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INTRODUCTION |
Endogenous and synthetic
-opioids have been shown to modulate
T-cell proliferation, cytokine production, and calcium mobilization through the
-opioid receptor
(DOR)1 on T cells (1-4). For
example,
-endorphin was shown to enhance intracellular calcium
mobilization in murine splenic T cells, which was inhibited by
naltrindole, a selective DOR antagonist, whereas the selective
µ-opioid receptor antagonist was ineffective (2). In addition, the
enhancement of human T-cell proliferation by certain
methionine-enkephalin analogs could be completely abolished by naloxone
and selective DOR antagonists (3). It was also reported that DOR
agonists such as deltorphin and SNC-80 could concentration-dependently suppress the expression of human
immunodeficiency virus-1 in DOR-transfected human T cells (4).
DOR transcripts and DOR protein have been detected in mouse splenic and
thymic T cells, as well as in some human or murine T-cell lines (5).
Considerable evidence indicates that the transcription of the
dor gene is correlated with both the expression of DOR on T
cells and the capacity of DOR agonists to affect the functions of the T
cell (6-10). Thus, understanding the molecular mechanism underlying
the transcriptional regulation of the dor gene in T cells
may raise the possibility of regulating the immunomodulatory effects of
-opioids on T cells by manipulation of the expression of DOR.
Previously, we analyzed a 1.3-kilobase pair DNA fragment immediately
upstream of the translation start site (
1300 to +1 base pair, with
the translation start site designated as +1) of the mouse
dor gene in a mouse neuronal cell line and identified a minimum promoter region (
262 to
141); a GC box (
226 to
221) and
a composite Ets-1-binding site/E box (
192 to
180) were found crucial for the promoter activity (11, 12). Subsequent studies revealed
that the minimum dor promoter was also sufficient to confer
constitutive dor promoter activity in EL-4 cells, a mouse T
cell line that constitutively expresses DOR. In addition, increased binding activity of Ikaros (Ik) at an Ik-binding site (
378 to
374)
was demonstrated to account for the significantly enhanced dor promoter activity in phytohemagglutinin (PHA)-activated
EL-4 cells (13). In the present study, further analyses were carried out in EL-4 cells. Through both in vivo and in
vitro experiments, we have demonstrated that Ik-2 homodimers bind
to the
378/
374 Ik-binding site and exerts a
position-dependent trans-activation effect on the
dor promoter. Moreover, the E box (
185 to
180), which
binds upstream stimulatory factor (USF), is essential for the
dor promoter activity in both resting and PHA-activated T cells. Furthermore, we have demonstrated that Ik-2 and USF synergize in
trans-activating the dor promoter via the putative
Ik-binding site and the E box, respectively.
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MATERIALS AND METHODS |
Plasmid Construction--
The luciferase fusion plasmids pD1300
and pD400 were constructed as described previously (13). The mutant
constructs pD400MIK2 and pD400ME were constructed using the Altered
Sites II in vivo mutagenesis system (Promega) according to
the instructions of the manufacturer. The Ik-2 expression vector was
created by polymerase chain reaction (PCR) using the reverse
transcription products from the total RNA of the EL-4 cell. The upper
primer bears the essential Kozak sequence and the lower primer bears
the XbaI site. The PCR product was inserted into the
EcoRV and XbaI sites of pcDNA3 vector
(Invitrogen). The pD1300IK and pD400AS constructs were generated by
PCR. All of the correct clones were confirmed by sequencing.
Cell Culture--
Mouse lymphoma EL-4 cells were grown in
Dulbecco's modified Eagle's medium with 10% fetal calf serum, 4 mM L-glutamine, and 4.5 g/liter glucose.
The cells were incubated at 37 °C in an atmosphere of 10%
CO2 and 90% air.
Transient Transfection and Reporter Gene Activity
Assay--
EL-4 cells were transfected using SuperFect transfection
reagent (Qiagen) according to the instructions of the manufacturer. Briefly, cells were transfected with equimolar amount of each plasmid.
