Molecular Mechanisms of Promoter Regulation of the gp34 Gene That
Is Trans-activated by an Oncoprotein Tax of Human T Cell Leukemia Virus
Type I*
Kiyoshi
Ohtani
,
Atsumi
Tsujimoto
,
Tomonori
Tsukahara§,
Noboru
Numata¶,
Shigeto
Miura
**,
Kazuo
Sugamura
, and
Masataka
Nakamura

From the
Human Gene Sciences Center and the
§ Department of Immunotherapeutics, Medical Research
Division, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8510, the ¶ Department of Microbiology,
Sendai Municipal Institute of Public Health, 2-5-10 Oroshimachi-higashi, Wakabayashi-ku, Sendai 983-0002, the
Department of Microbiology and Immunology, Tohoku University
School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, and
** Core Research for Evolutional Science and Technology, Japan Science
and Technology Corp., 4-1-8 Honcho, Kawaguchi 332-0012, Japan
 |
ABSTRACT |
We investigated the molecular mechanism of
transcriptional activation of the gp34 gene by the Tax oncoprotein of
human T cell leukemia virus type I (HTLV-I). gp34 is a type II
transmembrane molecule belonging to the tumor necrosis factor family
and is constitutively expressed on HTLV-I-producing cells but not
normal resting T cells. The transcriptional regulatory region of the gp34 gene was activated by HTLV-I Tax in the human T cell line Jurkat,
in which endogenous gp34 is induced by Tax. Sequence analysis demonstrated that two NF-
B-like elements (1 and 2) were present in
the regulatory region. Both NF-
B-like elements were able to bind to
NF-
B or its related factor(s) in a Tax-dependent manner. Chloramphenicol acetyltransferase assays indicated that NF-
B-like element 1 was Tax-responsive, although the activity was lower than that
the native promoter. NF-
B-like element 2 elevated promoter activity
when combined with NF-
B-like element 1, indicating cooperative function of the elements for maximum promoter function. Unlike typical
NF-
B elements, the NF-
B-like elements in gp34 were
not activated by treatment of Jurkat cells with phorbol ester despite induction of the NF-
B-like binding activity. Chloramphenicol acetyltransferase reporter assays using the region upstream of the
NF-
B-like elements identified an upstream region that reduced transcription from cognate and noncognate core promoters in a Tax-independent manner. Our results imply complex regulation of expression of the gp34 gene and suggest implication of gp34 in proliferation of HTLV-I infected T cells.
 |
INTRODUCTION |
Human T cell leukemia virus type I
(HTLV-I)1 is an etiologic agent
of adult T cell leukemia (1-3) and HTLV-I-associated
myelopathy/tropical spastic paraparesis (4, 5). At least two regulatory
molecules, Tax and Rex, are encoded by the HTLV-I genome (6). Tax was initially found to be a trans-acting transcriptional activator of viral
gene expression (7-9) and was subsequently shown to induce or enhance
the expression of a myriad of cellular genes, most of which are
implicated in cell growth. These include genes for growth factors
(interleukin 2 (IL-2) and granulocyte/macrophage colony-stimulating
factor) (10-14), growth factor receptors (IL-2 receptor
and
subunits) (15-17), a cytoplasmic signal mediator (lyn) (18), and
nuclear transcription factors (c-fos and c-jun) (19-21). Modulation of
expression of these genes is thought to be closely associated with
disease. Nevertheless, the mechanisms of Tax-induced cellular gene
expression and involvement in disease onset are yet to be elucidated.
Analyses of target DNA sequences showed that three enhancer elements
that bound to cyclic AMP-responsive element binding factor (CREB/ATF)
(22-25), NF-
B (26-28), and serum-responsive factor (29) were
activated by Tax. Tax itself cannot bind directly to target DNA
sequences, although it activates via direct association with the
cellular transcription factors, cyclic AMP-responsive element binding
factor, NF-
B and serum-responsive factor (29-32), and an inhibitor
of NF-
B, I
B (33, 34).
We cloned a cellular gene encoding a type II membrane glycoprotein,
named gp34, which was expressed on HTLV-I-producing cells (35, 36).
gp34 has been also shown to bind OX40, a marker of activated T cells,
which is also expressed on HTLV-I-producing cells (37-39). Apart from
HTLV-I-producing T cells, gp34 and OX40 are also expressed on activated
normal T and B cells (38-40). Transcriptional transactivation of the
gp34 and OX40 genes by Tax has been demonstrated (36, 41). OX40 is a
member of the tumor necrosis factor (TNF) receptor family, which
includes FAS and CD40 (42, 43), and gp34 belongs to the TNF family (39,
44). Functions of the TNF receptor/TNF and FAS/FAS ligand systems are
well documented with regard to cell proliferation and apoptosis (43).
