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INTRODUCTION |
MRG11 and MSG1 are the
members of a new family of transcription factors, which share a
conserved C-terminal acidic domain (the CR2 domain) that accounts for
their transcriptional activity. Through CR2 (1-4), MRG1 and MSG1
interact with the CH1 domain of the p300/cAMP response element-binding
protein (CBP) complex, nuclear proteins that function as coactivators
for basal transcription complexes (5-7). MRG1 also interacts with a
LIM homeodomain transcription factor, Lhx2, to enhance
Lhx2-dependent transcription possibly through recruitment
of p300/CBP and TATA-binding proteins (8). The interaction of MRG1 with
Lhx2 is dependent on the N-terminal region of MRG1, a region not
conserved in MSG1. MSG1 interacts with Smad4 and enhances
Smad-mediated transcription in a p300/CBP-dependent manner
(4, 9). The Smad interaction domain resides in the N terminus of MSG1
and thus is unique to MSG1. Based upon these findings, it has been
speculated that MRG1 family proteins bind p300/CBP through their
conserved C-terminal regions (the CR2 region) while interacting with
DNA-binding proteins through the unique N-terminal regions, thus
regulating the p300/CBP-dependent transcriptional activation of different target genes (4).
Besides their structural differences, MRG1 and MSG1 show a distinct
pattern of expression that may also determine their functional specificity (1-3, 10). MSG1 transcripts are predominantly expressed in
cultured human and mouse epidermal melanocytes, whereas MRG1 transcripts are detected in all of the human and mouse cell lines and
adult tissues examined (1-3, 10). Similarly, MRG1 and MSG1 expression
profiles during early development are distinct from each other (2).
MSG1 is predominantly expressed in a subset of mesoderm derivatives,
whereas MRG1 transcripts are restricted to anterior visceral endoderm
prior to gastrulation. Interestingly, MRG1 is expressed during heart
development and its expression can be detected throughout embryogenesis
from 8.5 days post-conception, which is consistent with the
ubiquitous expression of this gene in adult tissues. These results
suggested that MRG1 and MSG1 might play different roles during mouse embryogenesis.
MRG1 was cloned in our laboratory as part of an effort to isolate
cytokine-inducible genes. Overexpression in Rat1 cells results in loss
of cell contact inhibition, anchorage-independent growth in soft agar,
and tumor formation in nude mice, suggesting that MRG1 is a
transforming gene (10). Bhattacharya et al. demonstrated that MRG1 is up-regulated by hypoxia and deferoxamine (3), and
represses HIF-1
-mediated transactivation through competitive interaction with the CH1 domain of p300/CBP. Elevated expression of
MRG1 in hypoxic cells has been postulated to negatively regulate the
cellular response to hypoxia, which is cytokine-regulated.
The promoter region of human MRG1 has been reported but not analyzed in
detail (11), and the transcription factors that regulate it have not
been identified. We have now isolated the mouse MRG1 genomic sequence
and analyzed the promoter activity by deletion mapping and transient
expression in cells. The basal mouse MRG1 promoter maps to region to
104 to +121, which is highly conserved in the human and mouse genes.
Deletion analysis in conjunction with site-directed mutagenesis shows
that an Ets-1 site at
97 to
94 and an Sp1 site at
51 to
46 are
critical for MRG1 promoter activity. Electrophoretic mobility shift
assays (EMSA) show that Sp1 and Sp3 bind the Sp1 element, whereas
transcription factors binding to the Ets-1 element are yet to be
identified. Cotransfection in insect cells that lack Sp and Ets factors
demonstrates that Sp1, Sp3, Ets-1, or Elf-1 are important for MRG1 gene
expression and therefore for the responses of cells to different
biological stimuli.