After a 24-hour culture with or without PHA (1.5 µg/ml), cells were
harvested and lysed with lysis buffer (Promega). A one-fifth molar
ratio of PCH110 plasmid (Amersham Pharmacia Biotech) containing the
-galactosidase gene driven by an SV40 promoter was included in each
transfection for normalization.
Recombinant Protein Purification--
Bacterially expressed
recombinant human USF-1 and mouse Ik-2 were prepared as described by
Pognonec et al. (14). Briefly, competent JM109 bacteria
cells were transformed with human USF-1 or mouse Ik-2 expression
vectors. The cultures were grown and induced at 28 °C overnight. The
bacteria pellet was lysed by sonication in ice-cold lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1% (v/v) aprotinin (Sigma), 0.1% Nonidet P-40).
The sonicated sample was centrifuged at 4 °C for 10 min at
10,000 × g. Then, saturated ammonium sulfate was added
dropwise to the supernatant to a final concentration of 33% (v/v).
After 15 min on ice, the sample was centrifuged at 4 °C for 10 min
at 10,000 × g. The pellet was resuspended in lysis
buffer and centrifuged again. Then the supernatant was diluted with
lysis buffer devoid of NaCl. The partially purified recombinant protein
products, proved to comprise predominantly the desired recombinant
proteins by 10% SDS-polyacrylamide gel electrophoresis plus Coomassie
Blue staining, were used in subsequent gel retardation assays in which the purification product from mock-transformed bacteria cells was
employed to incubate with the radiolabeled probe as a control.
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear
extracts were prepared from resting or PHA-activated EL-4 cells using
the method described by Johnson et al. (15). The plasmids
pD400, pD400MIK2, and pD400ME were digested with KpnI,
dephosphorylated with calf intestinal alkaline phosphatase, end-labeled
with [
-32P]ATP, and then digested with NcoI
to generate the 5'-labeled 400-base pair probes, which were purified by
polyacrylamide gel electrophoresis. The double-stranded oligonucleotide
D198/169 was 5'-end labeled with [
-32P]ATP. The probes
were incubated with EL-4 nuclear extracts or the indicated amounts of
recombinant protein(s) in EMSA buffer (10 mM Tris, pH 7.5, 5% glycerol, 1 mM EDTA, pH 7.1, 50 mM NaCl, 1 mM dithiothreitol, and 0.1 mg/ml poly(dI-dC)). For
competition analysis, a 75-fold molar excess of cold probe was added to
the mixture and incubated at room temperature for 30 min. For
supershift assays, 2 µg of anti-Ik, anti-USF-1, or anti-USF-2
antibody (Ab) (Santa Cruz Biotechnology) was added to the mixture. The
reaction was then incubated on ice for 1 h. Protein-DNA complexes
and free DNA were fractionated on 5% polyacrylamide gels in 1× Tris
borate-EDTA electrophoresis buffer at 4 °C and visualized by autoradiography.
Western Blot Analysis--
16 µg of nuclear extracts prepared
from an equal amount of unstimulated or PHA-activated EL-4 cells was
loaded onto 10% SDS polyacrylamide gels. Proteins were blotted onto a
polyvinylidene difluoride microporous membrane (Millipore). Membranes
were incubated for 1 h with a 1/1000 dilution of anti-USF-1 or
anti-USF-2 Ab and then washed and revealed using anti-rabbit IgG
horseradish peroxidase conjugate (1/5000, 1 h). Peroxidase was
revealed with an Amersham Pharmacia Biotech ECL kit. Proteins were
quantified before being loaded onto the gel, and equal loading of
extracts was verified by Ponceau coloration.