Defects of the CD40 ligand are associated with hyper-IgM syndrome
(45-47). Interestingly, unlike those molecules, gp34 and OX40 are not
detected on the cell surface until lymphocytes are activated,
suggesting that they function at later stages of lymphocyte activation
and proliferation. However, little is known about the function of the
gp34/OX40 system. Recent studies indicate that both gp34 and OX40
transmit signals into the cytoplasm (38-40, 48). Unlike other cellular
genes transactivated by HTLV-I Tax, gp34 is not induced on T cells by
stimulation with mitogens, phorbol ester, or IL-2 (35). These
observations imply that the gp34/OX40 system may be involved in growth
regulation of T cells during immune responses and in growth of
HTLV-I-infected T cells. Thus, elucidation of the molecular mechanisms
of gp34 and OX40 expression would be useful in
understanding normal and cancerous proliferation of T cells.
In this study, we analyzed the mechanism of Tax-induced transcriptional
regulation of gp34. Tax-induced gp34
transactivation was mediated through a unique NF-
B-like element that
was not activated by phorbol ester. We detected a region upstream of
the NF-
B-like element that suppressed transcription from cognate and
noncognate core promoters. The array of these regions may contribute to
the strict and complex regulation of gp34 expression in T
cells.
 |
EXPERIMENTAL PROCEDURES |
Cells--
TL-Mor (49) and MT-2 (50) are HTLV-I-producing human
T cell lines. Jurkat (51) is a human acute lymphocytic leukemia T cell
line negative for HTLV-I. JPX-9 is a Jurkat subline that stably carries
Tax expression plasmid, in which expression of Tax is inducible by the
addition of CdCl2 (20, 52). Cells were cultured in RPMI
1640 medium supplemented with 10% fetal calf serum, 10 mM
L-glutamine, and antibiotics at 5% CO2 in
air.
DNA Cloning and Sequencing--
A human genomic DNA library was
constructed by introduction of Sau3AI partially digested
genomic DNA from normal peripheral blood lymphocytes into the
BamHI site of phage vector EMBL3. The library (5 × 105 plaques) was screened by plaque hybridization using the
5'-most EcoRI fragment of gp34 cDNA as a
probe. Nucleotide sequencing was performed by the dideoxy chain
termination method using Sequenase 2.0 (United States Biochemicals)
according to the protocol recommended by the manufacturer.
S1 Nuclease Mapping and Primer Extension--
Total RNA was
isolated from MT-2 and Jurkat cells by the guanidium thiocyanate method
as described previously (53). The probe used for S1 mapping was the
499-base pair (bp) AluI genomic DNA fragment. The
end-labeled AluI fragment was annealed with 20 µg of RNA
in 20 µl of solution containing 80% formamide, 20 mM
PIPES (pH 6.4), 0.5 mM EDTA, and 200 mM NaCl at
52 °C for 3 h after initial incubation at 85 °C for 5 min.
The annealed products were digested with S1 nuclease (100 units) in 100 µl of solution containing 270 mM NaCl, 1 mM
ZnSO4, 30 mM sodium acetate (pH 4.6) at
37 °C for 30 min. The reaction was terminated by phenol/chloroform extraction. DNA was ethanol-precipitated and analyzed by
electrophoresis in an 8% acrylamide sequencing gel with 7 M urea. The primer used for primer extension was a 30-mer
oligonucleotide (CTTGTTCCTCTCGAATCTTGGCCTGGCTGC) corresponding to the
5'-end of the minus strand of the S1 mapping probe. End-labeled primer
was annealed to 20 µg of RNA in 30 µl of solution containing 80%
formamide, 100 mM Tris-HCl (pH 8.0), 120 mM
KCl, and 20 mM MgCl2 at 30 °C for 8 h
after initial incubation at 85 °C for 5 min. The annealed product
was ethanol-precipitated and resuspended in 20 µl of solution
containing 50 mM Tris-HCl (pH 7.6), 60 mM KCl,
10 mM MgCl2, 1 mM dNTPs, 1 mM dithiothreitol, and 1 unit/µl human placental
ribonuclease inhibitor. The extension reaction was initiated by adding
50 units of reverse transcriptase and incubated at 37 °C for 2 h. The products were analyzed by electrophoresis along with the S1
mapping product. Size markers were sequencing ladders of the
gp34 genomic DNA fragment using a 17-mer primer
corresponding to the 5'-end of the 30-mer oligonucleotide used for
primer extension analysis.
Plasmids--
The 9-kbp fragment (Sau3AI (
9000)
-AvaII (+27)) upstream of the first exon of the gp34 gene
was inserted in front of the chloramphenicol acetyltransferase (CAT)
gene in a reporter plasmid, pSV0CAT (54), to generate pGP(
9000)CAT.