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EXPERIMENTAL PROCEDURES |
Cell Lines and Reagents--
NIH3T3, Rat1, and HepG2 cells were
maintained in Dulbecco's modified Eagle's medium with 10% fetal calf
serum. Schneider's Drosophila cell line 2 (SL2) was
maintained in Schneider's Drosophila medium supplemented
with 10% fetal bovine serum at room temperature with atmospheric
CO2. Cell culture reagents were obtained from Life
Technologies Inc. (Gaithersburg, MD). Antibodies to nuclear proteins
Sp1, Sp3, and STAT3 were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
Isolation of Mouse Genomic DNA Clone--
A 129/SvJ mouse liver
genomic library in Lambda FIXII (Stratagene) was screened using the
2.1-kb MRG1 cDNA as the probe. The probe was random-labeled with
[
-32P]dCTP using the Prime-It kit (Stratagene). A 5-kb
HindIII fragment from one of the positive clones was
subcloned into the HindIII site of a cloning vector, pUC19,
resulting in plasmid pUCHi. Sequence analysis showed that the fragment
contains a 2.7-kb 5'-flanking region, the first and the second exons,
the introns, and most of the third exon. To identify putative
cis-regulatory elements, 1 kb of the mouse MRG1 gene
5'-flanking region was analyzed with the Mat Inspector program (12).
The transcription start site was predicted by the neural network
promoter prediction (NNPP) program (13).
Plasmid Construction--
The
1036 to +121 region relative to
the transcription start site was PCR-amplified using primers: P-1036,
5'-CCCAAGCTTCCCAACCCGTCAGGCAAGA; and P+121,
5'-GGGGTACCCGAGGTTGGCGCCGAGGTCTT. The PCR product was digested with
HindIII and KpnI and inserted in a promoterless luciferase reporter vector pXP2 (14), resulting in plasmid
pXP-1036/+121. To construct pXP-2700/+121, a 2.5 kb
HindIII/BamHI fragment digested from genomic
subclone pUCHi and 0.21kb BamHI/KpnI fragment
from
1036/+121 PCR product was ligated and inserted between
HindIII and KpnI site of pXP2. pXP-762/+121,
pXP-338/+121, pXP-92/+121 were generated by means of restriction enzyme
digestion. PXP-200/+121, pXP-104/+121, pXP-72/+121, pXP-56/+121, and
pXP-44/+121 were generated by PCR using P+121 as 3'-end primer
and the following 5'-end primers: P-200, 5'-CCAAGCTTCGGTCCGGAGACCTGCT;
P-104, 5'-CCAAGCTTGCTGATGTTCCGGGATC; P-72, 5'-TTAAGCTTGCCGCCGGGGAGGC;
P-56, 5'-TTAAGCTTCGCTCCGCCCTTCC; P-44, 5'-CCAGCTTCCTGAGATCCTTATAT,
respectively. Constructs generated by PCR were verified by sequencing.
pXP-56mSp1 and pXP-104mEts were generated by PCR using P+121 as 3'-end
primer and the following primers as 5'-end primers: P-56mSp1,
5'-TTAAGCTTCGCTTTGCCCTTCC; P-104mEts, 5'-CTAAGCTTGCTGATTAAGGGATCCGTGT. Specific mutation for the Sp1 site in the
104/+121 region was generated by PCR. Briefly, two simultaneous PCR reactions, using pXP-2700/+121 as template were performed. The first one used primer P-104 or P-104mEts as a 5'-end primer and PmSp1B
(5'-GATCTCAGGAAGGGCAAAGCGACAGCCTCC) as a 3'-end primer; the second one
used PmSp1A (5'-GGGAGGCTGTCGCTTTGCCCTTCCTGAGAT) as a 5'-end primer and
P+121 as a 3'-end primer. Amplified fragments from each PCR reaction
were purified, mixed, and subjected to a second round of PCR using two
external primers (P-104 or P-104mEts with P+121). The amplified PCR
product was inserted into pGEM-T vector (Promega) and verified by
sequencing. The MRG1 promoter fragment was released by digestion with
HindIII and KpnI and inserted into the
HindIII and KpnI sites of pXP2.
Transfection and Luciferase Assays--
One day prior to
transfection, NIH3T3 or Rat1 cells were seeded onto 6-well
plates at 2 × 105 cells/well. Cells were transfected
with 1 µg of reporter plasmid and 0.25 µg of pCMV
(CLONTECH) using Superfect Transfection Reagents (Qiagen). 48 h after transfection, cell extracts were prepared, and luciferase activity was determined with Luciferase assay systems (Promega).
-Galactosidase assays were performed as described previously by Sambrook et al. (15).