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RESULTS |
Ik-2 Homodimers Bind to the
378/
374 Ik-binding Site
and Trans-activates the dor Promoter--
Previously we reported that
in parallel with the augmented expression of nuclear Ik proteins, the
increased binding of Ik family members at an Ik-binding site (
378
to
374) in the mouse dor promoter enhanced the
dor promoter activity in PHA-activated EL-4 cells, a mouse T
cell line that constitutively expresses DOR (13). As only Ik-1 and Ik-2
are the predominant Ik isoforms capable of DNA binding in the nucleus
(16, 17), we employed the mouse Ik-1 and Ik-2 expression vectors in the
present study to determine the individual roles of these proteins in
trans-activating the dor promoter. The Ik-1 or Ik-2
expression vector was transfected into EL-4 cells with pD400, a mouse
dor promoter/luciferase fusion plasmid encompassing the
dor promoter sequence from
400 to +1, or pD400MIK2, a
mutant of pD400 with a point mutation in the core binding motif of the
378/
374 Ik-binding site (Fig.
1A). As shown in Fig.
1B, pD400 and pD400MIK2 displayed similar promoter
activities in resting EL-4 cells. Overexpressed Ik-2 enhanced the
promoter activity of pD400 by ~2-fold, almost to the level of that
activated by PHA, while showing no effect on pD400MIK2. In addition,
overexpressed Ik-1 exerted no detectable effect on the promoter
activity of pD400. These results indicate that Ik-2 but not Ik-1 can
trans-activate the dor promoter via the
378/
374
Ik-binding site.

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Fig. 1.
Ik-2 trans-activates the dor
promoter via the 378/ 374 Ik-binding site. A,
the luciferase reporter construct pD400 contains the dor
promoter sequence from 400 to +1. The mutant construct pD400MIK2 was
made by introducing a point mutation into the core binding motif of the
378/ 374 Ik-binding site in pD400. The putative Ik-binding site in
pD400 is underscored, and the mutant nucleotide in pD400MIK2
is shown in boldface. B, EL-4 cells were
transfected with 2.0 µg of pD400 or pD400MIK2 and 0.5 µg of the
Ik-1 or Ik-2 expression vector. In addition, pD400 was transfected into
EL-4 cultures with or without PHA activation (1.5 µg/ml). Luciferase
activities were normalized to -galactosidase activity from a
co-transfected LacZ vector (pCH110) and expressed as fold activation to
the luciferase activity of pD400, which is defined as 1. Results are
means of three independent experiments. Error bars indicate
the range of standard errors. The empty expression vector
(pcDNA3, Invitrogen) was added to make an equal amount (2.5 µg)
of DNA for each transfection.
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To determine the binding activity of individual Ik proteins at the
putative Ik-binding site, EMSAs were performed with recombinant Ik-1
and Ik-2. Two DNA fragments, D400 and MIK2, corresponding to the
dor promoter sequences from
400 to +1 in constructs pD400 and pD400MIK2, respectively, were employed as probes (Fig.
2A). As shown in Fig.
2B, 50 ng of Ik-2 readily formed a complex with D400
(lane 4), which was completely abolished by molar
excess of unlabeled D400 (lane 6). In contrast, the cold
competitor MIK2 with a mutation in the
378/
374 Ik-binding site was
not able to reduce the Ik-2·D400 complex (lane 7).
In addition, anti-Ik Ab shifted the complex to a higher position
(lane 9). Together these results demonstrate that Ik-2 can
specifically bind to the
378/
374 Ik-binding site in the
dor promoter. Interestingly, it was observed that no
detectable DNA-protein complex was formed using 50 ng of Ik-1
(lane 2), and a higher concentration of Ik-1 (250 ng) formed
only a faint band with D400 (lane 3). Moreover, the
Ik-2·D400 complex apparently was reduced in the presence of 50 ng of
Ik-1, without the formation of any other detectable DNA-protein complex
(lane 5). As the Ik isoforms bind to DNA through
dimerization (16, 17), these results demonstrate that both Ik-1
homodimers and Ik-1/Ik-2 heterodimers have a very low affinity for the
378/
374 Ik-binding site, whereas Ik-2 homodimers can exert
efficient binding to the putative Ik-binding site. This is consistent
with the results in the functional assays that overexpression of Ik-2
but not Ik-1 significantly increased the dor promoter
activity via the
378/
374 Ik-binding site (Fig. 1). Collectively,
these results indicate that Ik-2 binds to the
378/
374 Ik-binding
site and trans-activate the dor promoter mainly in the form
of Ik-2 homodimers.