Deletion mutants of both 5'- and 3'-ends of the genomic fragment were
generated by digestion from restriction sites with exonuclease III and
mung bean nuclease. The 5' deletion mutants were derived from
pGP(
9000)CAT. The 3' deletion mutants were prepared by insertion of a
series of 3' deletion mutants of the
823 to
54 fragment into the
31 CAT reporter plasmid, pGP(
31)CAT, which contained the 58-bp
(
31 to +27) genomic fragment. Plasmids with mutant fragments were named according to the numbers of the 5'- or 3'-end of the fragments. pGP(
31)CAT was also used as a backbone vector to be introduced with
the
100 to
54 fragment, NF-
B-like elements 1 and 2, and the
HTLV-I enhancer fragment. The
100 to
54 fragment was introduced into the pGP(
31)CAT plasmid in single form (pGP(
100~
54) CAT) and four tandem repeat form (pGP(
100~
54)x4CAT). Similarly,
NF-
B-like elements 1 and 2 were inserted into the pGP(
31)CAT
plasmid in single and four tandem repeat forms, yielding pGP
B1CAT,
pGP
B2CAT, pGP
B1×4CAT, and pGP
B2×4 CAT, respectively.
pHE(
31)CAT is a derivative of the pGP(
31)CAT plasmid carrying the
267-bp HTLV-I enhancer fragment (55). pdHE4 is a CAT plasmid
containing the HTLV-I core promoter (55). pdHE
B-4 carries four
tandemly repeated SV40 NF-
B sites in pdHE4(56).
pdHE(
823~
54)CAT is a derivative of the pdHE4 having the
823 to
54 fragment of the gp34 gene promoter. pMAXneo is a Tax expression
vector in which the Tax gene is regulated by the mouse metallothionein
promoter, whereas pMAXneo/M is a nonfunctional Tax mutant (55).
Reexamination of the DNA sequence for the Tax mutant demonstrated that
the mutant gene has a 3-base insertion at the MluI site in
the coding region, rather than a 4-base insertion as reported initially
(55), resulting in a mutant with an additional Arg residue between
amino acids 62 and 63. Tax mutants TaxM22, Tax703, and Taxd3, which are
not effective in activation of the NF-
B, serum-responsive factor, and CREB/ATF binding sites, respectively, have been described previously (32, 57, 58). Wild type Tax and these Tax mutant genes were
cloned into pH
APr-1-neo, which has a
-actin promoter (59).
CAT Assay--
Plasmid DNA was transfected into Jurkat cells by
the DEAE-dextran method and assayed for CAT activity as described
previously (55). CAT activities were shown as percent acetylation, and transactivation was expressed as fold induction of CAT activity with
pMAXneo compared with that with pMAXneo/M. In CAT assays with
12-O-tetradecanoylphorbol-13-acetate (TPA), TPA was added to
cells transfected with CAT plasmids at a concentration of 20 nM, 12 h after transfection, and incubation was
continued for a further 36 h. We also compared CAT activity with
TPA to that without TPA. All CAT assays were carried out at least three
times in duplicate. Repeated assays showed similar results, and
representative data are presented.
Gel Shift Assay--
The gel shift assay was performed as
described previously (56). DNA probes were end-labeled with
polynucleotide kinase and [
-32P]ATP (5000 Ci/mmol;
Amersham). Nuclear extracts were prepared from TL-Mor, Jurkat, and
JPX-9 cells as described previously (56).
Oligonucleotides used as probes and competitors were as follows:
bases
100~
54.
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NF-
B-like element 1.
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|
NF-
B-like element 1 mutant.
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|
NF-
B-like element 2.
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|
NF-
B-like element 2 mutant.
|
|
Typical NF-
B.
|
|
HTLV-I C26.
|
|
HTLV-I 21-bp motif.
|
|
Antibodies used for gel mobility supershift assay were
anti-NF-
B p65 (sc-372X), anti-NF-
B p50 (sc-114X), anti-c-Rel
(sc-1827X), anti-RelB (sc-226X), and anti-NF-
B p52 (sc-298X)
antibodies (Santa Cruz Biotechnology).