For transfection of HepG2 cells, cells were plated onto 24-well plates
at 4.0 × 104 cells/well and transfected with 0.6 µg
of the reporter plasmid and 0.2 µg of pCMV
using the calcium
phosphate method as described previously (15). The medium was changed
24 h after transfection. After another 24 h, cell extracts
were prepared and luciferase and
-galactosidase assays were
performed as described above.
For transfection of SL2 cells, 1 day prior to transfection, cells were
plated onto 6-well plates at 2.0 ×106 cells/well and
transfected by the calcium phosphate method as described (16). Each
well received 10 µg of DNA, including 5 µg of indicated luciferase
reporter construct and varying amounts of expression plasmid such as
pPacSp1 (17), pPacUSp3 (16), pPacUEts-1, or pPacUElf-1 (18). Variable
amounts of the expression plasmids were adjusted with the control
plasmid pPacO. The medium was changed 24 h after addition of DNA,
and the cells were harvested for luciferase assays 48 h after
transfection. Luciferase values were normalized against total protein
concentrations determined by protein assay (Bio-Rad).
Nuclear Extracts and EMSA--
Nuclear extracts from Rat1 cells
were prepared as described (19). Synthesized double-stranded
oligonucleotides were end-labeled with [
-32P]ATP and
T4 polynucleotide kinase. Approximately 0.2 ng of the labeled
oligonucleotide (20,000 cpm) was added to 10 µg of nuclear extracts
in a final volume of 20 µl containing 1 µg of poly(dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM EDTA, 1 mM MgCl2, 4% glycerol,
and 1 mM dithiothreitol and incubated for 15 min at room
temperature. Subsequently, the DNA·protein complexes were separated from the free probe by electrophoresis through a 5% nondenaturing polyacrylamide gel. Gels were dried and subjected to
autoradiography in the presence of intensifying screens (DuPont) at
80 °C. For supershift analysis, 1 µl of antibody was incubated with nuclear extracts at 4 °C for 1 h in binding buffer,
followed by an additional incubation for 30 min at room temperature
with labeled oligonucleotides. For competition analysis, unlabeled DNA
was incubated with nuclear extracts at 4 °C for 20 min before the
addition of the labeled probe.
The oligonucleotides used as EMSA probes were annealed prior to
labeling. The sequences of the upper strands of the oligonucleotides used were as follows: MRG1 Sp1 (
56/
42), 5'-TTAAGCTTCGCTCCGCCCTTCC; MRG1 Ets-1 (
101/
85), 5'-GATGTTCCGGGATCCGT. The following
oligonucleotides were used for competition analysis: mutated MRG1 Sp1
(
56/
42), 5'-TTAAGCTTCGCTTTGCCCTTCC; mutated MRG 1 Ets-1
(
101/
85), 5'-GATGTTAAGGGATCC; consensus ETS/PEA3 oligonucleotide,
5'-CTGAACTTCCTGCTCGAGATC (Santa Cruz Biotechnology); MRG1
56/
34,
5'-TCGTCCCGCCTTCCTGAGATCC; and SIE (sis-interacting
element), 5'-GTCGACATTTCCGTAA ATCTTGT (20).
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RESULTS |
Isolation and Characterization of Mouse MRG1 Genomic
Sequence--
To understand the molecular basis for the ubiquitous
expression of MRG1, we analyzed its promoter activity. A 5-kb
HindIII fragment from a mouse MRG1 genomic clone, which
contains 2.7 kb upstream sequence from the transcription start site and
all of the coding regions, was subcloned into the pUC19 vector. Similar to the human MRG1 gene (11), the mouse gene consists of three exons,
separated by two small introns (data not shown). The transcription start site was predicted according to the neural network promoter prediction (NNPP) program (13). Potential regulatory elements have been
identified through the Mat Inspector program (12). These include C/EBP
sites at
998 and
758; Sp1 sites at
748,
698,
648,
348,
178, and
51; AP4 site at 718; AP2 site at
218; Ets binding sites
at
278,
97, and
44; MZF-1 site at
65; and an E-box at
148
(Fig. 1A) (the corresponding
GenBankTM accession number for mouse MRG1 gene is AF295547).
Extensive identity exists between mouse and human proximal promoter
regions (Fig. 1B), introns and exons (data not shown),
indicating that the MRG1 gene is well conserved during evolution. As
shown in Fig. 1B, there is an overall 80% identity between
mouse and human MRG1 genes in the proximal promoter region between
190 and +121. Sequences further upstream are more diverse, suggesting
190 to +121 may play an important role in MRG1 expression.