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Fig. 2.
DNA binding activities of Ik proteins at the
378/ 374 Ik-binding site in the dor promoter.
A, two DNA fragments were used in EMSAs. D400 contains the
dor promoter sequence from 400 to +1, encompassing the
378/ 374 Ik-binding site (underscored). MIK2 contains the
same sequence as D400 except for a point mutation (indicated in
boldface) in the putative Ik-binding site. B,
EMSAs were performed by using D400 as the probe in the presence of
recombinant Ik-1 and/or Ik-2 as indicated. Lane 1, control
reaction (as described under "Materials and Methods"); lane
2, 50 ng of recombinant Ik-1; lane 3, 250 ng of
recombinant Ik-1; lane 4, 50 ng of recombinant Ik-2;
lane 5, 50 ng of recombinant Ik-1 plus 50 ng of recombinant
Ik-2. Subsequently, 50 ng of recombinant Ik-2 was used in the presence
of different unlabeled competitors (lanes 6 and
7), control serum (lane 8), or anti-Ik Ab
(lane 9).
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A USF-binding E box Is Essential for dor Promoter
Activity--
We previously reported the identification of a
minimum mouse dor promoter (
262 to
141) in mouse
neuronal cell lines; this region contains a GC box (
226 to
221) and
a composite Ets-1-binding site/E box (
192 to
180) that contribute
to the constitutive dor promoter activity (11, 12). The
minimum dor promoter was found sufficient to confer the
constitutive promoter activity in resting T cells as well (13).
Mutation in the GC box or the Ets-1-binding site did not result in a
significant decrease in the dor promoter activity in either
resting or PHA-activated EL-4 T cells (data not shown). However,
mutation in the E box as present in pD400ME (Fig.
3A) almost completely
abolished the promoter activity of pD400 in both resting and
PHA-activated EL-4 cells (Fig. 3B), indicating that the E
box is required for the basal dor promoter activity in T
cells.

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Fig. 3.
Functional analysis of the E box in the
dor promoter. A, the luciferase
reporter construct pD400 contains the dor promoter sequence
from 400 to +1. The mutant construct pD400ME was made by replacing
the core binding motif of the E box in pD400 with a HindIII
site. The E box in pD400 is underscored, and the mutant
sequence in pD400ME is shown in boldface. B,
luciferase activities of 2.0 µg of pD400 or pD400ME in both
unstimulated and PHA-activated (1.5 µg/ml) EL-4 cells are expressed
as luciferase/ -galactosidase activity ratio. The
histograms represent mean values of four independent
transfection experiments with two different plasmid preparations.
Error bars indicate the range of standard errors.
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EMSAs were performed to determine the protein binding activity at the E
box. Oligonucleotides D198/169 and Dm185, synthesized as probes,
correspond to the dor promoter sequence from
198 to
169
in constructs pD400 and pD400ME, respectively (Fig.
4A). As shown in Fig.
4B, nuclear extracts from resting EL-4 cells formed a major
complex with D198/169 (lane 2). Molar excess of unlabeled
D198/169 abolished the complex formation (lane 3). In contrast, the cold competitor Dm185 with a mutation in the E box was
not able to reduce the complex formation (lane 4). In
addition, both anti-USF-1 Ab and anti-USF-2 Ab shifted the complex to a higher position (lanes 6 and 7). These results
demonstrate that USF family members specifically bind to the E box in
the dor promoter in EL-4 cells. Moreover, it was noted that
the USF-binding activity at the E box basically did not change in
PHA-activated EL-4 cells (lanes 8 and 9),
consistent with the results from Western blot analysis that the
expression of USF-1 or USF-2 is not changed in PHA-activated EL-4 cells
compared with that in the unstimulated cells (Fig. 4C).
