
phosphorylation is not required for Me2SO-mediated
induction of NF-E2 activity. Consistent with this finding,
preincubation of a nuclear extract of Me2SO-treated DS
cells with calf intestine phosphatase decreased NF-E2 binding activity
in a dose-dependent fashion (Fig. 4B). This
inhibition occurred in the absence of any change in the level of p45
protein in both cells and nuclear extracts (Fig. 4C).
However, this finding is in contrast to a previous report (15) which
found no significant effect by phosphatase treatment. The reason for
the observed discrepancy is unclear, and it is not possible to trace it
because no detail was provided in the previous report (15).

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Fig. 4.
Effect of protein kinase inhibitors on the
enhancer activity of the NF-E2 site and NF-E2 binding activity.
Panel A, DS cells were incubated in the presence of 1.5%
Me2SO (DMSO) for 67 h. Cells were then
incubated with 10 mM 2-AP or 100 µM genistein together with Me2SO for another 5 h. Gel mobility
shift assays using an oligonucleotide containing three NF-E2 sites were
performed as described under "Materials and Methods." Panel
B, 5 µg of nuclear extract from Me2SO-treated DS
cells were incubated calf intestine phosphatase (CIP) for 15 min before the addition of 32P-labeled probe. Panel
C, DS cells were treated as described above, then immunoblot
analysis was performed with anti-p45 antiserum. Panel D, DS
cells were transfected with a luciferase (LUC) reporter plasmid with or without NF-E2 binding sites. Cells were then incubated with 10 mM 2-AP or 100 µM genistein together
with 1.5% Me2SO for 12 h. The results are expressed
as the ratio of the luciferase activities of cells to that obtained
with a reporter that did not contain NF-E2 sites, based on the mean of
two determinations.
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2-AP treatment of cells also suppressed Me2SO-mediated
induction of NF-E2 site-dependent promoter activity (Fig.
4D), suggesting that serine/threonine phosphorylation may be
involved in both the induction of NF-E2·DNA binding and the
cis-regulatory activity of the NF-E2 site. Treatment of
cells with genistein even increased NF-E2 site-dependent
promoter activity (~2-fold compared with Me2SO-treated
cells), although the reason for this increase is not yet understood
(Fig. 4D).
Inhibition of Serine/Threonine Phosphorylation Also Inhibits the
Me2SO-mediated Increase in
-Globin and ALAS-E
mRNA Levels and Heme Content--
To examine the effect of
inhibition of serine/threonine phosphorylation on erythroid
differentiation, the effect of 2-AP on Me2SO-mediated
induction of
-globin and ALAS-E mRNAs and
heme content was examined. Combined treatment of cells with 2-AP and Me2SO for 48 h reduced the level of
-globin and ALAS-E mRNA levels to 13, and 75%,
respectively, compared with Me2SO treatment (Fig. 5). Addition of 2-AP to cells that had
been incubated with Me2SO for 48 h also decreased
significantly an increase of these indices in the subsequent 6 h
(Fig. 5). Heme contents in cells treated with Me2SO for
48 h were 21.36 ± 0.83 pmol of heme/106 cells
(mean ± S.D., n = 6), whereas they were decreased
to 15.74 ± 0.62 pmol of heme/106 cells in cells
treated with both Me2SO and 2-AP for 48 h. These findings indicate that inhibition of serine/threonine phosphorylation plays a critical role in erythroid differentiation of MEL cells.

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Fig. 5.
Effect of inhibition of serine/threonine
phosphorylation on erythroid differentiation. Upper left
panel, -globin mRNA. Lane 1, 1.5%
Me2SO; lane 2, 1.5% Me2SO + 10 mM 2-AP; lane 3, 1.5% Me2SO;
lane 4, 1.5% Me2SO + 10 mM 2-AP.
For cells in lanes 1-3, chemicals were added at time zero,
and cells were incubated for 48 h. For cells in lanes 3 and 4, cells were first treated with 1.5% Me2SO
for 48 h, and then 10 mM 2-AP was added. Incubation was continued for another 6 h. Northern blot analysis was
performed as described under "Materials and Methods." Lower
left panel, ALAS-E mRNA. Lane 1, 1.5%
Me2SO; lane 2, 1.5% Me2SO + 10 mM 2-AP; lane 3, 1.5% Me2SO;
lane 4, 1.5% Me2SO + 10 mM 2-AP.