 |
RESULTS |
Cloning of the gp34 Regulatory Region--
In order to analyze the
regulatory mechanism of gp34 gene transcription, we cloned the promoter
region of the gp34 gene. A human genomic DNA library constructed with
EMBL3 was screened with the 5'-most EcoRI fragment of the
gp34 cDNA. Several positive clones were identified, and
one clone, which contained sequences 9 kbp upstream of the ATG
initiation codon for gp34, was further analyzed. The promoter activity
of this region was examined by CAT assays. The 9-kbp 5'-flanking
fragment was isolated and ligated to the CAT reporter gene. CAT
activity was examined with or without a Tax expression vector, pMAXneo,
in the human Jurkat T cell line. As expected, in Jurkat cells, the
9-kbp fragment showed little or no promoter activity in the absence of
Tax (Fig. 1A). Tax expression induced promoter activity of this fragment. Thus, the promoter of the
gp34 gene was present in this region, and its activity was dependent on
Tax. Experiments using 5' deletion mutants of the 9-kbp fragment showed
that most, if not all, of the promoter activity was associated with the
850-bp KpnI-AvaII fragment in the 9-kbp
5'-flanking region. The nucleotide sequence of the 850-bp fragment was
then determined (Fig. 1B).

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Fig. 1.
The regulatory region of the gp34 gene.
A, Tax-dependent promoter activity of the gp34
gene. Jurkat cells were transfected with CAT reporter plasmids carrying
the 9-kbp Sau3AI-AvaII, 2.7-kbp
AvaII-AvaII, and 850-bp
KpnI-AvaII fragments with and without pMAXneo and
examined for CAT activity after 36 h of culture in the presence of
10 µM CdCl2. Restriction sites in the
5'-flanking region, first exon, and translation initiation codon are
indicated. B, nucleotide sequence of the 5'-flanking region.
The nucleotide sequence from the KpnI site to the initiation
codon (underlined) is shown. The transcription initiation
site determined from S1 nuclease mapping and primer extension is
numbered +1. NF- B-like elements 1 and 2 are
boxed. The AP-1 site-like sequence is double
underlined. The TATA-like sequence is indicated by a dashed
underline. C, transcriptional start site. RNA from
Jurkat (lanes 1 and 3) and MT-2 (lanes
2 and 4) cells was subjected to S1 nuclease mapping
(lanes 1 and 2) and primer extension (lanes
3 and 4). Size markers are sequencing ladders of the
5'-flanking region using the same primer as for primer extension.
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To confirm that the transcriptional start site was contained in this
fragment, S1 mapping and primer extension analyses were performed.
Total RNA was isolated from an HTLV-I infected T cell line (TL-Mor) and
an HTLV-I-unrelated T cell line (Jurkat). RNA was hybridized with an S1
mapping probe of the 497-bp AluI genomic fragment. A
224-base fragment protected from nuclease digestion was detected using
RNA from TL-Mor but not Jurkat cells (Fig. 1C). A product of
the same size was observed using TL-Mor but not Jurkat RNA by primer
extension analysis using a 30-mer primer with the same 5'-end as the
minus strand of the S1 probe. These results showed that the
transcriptional start site was located 154 bp upstream of the ATG
initiation codon and that most of the promoter activity resided in the
region extending from
824 to +27.
Sequence analysis showed that there were two NF-
B site-like
sequences (
87 to
77 and
64 to
54) and one AP-1 site-like sequence (
74 to
68) between the two NF-
B-like sequences (Fig. 1B). Although a typical TATA box sequence was not found
around 30 bp upstream of the transcriptional start site, it is possible that the TTAAA sequence located at
29 might be TATA-related.
Promoter Activities of Deletion Mutants--
In order to examine
the function of each subregion in the regulatory region, a series of
deletion mutants of the regulatory region was generated, and the
mutants were assayed for their promoter activities with or without
Tax.
A 5'-end deletion (up to
31) of the
824 to +27 fragment completely
abolished Tax responsiveness (Fig. 2). This
31 mutant retained the TATA-related sequence TTAAA and was thought to
contain the core promoter as evidenced by the fact that addition of the HTLV-I enhancer to the
31 mutant restored Tax responsiveness. Thus,
the core promoter of the gp34 gene seemed to be Tax-independent, and
the upstream region appeared to be responsible for
Tax-dependent activation. As expected, the
31 mutant
linked to the
823 to
54 fragment exhibited the same CAT activity in
response to Tax as did the
823 mutant. Combination of the
823 to
54 fragment with the HTLV-I core promoter also showed
Tax-dependent activation. These results indicate that the
region between
823 and
54 of the gp34 gene mediates transactivation
by Tax.

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Fig. 2.
Analysis of deletion mutants of the gp34 gene
regulatory region. Progressive 5' or 3' deletion mutants of the
gp34 promoter are schematically illustrated. Fragments of
the upstream region of gp34 gene are shown as solid boxes.
The HTLV-I enhancer and HTLV-I core promoter are indicated by and
, respectively. These reporter plasmids were co-transfected into
Jurkat cells with or without the Tax expression vector pMAXneo and
assayed for CAT activity.