Combined with the data from the mutational analysis (Fig. 3), these
results indicate that USF family members specifically binds to the E
box and confer basal dor promoter activity in either resting
or activated T cells. However, USF binding is not the rate-limiting
step for the dor promoter activity increment in activated T
cells. This is in agreement with our previous conclusion that the
increased Ik binding activity at the
378/
374 Ik-binding site
triggers the augmentation of dor promoter activity in
activated T cells (13).

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Fig. 4.
Protein binding activity at the E box in the
dor promoter. A, two oligonucleotides
were used in EMSAs. D198/169 contains the dor promoter
sequence from 198 to 169 including the E box. Dm185 contains the
same sequence as D198/169 except that the core binding motif of the E
box is replaced by a HindIII site. B, EMSAs were
performed by using D198/169 as the probe in the presence of nuclear
extracts from an equal amount of unstimulated (lanes 2-7)
or PHA-activated (lanes 8-9) EL-4 cells. Lane 1,
probe only; lane 2 and lane 8, normal reaction;
lanes 3 and 4, different unlabeled competitors as
indicated; lane 5, control serum; lanes 6 and
9, 2 µg of anti-USF-1 Ab; lane 7, 2 µg of
anti-USF-2 Ab. C, nuclear extracts from an equal amount of
unstimulated or PHA-activated EL-4 cells were analyzed by Western blot
using anti-USF-1 and anti-USF-2 Abs, respectively. The positions of
USF-1 and USF-2 are indicated.
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Ik-2 and USF Synergistically Trans-activate the dor
Promoter--
To determine whether Ik-2 could functionally
interact with USF, plasmid pD400, pD400MIK2 (Ik binding site mutated),
or pD400ME (E box mutated) was co-transfected into EL-4 cells with
expression vectors for mouse Ik-2 and/or human USF-1, which shows over
95% amino acid identity to murine USF-1. As shown in Fig.
5, overexpressed Ik-2 or USF-1 alone
elevated the promoter activity of pD400 slightly more than 2-fold or
1-fold, whereas the combined overexpression of Ik-2 and USF-1 resulted
in a more than 4-fold activation. The latter activation was abolished
either by mutation of the putative Ik-binding site (pD400MIK2) or the E
box (pD400ME). In addition, overexpressed Ik-2 could not rescue the
promoter activity that was abolished by the E box mutation, indicating
that Ik-2 needs to function through the E box-bound USF, which confers
the basal dor promoter activity. Similar results were
observed in co-transfection assays using Ik-2 and USF-2 (data not
shown). Taken together, these data demonstrate that Ik-2 synergizes
with USF in trans-activating the dor promoter via the
378/
374 Ik-binding site and the E box, respectively.

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Fig. 5.
Ik-2 and USF synergistically trans-activate
the dor promoter. EL-4 cells were transfected
with 2.0 µg of pD400, pD400MIK2, or pD400ME and 0.5 µg of the Ik-1
and/or USF-1 expression vector. Luciferase activities were normalized
to -galactosidase activity from a co-transfected LacZ vector
(pCH110) and expressed as fold activation to the luciferase activity of
pD400, which is arbitrarily defined as 1. Results are means of three
independent experiments. Error bars indicate the range of
standard errors. The empty expression vector (pcDNA3,
Invitrogen) was added to make an equal amount (2.5 µg) of DNA for
each transfection.
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The
378/
374 Ik-2-responsive Element Is Position- but
Not Orientation-dependent--
To further understand the
functional properties of the
378/
374 Ik-binding site, several
constructs were generated. Plasmid pD1300IK was created by inserting a
duplicate of the Ik-2-responsive element (
400 to
360) upstream of
1300 while silencing the original Ik-2-responsive element by mutating
the Ik-binding site at
378 to
374. pD400AS was generated by
inserting the Ik-2-responsive element upstream of
360 in the
antisense orientation (Fig.