Upper right panel, -globin mRNA levels normalized on the
basis of 28 S rRNA. Lower right panel, ALAS-E mRNA levels
normalized on the basis of 28 S rRNA. Conditions of incubation were
identical to those in the upper left panel. Experiments were
repeated twice using a nitrate salt of 2-AP and once using a free base
of 2-AP. Similar results were obtained in all of these
experiments.
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NF-E2 Function Is Regulated by the Ras Signaling
Cascade--
Various cell surface receptors transmit proliferation and
differentiation signals into cells through activation of signaling transduction cascades. The Ras-Raf-MAPKK cascade represents such an
important signal transduction cascade which ultimately phosphorylates serine/threonine residues of effector molecules, including various transcription factors (28). Thus we examined whether this cascade is
involved in the regulation of NF-E2 function. Coexpression of p45 and
MafK in NIH3T3 cells resulted in a small increase of the reporter gene
activity (~3-fold) which contained three copies of NF-E2 sites. When
a plasmid expressing the oncogenic form of H-ras
(H-RasV12) was cotransfected into NIH3T3 cells with p45 and
MafK expression plasmids, however, there was a marked increase in the
reporter gene activity (~25-fold) (Fig.
6A). In contrast, when a
deletion construct of p45 (p45
N), which lacked the amino-terminal
region thus defective in transcription (5), was cotransfected with a
MafK expression plasmid, the reporter gene activity remained suppressed, even in the presence of the H-RasV12 expression
plasmid (Fig. 6A). Hence it can be concluded that the
observed effect of H-RasV12 involved the functional
NF-E2.

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Fig. 6.
The Ras-Raf-MAPKK signaling cascade affects
the enhancer activity of the NF-E2 site. Panel A, NIH3T3
cells were transfected with luciferase (LUC) reporter pRBGP2
(0.5 µg), 1 µg each of p45 or p45 N (corresponding to 243-383
amino acids) effector molecules, and 0.2 µg each of MafK and
H-RasV12 expression plasmid. The results are expressed as
the ratio of luciferase activity to that generated from a pRBGP2 in the
absence of any effector plasmid. Panel B, NIH3T3 cells were
transfected with a reporter containing one NF-E2 site and various
combinations of p45 (1 µg) or MafK (0.2 µg) in the absence or
presence of a dominant positive MAPKK (ESES-MAPKK) expression plasmid.
The results are expressed as the mean of two separate
experiments.
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The effect of a constitutively active form of MAPKK was also examined
using a transactivation assay (Fig. 6B). In this experiment, we utilized a reporter plasmid pRBGP10, which contained a single NF-E2
site in front of a minimal promoter. Expression of activated MAPKK
itself had little effect on the reporter gene activity, indicating that
under these experimental conditions, this reporter construct is not a
suitable target for endogenous effectors such as AP-1. However, MAPKK
increased the reporter gene activity in the presence of both p45 and
MafK (~50-fold). As with the activated Ras, MAPKK also required the
amino-terminal region of p45 to achieve a high level of reporter gene
expression (data not shown).
The effect of H-RasV12 on the DNA binding activity of NF-E2
was also examined using a gel mobility shift assay. Although expression of p45 and MafK by themselves in NIH3T3 cells resulted in only weak DNA
binding activity, coexpression with H-RasV12 resulted in a
marked increase in p45-MafK heterodimer formation (Fig.
7). These results clearly show that the
Ras cascade has a significant effect on the DNA binding activity of
NF-E2. Because activation of this cascade is known to result in
serine/threonine phosphorylation of an effector molecule, these
findings are consistent with the results that serine/threonine
phosphorylation may play an important role in the regulation of NF-E2
function.

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Fig. 7.
The Ras signaling cascade increases NF-E2 DNA
binding. NIH3T3 cells were cotransfected with various combinations
of expression plasmids indicated in the figure. pSV- -galactosidase was also used to correct transfection efficiency. Gel mobility shift
assays were performed as described under "Materials and Methods."