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Fig. 1.
DNA sequence of the mouse MRG1 promoter and
its alignment with the human MRG1 promoter. A, mouse
MRG1 promoter sequence. Several putative regulatory elements are
identified and boxed. End points of 5'-deletion mutants are
indicated by brackets at 762, 338, 200, 104, 92,
72, 56, and 44. The arrow indicates the transcription
start site. B, alignment between mouse and human MRG1
proximal promoter sequences. Dotted lines indicate identical
nucleotide sequences between the two promoters. Horizontal
lines are introduced in the sequences to generate highest homology
between the two sequences.
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Identification of Transcription Elements Responsible for MRG1
Expression--
To assess the promoter activity of the MRG1
5'-flanking region, deletion mutants were generated using the firefly
luciferase reporter vector, pXP2 (14). Each resulting recombinant
plasmid DNA was then transiently transfected into Rat1 fibroblasts with pCMV
to monitor transfection efficiency. Among the constructs tested, pXP-104/+121 still had the full promoter activity, whereas pXP-44/+121 lost most of the promoter activity (Fig.
2A), indicating that the
region between
104 and
44 contains critical elements for MRG1
expression. This region contains an Sp1 site (CCGCCC) at
51, an Ets-1
site (TTCC) at
97, and an MZF-1 site (GGGA) at
65. Rat1 cells were
chosen, because we previously utilized this cell line to demonstrate
that MRG1 expression can be induced by serum, insulin, and
platelet-derived growth factor (10). The same experiments were also
performed in NIH3T3, and HepG2 cells, and the results were similar,
consistent with the ubiquitous expression of MRG1 in different cell
types (data not shown).

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Fig. 2.
Promoter activity of serial deletion
constructs of the mouse MRG1 gene. Rat1 cells were transiently
cotransfected with 1.0 µg of sequentially deleted reporter constructs
and 0.25 µg of pCMV construct. Luciferase results were normalized
by -galactosidase activity. The experiments were done in
triplicates. Data are the mean ± S.D. P represents the
promoterless luciferase vector pXP2. In A, promoter
activities of eight deletion constructs are shown. The promoter
activity of pXP-2700/+121 represents 100, and activities of other
constructs are compared with that of pXP-2700/+121. In B,
promoter activities of another three deletion constructs and a
site-directed mutant are shown. The promoter activity of pXP-104/+121
represents 100.
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We then constructed more detailed deletions within this region (Fig.
2B). As compared with pXP-104/+121, construct pXP-92/+121 with only 12-bp sequence deleted at the 5'-region had a significantly lower promoter activity. This 12-bp region contains an Ets-1 binding site. pXP-56/+121 still had about 26% promoter activity of
pXP-104/+121. However, further deletion of 12 base pairs from
56/+121
(pXP-44/+121), which contains the Sp1 binding site, greatly reduced the
promoter activity. Similarly, mutations of the Sp1 site in pXP-56/+121 from CCGCCC to TTGCCC decreased the promoter activity to the level of
pXP-44/+121 (Fig. 2B). It should also be noted that deletion of the region between
92 and
57 also resulted in a significant decrease in the promoter activity. This region contains an MZF-1 binding site (GGGA sequence at
66 to
62). These results
demonstrated that the Ets-1 site, Sp1 site, and the region between
92
and
56 are important for the mouse MRG1 promoter activity.
Contribution of Ets-1 and Sp1 Sites to the Proximal Promoter
Activity of Mouse MRG1 Gene--
To further define the specific
elements in the proximal
104 region that contribute to the proximal
promoter activity, we generated a series of MRG1 promoter constructs
with mutations in Ets-1 and Sp1 elements from pXP-104/+121. Ets-1 motif
TTCC was mutated to TTAA, whereas Sp1 motif CCGCCC was mutated to
TTGCCC. Upon transfection into Rat1 cells, constructs containing a
specific mutation of Sp1 or Ets-1 element resulted in about 90 and 50% decreases in the promoter activity, respectively (Fig.