6A). As shown in Fig.
6B, although overexpressed Ik-2 enhanced the promoter
activity of pD400, pD400AS and pD1300 by about 2-fold in EL-4 cells,
the promoter activity of pD1300IK was not affected. These results
demonstrate that the
378/
374 Ik-binding site functions in a
position-dependent, orientation-independent manner,
indicating that the Ik-2-responsive element acts as an upstream
promoter element rather than an enhancer for the dor
promoter (18, 19). Interestingly, similar results were observed when
the Ik-2 treatment was replaced by PHA activation in EL-4 cells (Fig.
6C), suggesting that the enhanced dor promoter
activity in PHA-activated EL-4 cells results from increased binding
activity of Ik-2 at the
378/
374 Ik-binding site.

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Fig. 6.
The 378/ 374 Ik-2-responsive element is
position- but not orientation-dependent. A,
luciferase reporter constructs pD1300 and pD400 contain the
dor promoter sequences from 1300 to +1 and from 400 to
+1, respectively. The mutant construct pD1300IK was made by inserting a
duplicate of the Ik-2-responsive element ( 400 to 360) upstream of
1300 while silencing the original Ik-2 responsive element by mutating
the Ik-binding site at 378 to 374. Mutant construct pD400AS was
generated by inserting the Ik-2-responsive element upstream of 360 in
the antisense orientation. B, EL-4 cells were transfected
with 2.0 µg of pD1300, pD400, pD1300IK, or pD400AS and 0.5 µg of
the Ik-2 expression vector. The empty expression vector (pcDNA3,
Invitrogen) was added to make an equal amount (2.5 µg) DNA for
each transfection. C, 2.0 µg of pD1300, pD400, pD1300IK,
or pD400AS was transfected into EL-4 cultures with or without PHA
activation (1.5 µg/ml). Luciferase activities were normalized to
-galactosidase activity from a co-transfected LacZ vector (pCH110)
and expressed as fold activation to the luciferase activity of pD400,
which is arbitrarily defined as 1. Results are means of three
independent experiments. Error bars indicate the range of
standard errors.
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DISCUSSION |
Previously, we reported that increased Ik binding activity at an
Ik-binding site (
378 to
374) in the mouse dor promoter is required for the enhanced dor promoter activity in
PHA-activated EL-4 cells, a mouse T cell line that constitutively
expresses DOR. In the present study, we further analyzed the mouse
dor promoter in EL-4 cells and have demonstrated that Ik-2
homodimers bind to the
378/
374 Ik-binding site and exerts a
position-dependent trans-activation effect on the
dor promoter. Moreover, an E box (
185 to
180) that binds
upstream stimulatory factor is essential for the dor
promoter activity in both resting and PHA-activated T cells.
Furthermore, we have demonstrated that Ik-2 and USF synergize in
trans-activating the dor promoter via the putative
Ik-binding site and the E box, respectively.
The Ikaros gene encodes a family of
hemopoiesis-specific zinc finger transcription factors by means
of alternative splicing. Three Ik isoforms, Ik-1, Ik-2, and Ik-3, are
capable of binding DNA. Ik-1 and Ik-2 are detected predominantly in the
nucleus, whereas Ik-3 is present mainly in the cytoplasm. Interactions between the three DNA-binding Ikaros isoforms generate six homo- and
heterodimeric complexes with distinct combinations of two DNA-binding
domains that can interact with a range of regulatory sequences with
different affinities (16, 17, 20). This is in agreement with our
observation that Ik-2 homodimers can bind efficiently to the
378/
374 Ik-binding site in the dor promoter, whereas
Ik-1 homodimers or Ik-1/Ik-2 heterodimers show very low affinity for
the same site (Fig. 2). In addition, although overexpressed Ik-1
exerted no effect on the dor promoter activity in EL-4
cells, the overexpression of Ik-2 elevated the dor promoter
activity almost to the level of that activated by PHA (Fig. 1). These
results, together with our previous observation that the expression of the nuclear Ik proteins was increased in PHA-activated EL-4 cells (13),
indicate that the increased Ik binding activity at the
378/
374
Ik-binding site in PHA-activated EL-4 cells is mainly due to the
augmented formation of Ik-2 homodimers, which readily bind to the
putative Ik-binding site and enhance the dor promoter activity.