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To examine whether these results were caused by changes in the level of
expression of NF-E2 subunits, we determined the accumulation of NF-E2
subunit proteins in transfected cells, using FLAG epitope-tagged p45 or
tagged MafK expression plasmids. These epitope-tagged proteins behaved
in a manner similar to that of the wild-type proteins in transfection
assays (data not shown). The levels of p45 were similar for p45
expression by itself (Fig. 8,
fourth lane from left), and expressed together
with MafK (Fig. 8, sixth lane). Similar results were also
observed when exogenous H-RasV12 was expressed additionally
(Fig. 8, fifth and seventh lanes). In contrast,
the amount of MafK increased slightly in response to exogenous
H-RasV12 in both the absence of p45 (Fig. 8,
second and third lanes) and the presence of p45
expression plasmid (Fig. 8, sixth and seventh lanes). This change in MafK protein level, however, did not
influence NF-E2 function (discussed below). These findings indicate
that an effect of the Ras cascade on NF-E2 function is not caused by a
change in the level of NF-E2 subunit expression.

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Fig. 8.
The Ras signaling cascade has no effect on
p45 but increases MafK protein levels slightly. p45 and MafK were
tagged with the FLAG epitope at the NH2 termini. NIH3T3
cells were transfected with various combinations of expression
plasmids. Transfection efficiency was corrected based on the
-galactosidase activity. Total cell extracts were prepared,
separated by SDS-polyacrylamide gel electrophoresis, transferred onto a
membrane, and reacted with anti-FLAG antibody.
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The NH2-terminal 83-Amino Acid Region of p45 Contains
an Activation Domain That Is Responsible for Ras-mediated Induction of
Transactivation--
To understand the molecular mechanism of
regulation of NF-E2 function, it is essential to define the function of
each subunit with respect to its trans-regulation. To
examine trans-regulatory function of p45 independent of DNA
binding, we fused various regions of p45 to the DNA binding domain of
GAL4 and transfected their expression plasmids into cells together with
a reporter gene plasmid. The results demonstrated that the
NH2-terminal region of p45 contains a transcription
activation domain (Fig. 9A).
When fused to the GAL4 DNA binding domain, 1-272 amino acids, or 1-83
amino acids of p45, showed marked transactivation of the reporter gene
in DS cells. In contrast, G4-p45 (86-272 amino acids) showed a minimal luciferase activity. G4-p45 (39-83 amino acids) increased luciferase activity moderately, whereas G4-p45 (1-36 amino acids) had little effect (Fig. 9A). These results indicate that the
NH2-terminal 83-amino acid region is required for a full
transactivation activity.

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Fig. 9.
The NH2-terminal region of p45
which contains transactivation domain is required for the Ras-mediated
induction of p45 transactivation potential. Panel A,
G4-p45(1-272) which contains the 5'-portion of mouse p45
cDNA (corresponding to 1-272 amino acids) linked to a
GAL4 DNA binding domain (G4DBD) and deletion constructs G4-p45(1-83),
G4-p45(86-272), G4-p45(1-36), G4-p45(39-83), corresponding to
1-83, 86-272, 1-36, 39-83 amino acids, respectively, were prepared.
One µg of each construct was cotransfected into DS cells with a
5 × GAL4 site-luciferase construct (G4LUC), as described under
"Materials and Methods." Results are expressed as the mean ± S.E. of four determinations. Panel B, 0.4 µg of G4-p45(1-272), G4-p45(1-83), or G4-p45(86-272) was cotransfected with 0.2 µg of G4LUC into NIH3T3 cells in the absence or presence of
H-RasV12 expression plasmid. The results are expressed as
the ratio of luciferase activity to that generated from G4LUC in the
absence of an effector plasmid. Results are expressed as the mean of
triplicate determinations.
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Using transactivation assays of p45 fusion constructs with the GAL4 DNA
binding domain, we investigated next whether this region contains the
Ras-responsive activation domain. As shown in Fig. 9B,
expression of H-RasV12 augmented reporter gene activity
markedly in the presence of p45 that contained the 83-amino acid
NH2-terminal region, whereas it had little effect on
reporter gene activity when a p45 fragment lacking the amino-terminal
region was used (Fig. 9B). These results indicate that the
activity of the activation domain of p45 can be regulated by the Ras
cascade. Hence, it can be concluded that the Ras cascade also has a
direct stimulatory effect on p45 transactivation potential.