3). The results were similar in the
NIH3T3 fibroblast cell line. In HepG2, disruption of the Sp1 site
decreased 75% of the promoter activity. In all cases, mutations of
both sites resulted in a 90-98% loss of the proximal promoter
activity (Fig. 3), demonstrating that both Ets-1 and Sp1 elements are
important for the MRG1 proximal promoter activity.

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Fig. 3.
Effect of site-specific mutations on mouse
MRG1 proximal promoter activity. Site-specific mutations were
performed on pXP-104/+121 (WT). Rat1 or NIH3T3 cells were
cotransfected with 1.0 µg of MRG1 promoter construct and 0.25 µg of
pCMV . HepG2 cells were cotransfected with 0.6 µg of MRG1 promoter
construct and 0.2 µg of pCMV . Luciferase activity was normalized
against -galactosidase activity. The activity of the WT construct
represents 100, and activities of mutated constructs were compared with
the activity of WT. The solid circles represent a wild type
sequence, whereas the circles with a cross
represent a mutated sequence. Experiments were performed in
triplicates. Shown are mean ± S.D.
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Identification of Transcription Factors Binding to Sp1 or Ets-1
Element in MRG1 Proximal Promoter--
To determine the biochemical
composition of protein complexes binding to the proximal Sp1 or Ets-1
element of the MRG1 promoter, nuclear extracts from Rat1 fibroblast
cells were analyzed by EMSA using a labeled Sp1 or Ets-1
oligonucleotide probe. As illustrated in Fig.
4A, three DNA·protein
complexes (A, B, and C with
arrows) were detected using labeled Sp1 probe (lane
2). These DNA·protein complexes were specific, because they
disappeared in the presence of excess amounts of unlabeled Sp1
competitors (lanes 3 and 4), and inclusion of an
excess amount of unlabeled mutated Sp1 oligonucleotide did not affect
the formation of these complexes (lane 5). Because members
of the Sp1 multigene family share the same binding consensus sequence
(GC or GT box) (21), we performed supershift experiments to determine
the identity of the three complexes. As shown in Fig. 4B,
anti-Sp1 antibody inhibited complex A formation (lane 2),
whereas complexes B and C were supershifted in
the presence of anti-Sp3 antibody (lane 3). In the presence
of both antibodies, complexes B and C were
supershifted and the intensity of complex A decreased
(lane 4). Anti-STAT3 was included as a negative control and
was found not to affect the mobility of these complexes (lane 5). The gel shift pattern was similar when performed using nuclear extracts from NIH3T3 cells (data not shown). These results indicated that Sp1 is a component of complex A and complexes
B and C contain Sp3, a typical gel shift pattern
as previously described for the Sp1 site (21).

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Fig. 4.
EMSA analysis of nuclear proteins bound to
the mouse MRG1 Sp1 binding site. A, gel mobility shift
assay for Sp1 site. 10 µg of Rat1 nuclear extract was incubated with
Sp1 ( 56/ 42) probe alone (lane 2), or in the presence of
unlabeled Sp1 (lane 3, 50-fold excess; lane 4,
100-fold excess) or mutated Sp1 (lane 5, 100-fold excess)
oligonucleotide as competitors. Lane 1 shows the mobility of
Sp1 probe alone without nuclear extract. Three Sp1-specific
DNA·protein complexes are indicated as A, B,
and C. B, gel mobility supershift assay for the
Sp1 site. Mouse MRG1 Sp1 probe was incubated with 10 µg of Rat1
nuclear extracts either alone (lane 1) or in the presence of
anti-Sp1 (lane 2), anti-Sp3 (lane 3),
anti-Sp1+anti-Sp3 (lane 4), or anti-STAT3 (lane 5).
C, gel mobility shift assay for the Ets-1 site. Gel mobility shift
assay was performed using Ets-1 ( 101/ 85) probe and Rat1 nuclear
extract (15 µg) (lane 1). Competition analysis was
performed in the presence of 100-fold excess of unlabeled Ets-1
( 101/ 85) (lane 2), mEts-1 ( 101/ 85) (lane
3), MRG1 56/ 34 (lane 4), ETS/PEA3 consensus
sequence (lane 5), or SIE oligonucleotide (lane
6). The sequences of the oligonucleotides used are shown at the
bottom of the figure. Three Ets-1-specific DNA·protein complexes are
indicated as A, B, and C.