On the other hand, a USF-binding E box (
185 to
180) is essential
for dor promoter activity. The E box mutant exhibited almost total loss of dor promoter activity in both resting and
PHA-activated EL-4 cells (Fig. 3), suggesting that the USF-bound E box
functions as the basal dor promoter. This proposition
is supported by several lines of evidence from other reports. First,
USF has been shown to be able to interact with TFIID (21, 22),
increases the rate or stability of TFIID binding (23), and stabilize
formation of the preinitiation complex (24). Second, USF is able to
interact with other basal transcription factors, including
TAFII55 (25), TFII-I (26, 27), and transcriptional co-factor PC5
(28). Third, the USF-bound E box has been found to function in a
manner similar to the initiator element (27) and to direct
transcription in a TATA- and initiator-less promoter (29). In addition,
multiple transcription initiation sites are present within the 40-base pair region immediately downstream of the USF-binding E box in the
TATA- and initiator-less dor promoter (30). Thus, we propose that the E box-bound USF can recruit TFIID and/or other components of
the basal transcription machinery to the dor promoter and
facilitate the assembly of the preinitiation complex.
USF consists of two ubiquitous polypeptides (31), USF-1 (43 kDa) and
USF-2 (44 kDa), both of which bind the E box (Fig. 4) and interact
functionally with Ik-2 in trans-activating the dor promoter
(Fig. 5). Interestingly, overexpressed Ik-2 could not rescue the
promoter activity that was abolished by the E box mutation, in
agreement with the notion that the USF-bound E box may functions as the
basal promoter. It was observed that USF expression as well as USF
binding activity at the E box basically did not change in PHA-activated
EL-4 cells compared with the unstimulated cells (Fig. 4). This
indicates that USF binding at the E box is not rate-limiting for the
dor promoter activity increment in PHA-activated T cells,
consistent with our conclusion that increased binding activity of Ik-2
homodimers triggers the augmentation of the dor promoter
activity in PHA-activated EL-4 cells.
The
378/
374 Ik-binding site functions in a
position-dependent manner (Fig. 6), indicating that the
Ik-2-responsive element does not act as an enhancer (18, 19) but an
upstream promoter element. Although the functional synergy between Ik-2
and USF in trans-activating the dor promoter was obvious in
EL-4 cells (Fig. 5) and some other mouse T cell lines (data not shown),
no direct interaction between Ik-2 and USF was detected in this study (data not shown). Thus, Ik-2 probably exerts its trans-activation effect on the dor promoter by promoting the assembly of the
basal transcription machinery initiated via the E box-bound USF through interaction with components of the preinitiation complex. Collectively, our data support such a model; the USF-bound E box confers the constitutive dor promoter activity in resting T cells,
wherein the binding activity at the
378/
374 Ik-binding site is weak (13) because of inadequate Ik-2 homodimers. However, in activated T
cells, the augmented expression of nuclear Ik proteins results in
increased formation of Ik-2 homodimers, which in turn leads to the
increased binding activity at the putative Ik-binding site and enhances
the dor promoter activity via functional synergy with the E
box-bound USF.
T lymphocytes are exposed to endogenous opioid peptides in
vivo (32, 33). Because Ik has been reported to set
threshold for T-cell activation (34) and to play an important role in T-cell homeostasis (35), the link between Ik-2 and the transcriptional regulation of the dor gene in T cells implies an active role
for endogenous opioids in modulating the functions and homeostasis of T
cells in different physiological settings. In addition, as Ik proteins
are specific to the hemopoietic system, particularly T cells (16, 17,
36), this study also provides insight into the tissue-specific
transcriptional regulation of the dor gene.