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Note that the
31 mutant showed higher CAT activity than the
823
mutant in the absence of Tax. Similarly, the
823 to
54 fragment,
when linked to the HTLV-I core promoter, significantly reduced CAT
activity in the absence of Tax, although it endowed Tax-dependent activation. These results indicate that in
addition to a region enhancing transcription in response to Tax, there is a region in the
823 to
54 fragment that suppresses basal transcription in the absence of Tax.
The Tax-responsive region was further localized by CAT assays using 5'
and 3' deletion mutants of the 5'-flanking fragment. A mutant with a
106 deletion at the 5'-end retained Tax responsiveness although it
showed an increased inherent CAT activity without Tax. This result
suggests that an element responsible for Tax-dependent activation is retained in a region downstream of
106 and that the
suppressive activity is associated with a region upstream of
106.
To determine the 3'-border of the Tax-responsive region, a set of 3'
deletion mutants of the
823 to
54 fragment were linked to the
31
mutant and assayed for their Tax-dependent activation. Addition of the
823 to
54 fragment to the
31 core CAT vector gave
as high an activation (more than 30-fold induction) in response to Tax
as did the native
823 to +27 fragment (Fig. 2). A 3' mutant deleted
to
57 exhibited a profoundly reduced response to Tax with induction
of only 7.7-fold. Further deletion to
118 completely abolished
Tax-dependent activation. Collectively, these results indicate that a Tax-responsive element is present in the region between
106 and
54.
Interestingly, all 3' deletion mutants exhibited reduced CAT activity
as compared with the
31 mutant in the absence of Tax (Fig. 2). This
implies that suppressive activity is associated with a region upstream
of
118, consistent with observations from the 5' deletion
mutants.
Tax-responsive Element--
To identify an element responsible for
Tax-dependent activation, an isolated sequence from
106
to
54 was assayed for its ability to mediate
Tax-dependent activation. The nucleotide sequence spanning
bases
100 to
54 was synthesized and linked to the
31 mutant as
either a single copy or four tandem repeats in the sense orientation,
yielding pGP(
100~
54)CAT and pGP(
100~
54)x4CAT, respectively.
Reporter plasmids were introduced into Jurkat cells and examined for
CAT activity in the absence or presence of Tax. Introduction of one
copy of the synthetic sequence reproducibly induced slight but
significant activation in response to Tax with a 4-fold activation
(Fig. 3A). Four tandem repeats
mediated profound (up to 90-fold) Tax-dependent activation.
Introduction of three tandem repeats in the antisense orientation
induced 6-fold activation, demonstrating that the sequence from
100
to
54 contains an element(s) that functions as an enhancer and is
transactivated by Tax.

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Fig. 3.
Involvement of NF- B pathway in
Tax-mediated activation of the gp34 promoter. A,
Tax-responsive region in the gp34 gene regulatory region. CAT reporter
plasmids were transfected into Jurkat cells together with or without
pMAXneo. Cells were then cultured in the presence of CdCl2
for 36 h and CAT activity in the cell lysates was determined. The
reporter plasmids carried the isolated 100 to 54 fragment and
synthesized NF- B-like elements 1 and 2 in single and tandem repeat
forms in a CAT reporter plasmid pGP( 31)CAT with the gp34
core promoter. Mutant fragments of NF- B-like elements 1 and 2, indicated by dotted arrows, were similarly tested. CAT
activities in cells with and without pMAXneo were compared.
B, responsiveness of the gp34 promoter to Tax
mutants. pGP( 823)CAT was cotransfected along with vectors for wild
type Tax (pMT2Tax) (WT) or Tax mutants TaxM22, Tax703 and
Taxd3 into Jurkat cells, and CAT activity in the cell lysates was
determined. pH APr-1-neo was used as a control.
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Sequence analysis revealed that there were two NF-
B binding
site-related sequences in the region. These sequences were therefore possible candidates for the Tax-responsive element. We thus examined the NF-
B-like sequences for their ability to be activated by Tax.
The wild type oligonucleotides (NF-
B-like elements 1 and 2; see Fig.
1B and Table I) and their
mutants (NF-
B-like element 1 and 2 mutants) were introduced into the
31 core CAT plasmid in the sense orientation in single copy and four
tandem repeat form. Introduction of one copy of NF-
B-like elements 1 and 2 induced little or no activation in response to Tax. The tandem repeat form of NF-
B-like element 1 induced 14-fold activation. Mutation of NF-
B-like element 1 abolished Tax-dependent
activation. Unexpectedly, four tandem copies of NF-
B-like element 2 exhibited CAT activity as low as its corresponding mutant in the
presence of Tax. These results showed that NF-
B-like element 1 is,
at least in part, a Tax-responsive element of the gp34 gene promoter. However, CAT activity induced by NF-
B-like element 1 in the presence of Tax was less than that induced by the original
100 to
54 fragment, suggesting that NF-
B-like element 1 is not sufficient for
full Tax-mediated activation and that another sequence or proper
organization of elements in the region may be required for full
activity (see below and under "Discussion").