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The potential interaction of the Ets-1 element with nuclear proteins
was also evaluated by EMSA (Fig. 4C). Transcription factors in the Ets family recognize the sequence
(A/G)(T/A)(G/A)(A/T)TCC(G/T)(G/C)Y with a similar structure in the DNA
binding domain. The TCC sequence is absolutely essential for Ets family
members to bind DNA, with the specificity between different members
determined by surrounding nucleotides (22, 23). The
104/+121 region
contains two Ets core consensus sequences: one at
97 to
94 (most closely matched Ets-1 recognition sequence) and the other one
at
45 to
42. With MRG1 Ets-1 element (
101/
85) as a probe,
three complexes were formed (lane 1) and could be competed
by unlabeled
101/
85 oligonucleotide (lane 2), suggesting
they are specific. Conversely, unlabeled oligonucleotide bearing a
mutated Ets-1 element (TTCC mutated to TTAA) was unable to compete for
binding (lane 3), indicating the core TTCC motif is critical
for the formation of DNA·protein complexes. However, the unlabeled
sis-interacting element (SIE) oligonucleotide (20),
containing a core TTCC motif, could not compete for the complex
formation (lane 6), suggesting that flanking sequences are
also important for the formation of DNA·protein complexes.
Interestingly, the other TTCC-containing oligonucleotide from the MRG1
promoter (
56 to
34) could compete for the formation of complex
A but not complexes B and C. Mutations
of the TTCC motif in region
56 to
34 did not significantly affect
the promoter activity in Rat1 cells (data not shown). Most importantly,
the addition of a 100-fold excess of unlabeled Ets/PEA3 binding
oligonucleotide (lane 5) inhibited the formation of all
three complexes almost as effectively as did MRG1-101/
85
oligonucleotide. These results strongly suggested that
101/
85
contains an Ets-1 binding site. However, addition of monoclonal
antibodies directed against various members of Ets factor family
(Ets-1, Ets-2, PU.1, Erg-1, Fli-1, and Elk-1) did not change the gel
shift pattern (data not shown). The exact identity of proteins in each
of the DNA·protein complexes therefore remains to be determined.
Cooperative Activation of Mouse MRG1 Promoter by Sp1- and
Ets-related Proteins--
To directly determine whether Sp1- and
Ets-related proteins could functionally modulate MRG1 promoter
activity, Drosophila SL2 cells, which are deficient in Sp1-,
Sp3-, and Ets-related proteins (16, 17) were utilized. The reason for
using insect instead of mammalian cells is because Sp1-, Sp3-, and
Ets-related factors are expressed in virtually all mammalian cells,
which could affect the interpretation of cotransfection experiments. We
introduced MRG1 proximal promoter construct pXP-104/+121 along with
Drosophila expression vectors pPacSp1 or pPacUSp3 into
Drosophila SL2 cells. As shown in Fig.
5A, both pPacSp1 and pPacUSp3
stimulated pXP-104/+121 promoter in a dose-dependent
manner, with pPacSp1 being the more potent activator. Stimulation was
about 35-fold with 2.5 µg of pPacSp1. To further investigate whether
Sp3 could enhance transcriptional activation by Sp1, pXP-104/+121 (5 µg), pPacSp1 (1 µg), and pPacSp3 (1-4 µg) were cotransfected
into Drosophila SL2. As shown in Fig. 5B,
transcriptional activation by Sp1 could clearly be enhanced by Sp3.
Moreover, when the Sp1 binding site was mutated, no potentiation was
observed with Sp1, Sp3, or the combination of Sp1 and Sp3. These
results indicated that the Sp1 element is required for mediating the
transactivation effect of both Sp1 and Sp3 on the MRG1 promoter (Fig.
5C).

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Fig. 5.
Sp1- and Ets-related proteins activate mouse
MRG1 promoter activity in Drosophila SL2 cells.