Our previous studies demonstrated that MafK repressed the NF-E2
site-dependent promoter activity in the absence of p45 and that the presence of p45 reversed MafK transcription repression activity (5). Therefore we examined in this study the effect of MafK on
NF-E2 activity stimulated by the Ras cascade. First, we titrated the
effect of the MafK expression plasmid on reporter gene activity in the
absence or the presence of MAPKK. As shown in Fig.
10A, the reporter gene
activity showed a biphasic response to an increasing amount of the MafK
plasmid in the presence of a fixed amount of p45 expression plasmid.
For example, MafK plasmid transfection strongly augmented reporter gene
expression at low levels, whereas it suppressed at levels higher than
0.2 µg. This suppression can be accounted for by the fact that an
excess amount of MafK homodimer is known to compete against p45-MafK
heterodimers for binding at the NF-E2 site (3, 5). These results thus indicate that in the Ras-mediated regulation of NF-E2 activity, MafK
exhibits in the presence of p45 a positive transcriptional effect at
low levels of expression, whereas it represses transcription when
expressed in excess. Because 0.2 µg of MafK expression plasmid was
used in all of these experiments, it can be concluded that the
induction of NF-E2 activity by the Ras cascade (Fig. 6) is not caused
by an increase in the amount of MafK protein. Thus, the Ras signaling
cascade stimulates NF-E2-dependent transcription by
eliciting a qualitative, rather than a quantitative, change in the
NF-E2 subunits.

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Fig. 10.
Excess of MafK homodimer formation has an
inhibitory effect on the Ras-mediated stimulation of the NF-E2 site
enhancer activity. Panel A, transfection was carried out
with a fixed amount of p45 (0.4 µg) and increasing amounts of MafK
expression plasmid, with or without a MAPKK expression plasmid (0.2 µg) in NIH3T3 cells. Panel B, QT6 cells were cotransfected
with 1 µg of G4LUC and 1 µg each of various MafK expression
vectors, G4-MafK, G4MafK N, and G4MafK C, corresponding to 1-157,
8-157, and 1-123 amino acids, respectively, which were linked to
G4DBD. Results are expressed as the mean ± S.E. of three
determinations.
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Domain analysis of MafK using fusion constructs with GAL4DBD revealed
an additional mechanism for the repression of reporter activity by an
excess amount of MafK. Namely, the G4DBD-MafK fusion protein repressed
the reporter activity in QT6 fibroblasts (Fig. 10B). Because
such repression was observed with a GAL4 fusion product but not with
wild-type MafK or with GAL4DBD, the results indicate that MafK
possesses direct trans-repression activity, which does not
involve competition for a binding site. MafK derivatives that lacked an
NH2-terminal or COOH-terminal region also suppressed the
reporter gene activity potently (Fig. 10B), indicating that the trans-repression activity of MafK is confined to the
b-zip domain. A similar experiment in MEL cells was not possible,
however, because a basal reporter activity in these cells was too low. Nonetheless, these results taken together indicate that MafK exerts transcription repression not only by competition for its binding site
but also by direct trans-repression.
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DISCUSSION |
Previous studies using MEL cells suggested that NF-E2 may provide
the major enhancer function for globin gene expression (6, 7). For
example, the DNA binding activity of NF-E2 was shown to be increased
during erythroid differentiation of MEL cells (8-10); however, the
exact mechanism of regulation of NF-E2 activity remained unclear. The
level of p45 mRNA itself was reported either to increase
(29) or not to change (15) during erythroid differentiation of MEL
cells. Our results in this study demonstrated that the levels of
p45 mRNA, p45 protein, and the NF-E2·DNA complex were all
clearly increased in DS cells during erythroid differentiation (Figs. 1
and 2). The reason for this difference is unclear, but it may be
related to different clones of MEL cells used in these studies. It
should be noted that the clone of DS (DS-19) cells used in this study
is one of the most efficiently differentiating MEL cell clones (16). It
is also known that there is a significant increase in p45 protein
levels in erythroid differentiation of normal hematopoietic progenitor
cells which precedes the expression of erythropoietin receptor (30).
These changes of NF-E2 activity were absent in DR cells, which also
fail to undergo erythroid differentiation (Figs. 1 and 2). These
findings suggest that the up-regulation of NF-E2 activity is an
important regulatory event in erythroid differentiation.