To test this possibility, we introduced a mutation into either element
1 or element 2 in the
100 to
54 fragment. Neither CAT reporter
constructs with element 1 mutant or element 2 mutant were activated by
Tax (Fig. 3A). The results indicate that element 2 is
critically involved in Tax-induced full activation, consistent with the
results with the
823 to
69 fragment (Fig. 2).
In addition, Tax mutants differing in properties in activation of
target elements, NF-
B, CREB/ATF, and serum-responsive factor binding
sites, were examined for their ability to activate the gp34
promoter. The CAT reporter plasmid (
823) was transfected into Jurkat
cells along with Tax mutant expression vectors TaxM22 (M22), Tax703
(703), and Taxd3 (d3), which are not effective in activation of the
NF-
B, serum-responsive factor, and CREB/ATF binding sites,
respectively. Tax mutants 703 and d3, but not M22, were able to
activate the gp34 promoter (Fig. 3B), as
expected, suggesting the implication of the NF-
B activation pathway
in gp34 gene expression.
Cellular Factor(s) Bound to the Tax-responsive Element--
To
investigate the cellular factor(s) that bind to the Tax-responsive
element and to examine effects of Tax on such factor(s), we performed
gel mobility shift assays using nuclear extracts from HTLV-I-expressing
TL-Mor cells and HTLV-I-unrelated Jurkat cells. When the
100 to
54
fragment was used as a probe, a clear shifted band was observed with
TL-Mor nuclear extract but not Jurkat nuclear extract (Fig.
4A). The shifted complex was
specific to the fragment because complex formation was competed away by addition of excess cold probe but not by unrelated competitors such as
the 21-bp motif sequence in the HTLV-I enhancer. As expected, addition
of either a typical NF-
B consensus sequence or NF-
B-like element
1 abolished complex formation, suggesting that the factor(s) that
interacts with the
100 to
54 fragment is an NF-
B-related molecule(s). Surprisingly, this complex formation was also competed away by NF-
B-like element 2, which was not Tax-responsive by itself
in CAT assays (Fig. 4A).

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Fig. 4.
Tax-dependent binding of
NF- B-like factor(s) to the Tax-responsive elements. A and
B, complex formation between the Tax-responsive element and
NF- B-like factor(s). Nuclear extracts of TL-Mor (lanes
2-10) and Jurkat (lanes 11 and 12) were
mixed with the 100 to 54 fragment (A), NF- B-like
element 1, or NF- B-like element 2 (B). Lane 1,
no nuclear extract. The protein-DNA fragment complexes were separated
on a 4% nondenaturing polyacrylamide gel. Competitors used were the
100 to 54 fragment, NF- B-like element 1, NF- B-like element 1 mutant, NF- B-like element 2, and NF- B-like element 2 mutant. The
C26 element and 21-bp motif of the long terminal repeat of HTLV-I were
also used as competitors. Competitor fragments were added at a 100-fold
molar excess. Specific bands are indicated by arrows.
C, Tax-dependent binding activity to the
NF- B-like elements. Nuclear extracts of JPX-9 cells treated with or
without CdCl2 (20 nM CdCl2 for
24 h) were mixed with NF- B-like element 1 or 2. The same
element was used as a competitor for each probe. The band with the same
mobility as that with TL-Mor extract is indicated by an
arrow. D, the same mobility of complexes with
NF- B-like elements as with typical NF- B site. TL-Mor extract was
mixed either with NF- B-like element 1 or 2 or with a typical NF- B
binding site. The same element was used as a competitor for each probe.
The band with the same mobility is indicated by an arrow.
E, gel mobility supershift assay with antibodies against
NF- B family members. Gel shift assay reaction mixtures containing
TL-Mor extract and indicated antibodies were incubated for 3 h, and
then a probe, NF- B-like element 1 or 2, or a typical NF- B binding
site was added. Supershifted bands with anti-NF- B p65, anti-NF- B
p50, and anti-RelB antibodies are indicated by arrows with
a, b, and c, respectively.
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Gel shift assays were similarly performed using NF-
B-like elements 1 and 2 as probes. Combination of NF-
B-like element 1 and TL-Mor
nuclear extract, but not Jurkat nuclear extract, resulted in complex
formation (Fig. 4B). This binding was competed away by the
typical NF-
B sequence and also by NF-
B-like element 2. NF-
B-like element 2 essentially showed the same pattern of complex formation as NF-
B-like element 1 (Fig. 4B). Mutant
sequences of NF-
B-like elements 1 and 2 were not effective in
complex formation even with TL-Mor nuclear extract (data not shown).