A, Sp1 or Sp3 alone activates MRG1 promoter. SL2 cells were
cotransfected with 5.0 µg of pXP-104/+121 with varying amounts
(0.25-5.0 µg) of expression plasmids for Sp1 and Sp3. Cells were
incubated with calcium phosphate-precipitated DNA for 24 h, then
changed to fresh medium for another 24 h. The luciferase value was
normalized against protein concentration. Fold induction
represents the -fold increase in the luciferase activity against vector
pPacO-cotransfected control. Data shown are representative of three
experiments. B, Sp3 enhances Sp1-mediated transactivation of
the MRG1 promoter. SL2 cells were transfected with pXP-104/+121 with
1.0 µg of pPacSp1 and varying amounts of pPacUSp3 (0-4.0 µg). Data
are the mean ± S.D. from three experiments. C, Sp1/Sp3
activation of the MRG1 promoter is through the Sp1 site. SL2 cells were
transfected with pXP-104/+121 or pXP-104mSp1 with 1.0 µg of
expression plasmids for Sp1, Sp3, or Sp1+Sp3. Data shown are
representative of three experiments. D, Sp1- and Ets-related
proteins act in synergy in activation of MRG1. 5 µg of pXP-104/+121
or pXP-104mEts-1 construct was cotransfected into SL2 cells with 1 µg
of expression plasmids for Sp1, Ets-1, Sp1+Ets-1, Elf-1, and Elf-1+Sp1.
Luciferase assays were performed as described. All the experiments were
performed in triplicates. Data shown are mean ± S.D.
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To assay the functional effect of Ets-related proteins on MRG1
expression, pXP-104/+121 or pXP-104mEts-1 (5 µg) was introduced into
Drosophila SL2 cells together with the Drosophila
expression plasmids for Ets factors, pPacUEts-1 (1 µg), or pPacUElf-1
(1 µg). Results showed that Ets-1 or Elf-1 alone could increase
luciferase activity of pXP-104/+121, with Ets-1 being more potent than
Elf-1 (Fig. 5D). They had no effect on the Ets-1 mutant
construct pXP-104mEts-1, suggesting the transactivation by Ets-1 or
Elf-1 is dependent on the integrity of the Ets-1 element. Consistent
with these results, six copies of the
101/
85 region have been shown
to enhance the activity of a heterologous TK promoter in various cell
lines and to mediate the Ets-1 or Elf-1 transactivation in SL2 cells
(data not shown). Finally, to test the possible functional interplay between the Sp1- and Ets-related proteins, we performed cotransfection experiments using pXP-104/+121 or pXP-104mEts-1, together with plasmids
expressing different transcription factors. As shown in Fig.
5D, pPacUSp1 (1 µg) stimulated promoter activity by about 11-fold, whereas coexpression of Sp1 with Ets-1 stimulated pXP-104/+121 by about 35-fold. Cotransfection of Elf-1 with Sp1 also resulted in a
cooperative functional interaction on MRG1 promoter. This synergy was
dependent upon the integrity of the
101/
85 region and was not
observed on mutant promoter construct pXP-104mEts-1. These results
demonstrated synergistic activation of the MRG1 promoter by Sp1 and Ets
transcription factors.
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DISCUSSION |
MRG1 represents the founding member of a new family of
transcription factors that may be involved in many physiological
processes through its interactions with p300/CBP, TATA binding proteins and certain specific DNA-binding transcription factors (1-4, 8-10).
Sequence alignment of mouse and human (11) MRG1 genes showed extensive
identity in proximal promoter regions (Fig. 1B), exons and
introns, implicating high conservation of MRG1 during evolution.
Sequences upstream of
190 are more divergent, suggesting the proximal
promoter region might play an important role in MRG1 gene regulation.
Consistent with this inference, we mapped the basic promoter region of
mouse MRG1 to
104/+121. We further identified two elements, Sp1
element (
51 to
46) and Ets binding site (
97 to
94) within this
region to be essential for the promoter activity.
Sp1 is a well-characterized sequence-specific DNA-binding protein that
is important for transcription of many cellular and viral genes that
contain GC boxes in their promoters (17, 21). Three Sp1-related
transcription factors (Sp2, Sp3, and Sp4) have been cloned. Sp2 does
not recognize the same sequence as Sp1, and Sp4 expression is
restricted to the brain. Sp3, on the other hand, is ubiquitously
expressed and recognizes the same sequence as Sp1. Although Sp1 appears
to be almost exclusively an activating transcription factor, Sp3
contains a transcriptional repression domain and can act as a
transcriptional activator or repressor, depending on the promoter and
cell type studied (16, 24). We have shown that Sp1 is part of complex
A, whereas complexes B and C contain
Sp3 (Fig. 4). In addition, we have demonstrated that both Sp1 and Sp3
transactivate MRG1 promoter in Drosophila SL2 cells.