NF-E2 can be phosphorylated by a cAMP-dependent protein
kinase in vitro, and prolonged activation of this kinase
increases the amount of NF-E2·DNA complex in MEL cells (15). Our
findings in this study show that inhibition of serine/threonine
phosphorylation results in marked reduction of
Me2SO-mediated induction of a NF-E2·DNA complex as well
as the enhancer activity of the NF-E2 site (Fig. 4, A and
D), suggesting that NF-E2 activity may be regulated by serine/threonine phosphorylation. Inhibition of serine/threonine phosphorylation by 2-AP was also accompanied by marked reduction in the
levels of
-globin mRNA, ALAS-E mRNA
(Fig. 5) and heme content. These findings suggest that serine/threonine
phosphorylation plays a critical role in the expression of erythroid
genes in MEL cell differentiation.
In contrast, inhibition of tyrosine phosphorylation augmented the
enhancer activity of the NF-E2 site, without influencing DNA binding
activity of NF-E2 (Fig. 4, A and D). It is
unclear at present why genistein increased the enhancer activity of the NF-E2 site, but it is possible that transactivation activity of the
NF-E2 molecule might be inhibited directly or indirectly by tyrosine
phosphorylation. It is also possible that other factors that can
interact with the NF-E2 site might be activated by genistein.
Consistent with such a hypothesis, our results demonstrated that the
activity of NF-E2 reconstituted in fibroblasts is under the control of
the Ras signaling cascade. Namely, a constitutively active form of Ras
and MAPKK, a downstream protein kinase, potentiated DNA binding
as well as transactivation of NF-E2 (Figs. 6A and 7). Transactivation assays using GAL4DBD-p45 fusion constructs also
demonstrated that the active form of Ras stimulates the p45 transactivation domain (Figs. 7B and 8A). The
amino-terminal transactivation domain of p45 also possesses two
consensus sequences for phosphorylation by MAPK (31, 32). The insertion
of a 2-amino acid substitution into the consensus sequences, however,
did not affect the reporter gene activity in transactivation assays
(data not shown). These findings suggest that either coactivators
essential for the activation domain of p45 may be modified by
phosphorylation, or other phosphorylation sites of the p45 activation
domain may be involved in such regulation.
We showed previously that the small Maf family proteins can function as
transcriptional repressors of NF-E2 site-dependent transcription in the absence of p45 (3, 5). Because MafK possesses an
active transcription repression domain in its b-zip region (Fig.
10B), it can exert effects not only by competition for
binding sites but also by an active repression mechanism that might
involve protein interaction with other factors such as corepressors. Furthermore, the fact that an excess amount of MafK repressed the
MAPKK-, or Ras-induced cis-regulatory activity of the NF-E2 site even in the presence of p45 (Fig. 10A) suggests that
the amount of MafK homodimer may be crucial in determining the level of
NF-E2 site-dependent gene expression. In other words,
certain combinations of MafK and heterodimerizing partners may
predispose cells to a particular response upon stimulation of the Ras
cascade.
To provide a hypothesis for further testing, a model for the role of
the Ras signaling cascade in NF-E2 activation is presented in Fig.
11. This predicts that the Ras
signaling cascade is activated during erythroid differentiation,
resulting in the increase of the erythroid-specific gene expression,
which is caused in part by an increase in NF-E2·DNA binding and in
part by an increase in p45 transactivation activity. One feature is
that MafK functions as both a signal transducer and a signal blocker,
depending on its amount as well as on the presence of p45. The fact
that DR cells show constitutive expression of mafK mRNA
and no increase in p45 mRNA by Me2SO
treatment is consistent with the view that signal transduction
resulting in the activation of NF-E2 function by the Ras signaling
cascade may be inhibited by an excess amount of MafK homodimer in these
cells.

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Fig. 11.
Hypothetical scheme of the regulation of
NF-E2 function. Panel A, p45-MafK heterodimer formation.
Panel B, MafK homodimer formation.
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We are grateful to Drs. M. Nakafuku, Y. Gotoh
and E. Nishida for providing various expression plasmids and to Dr.
Nakafuku for helpful discussion. We also acknowledge gratefully the
technical assistance of Luba Garbaczewski.