That binding activity was observed only with nuclear extracts of TL-Mor
indicates that the binding activity was Tax-induced. This notion was
confirmed by observations with the Tax-inducible T cell line, JPX-9, in which induction of Tax expression resulted in induction of complex formation with NF-
B-like elements 1 and 2 (Fig. 4C).
Thus, Tax induces gp34 gene expression, at least in part, through
binding of NF-
B-like factor(s) to NF-
B-like elements in the
enhancer.
Bands representing the complex between elements 1 and 2 and
NF-
B-like factors showed very close migration by electrophoresis to
that with the typical NF-
B sequence (Fig. 4D).
Furthermore, to identify factors bound to gp34 gene NF-
B-like
elements, we performed gel shift assay using antibodies specific for
members of the NF-
B family. Supershifts were seen with anti-p65,
anti-p50, and anti-RelB antibodies in both elements 1 and 2, illustrating that the complex with elements 1 and 2 contains p50 and
p65 subunits of NF-
B and RelB, as shown with the consensus NF-
B
sequence (Fig. 4E).
Because a typical NF-
B binding site is activated by TPA in Jurkat
cells, we examined whether NF-
B-like element 1 in the gp34 promoter region was affected by TPA. Jurkat cells were
transfected with CAT reporter plasmids and, 12 h later, treated
with 20 nM TPA. The four tandem repeat form of NF-
B-like
element 1 did not respond to TPA (Fig.
5A), in contrast to responsiveness
of the same fragment to Tax, although treatment of Jurkat cells with TPA induced complex formation with elements 1 and 2 in gel shift assay
similar to TL-Mor (Fig. 5B). Consistent with this, the
native gp34 promoter fragment (
823 to +27) and the
100
to
54 fragment with the
31 core promoter linked to the CAT plasmid
did not show appreciable elevation of CAT activity in response to
TPA.

View larger version (25K):
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|
Fig. 5.
Effect of TPA on the gp34 gene regulatory
region. A, no activation of gp34 promoter by TPA.
CAT reporter plasmids were transfected into Jurkat cells. Cells were
cultured with 20 nM TPA for 36 h, and CAT activities
were compared between TPA-treated and untreated cells. B,
TPA-induced complex formation of Jurkat extract with NF- B-like
elements. NF- B-like element 1 or 2 was mixed with extracts from
TL-Mor and Jurkat cells treated with 20 nM TPA for 7 h. Specific binding with the same mobility as TL-Mor extract is
indicated by an arrow.
|
|
Upstream Suppressive Region--
Several lines of evidence as
shown above suggest that there is a negatively acting sequence upstream
of the NF-
B-like elements. To examine this notion, we tested whether
the isolated upstream sequence demonstrated suppressive effects. The
upstream fragment spanning bases
823 to
155 was introduced into the
113 and
105 deletion mutants (pGP(
113)CAT and pGP(
105)CAT),
which retained the NF-
B-like elements. The
113 mutant exhibited
CAT activities of 7.8 and 3.6% conversions with and without Tax,
respectively, showing 2.2-fold activation. Introduction of the upstream
fragment reduced CAT activities to 2.4 and 0.26% in the presence and
absence of Tax, respectively, resulting in 8.3-fold activation (Fig.
6). Similar results were observed with the
105 mutant construct, which showed an increase in fold activation
from 2.7 to 5.6 by introduction of the upstream sequence. The
31
mutant, carrying only the core promoter, showed basal promoter activity
irrespective of Tax; however, addition of the upstream sequence
significantly reduced the promoter activity. These results illustrated
that the upstream sequence, which lacks Tax responsiveness can suppress basal promoter activity.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Suppressive effects of the upstream region of
the gp34 gene regulatory region. The upstream fragment ( 823 to
154) was inserted into pGP( 113)CAT, pGP( 105)CAT, and
pGP( 31)CAT. These plasmids were transfected into Jurkat cells with
and without pMAXneo. Cells were cultured with CdCl2 for
36 h, and CAT activity was measured.
|
|
Thus, the upstream fragment reduced CAT activity to a greater extent in
the absence of Tax than in its presence, generating a higher activation
in response to Tax. Alternatively, Tax may be able to relieve the
suppressive effect of the upstream fragment.