Interestingly, our data also suggested that Sp1 and Sp3 function in a
cooperative manner for MRG1 promoter activity (Fig. 5). Sp1 has been
shown to associate directly with members of the basal transcription
machinery such as TFIID components and has been viewed as a
constitutive transcriptional activator that acts as a basal factor for
TATA-less promoters. Ubiquitous and constitutive expression Sp1/Sp3 may
account for high basal expression of MRG1 in various cell types and
tissues. In addition, despite its general role in transcription of
housekeeping genes, Sp1 has been demonstrated to be involved in induced
transcription of various genes responding to different biological
stimuli (25-29). While Sp1 and Sp3 are ubiquitous nuclear factors,
differences in expression level during different stages of development
(30, 31) or in various cell types (31) along with specific
post-translational modifications (32) are responsible for altering gene
transcription in a development-specific and cell-specific manner (33).
The correlation of Sp1 and MRG1 expression during development and responses to biological stimuli need to be further investigated.
The common structural feature of Ets family transcription factor
is the presence of a unique DNA binding domain that recognizes the core
GGAA/T motif in a 10-bp DNA sequence (22, 23). Different Ets proteins
exhibit low selectivity in binding site preference, in common with many
other transcription factor families, including the homeodomain
proteins. The residues flanking the GGA motif dictate whether a
particular Ets domain will bind the site. This extending family
contains proteins involved in cell growth, differentiation, and
transformation. We have identified Ets-1 site at
101/
85 and its
binding proteins to be essential for MRG1 expression. These binding
proteins appear to be relatively ubiquitous, because the Ets-1 element
served as an enhancer in HepG2, Rat1, NIH3T3, and HeLa cell lines (data
not shown). However, we failed in our initial attempts to identify
specific Ets factors involved in complex formation through gel
supershift assays. This is consistent with difficulties encountered by
others in the identification of specific Ets family members (18), in
part due to the contribution of autoinhibitory binding domains present
in each of the Ets factors (34-36). Nevertheless, two of the Ets
family members, Ets-1 and Elf-1, which have very different expression
patterns among tissues and cell lines, could activate transcription
through the MRG1 Ets-1 element in SL2 cells (Fig. 5D). These
data therefore strongly support the positive role of the MRG1 Ets-1
element in MRG1 expression. Given the fact that most cell types express
multiple Ets proteins and these proteins can interact with similar or
identical sequences, the existence of this functional Ets-1 site in
MRG1 promoter may also account for the ubiquitous nature of MRG1
expression. Furthermore, several Ets family transcription factors have
been shown to be nuclear targets of the Ras-Raf-MAPK signaling cascade
(37). In many instances, specific phosphorylation of Ets proteins
greatly enhances their ability to activate transcription and subsequent cross-talk of ubiquitous signaling cascades. Therefore, the transient up-regulation of MRG1 we reported previously (10) might in part be
contributed by the Ras-Raf-MAPK signaling pathway. Further experiments
are required to clarify this point.
It is well established that Ets factors bind DNA as monomers (except
for GABP
·GABP
complex) but can also associate with other
transcription factors bound to their cognate motifs in the vicinity of
the Ets binding site (22, 23). For example, Ets-1 and Sp1 interact to
activate synergistically the human T-cell lymphotropic virus long
terminal repeats (38). In addition, Sp1 activity can be modulated by
factors that recognize DNA elements flanking or overlapping a GC box
(39, 40). In the context of the mouse MRG1 promoter, the Ets-1 element
is located 40 nucleotides upstream of the Sp1 element, which provides a
close proximity for an Ets-related factor and Sp1 to interact.
In this regard, it is interesting to find that indeed Sp1 and Ets
factors synergistically activate the MRG1 proximal promoter in SL2
cells (Fig. 5D).
In summary, we cloned and functionally characterized mouse MRG1
promoter and identified two essential elements in the proximal promoter
region. We showed that Sp1, Sp3, and an Ets-related transcription factor act synergistically to activate the mouse MRG1 promoter. The
identification of these cis-elements and associated
transcription factors provide the first explanation for the ubiquitous
expression of MRG1 in cells and the possible mechanism of its
up-regulated expression by different biological stimuli.