 |
DISCUSSION |
Initial studies of gp34 demonstrated that its expression was
restricted to cells expressing HTLV-I. It was therefore speculated that
gp34 was a product of HTLV-I, because treatment with phytohemagglutinin (PHA), which is thought to mimic antigen-dependent
stimulation, was not effective in inducing gp34 expression in normal
human T cells and T cell lines (35). However, we previously
demonstrated that gp34 is encoded by a cellular gene that is a target
of the transcriptional transactivation function of HTLV-I Tax (36). Tax
activates several cellular genes, most of which are involved in
cellular signals for growth. gp34 differs from other
cellular genes transactivated by Tax in that other Tax-responsive
genes, such as the gene for IL-2 receptor
chain, the gene for IL-6, c-fos, and c-jun, are induced by mitogens,
phorbol ester, ionophores, and IL-2, whereas gp34 is not.
gp34 is expressed on normal T and B cells activated by antigen
stimulation and physically associates with OX40 (38-40 and unpublished
data). OX40 has also been shown to be transcriptionally activated by
Tax (41). Recently, gp34 and OX40 have been shown to transduce
intracytoplasmic signals upon association (38-40), implying that the
gp34/OX40 system may be involved in the growth of normal and malignant
T cells.
In this report, we examined the gp34 gene promoter region that is
transactivated by Tax. Our data provide important features of the
transcriptional regulatory mechanism of the gp34 gene. First,
transactivation of the gp34 gene by Tax is mediated through a 46-bp
sequence carrying two NF-
B-like elements. An increase in binding of
the NF-
B family members to the elements could be a mechanism for
transactivation. Second, the upstream region can suppress
promoter-driven basic transcription.
Two distinct NF-
B-like sequences exist in the Tax-responsive
region of gp34. Both sequences bound the NF-
B family
members in Tax-expressing cell extracts in vitro; however,
two elements seemed to be different in binding affinity of the NF-
B
family members: element 1 showed higher affinity than element 2. This may be one reason that NF-
B-like element 1 alone, when repeated, is
sufficient for conferring Tax responsiveness, whereas no Tax-induced activation was seen with NF-
B-like element 2, even with the tandemly repeated form. It is thus obvious that there is a functional difference between NF-
B-like elements 1 and 2. NF-
B-like element 1 behaves much like typical NF-
B sites in the SV40 early promoter and human immunodeficiency virus long terminal repeat (Table I), both of which
are transactivated by Tax via increased binding of NF-
B (27, 56).
NF-
B-like element 1 differs in sequence by three bases from the SV40
NF-
B site. NF-
B-like element 2 also has three base substitutions
from the SV40 NF-
B site, which are also distinct from those in
NF-
B-like element 1.
The NF-
B-like element 1-mediated response to Tax in a tandemly
repeated form was much lower than the native
100 to
54 fragment, which carries one copy of NF-
B-like elements 1 and 2. The three base
substitutions in NF-
B element 1 may reduce binding affinity to the
NF-
B family members (see Fig. 4D), resulting in a weaker response to Tax than the typical NF-
B site. This result suggests a
possible participation of another element(s) in Tax-induced gp34 activation. Coexistence of NF-
B-like elements 1 and
2 in the
100 to
54 fragment may stabilize the association between the factors and DNA elements, presumably efficiently, to render the
complex active. A particular configuration of NF-
B and its binding
sites may be required for active transcription. The location of
NF-
B-like elements 1 and 2, with a space between them, may be
adequate for formation of complexes of binding factors and DNA
elements. NF-
B-like element 2 is reminiscent of another NF-
B-like element, the C26 element in the HTLV-I enhancer (60). This element has
also been shown to bind an NF-
B-like factor but does not activate
transcription in response to Tax by itself, even in tandem repeat form,
whereas it can cooperate with Tax to augment transactivation of the
21-bp motif in the HTLV-I enhancer. The C26 element also differs by
three bases from the typical NF-
B sequence, and it competed
partially in complex formation with NF-
B-like elements 1 and 2 (Fig.
4B). Apart from NF-
B-like elements 1 and 2, there may be
other elements in the
100 to
54 fragment that are involved in
activation by Tax. An AP-1 site-like sequence between NF-
B-like elements 1 and 2 may be effective in formation of more stable configuration of the factor/DNA element complex. This may involve a
factor bound to the AP-1 site-like element. This notion is supported by
recent observations of functional interactions between NF-
B and
other cellular transcription factors, for example, C/EBP, the ATF
family, and Jun/Fos (61-63). NF-
B-like element 2 of the gp34 gene
and the C26 element in the HTLV-I enhancer may constitute a new
subfamily of NF-
B binding sites, which cannot activate transcription
despite being able to bind NF-
B and augment transcription by
cooperation with other enhancers.
Many transcriptional regulatory regions containing NF-
B binding
sites are activated by TPA. In this context, the NF-
B-like elements
of the gp34 gene are unique in that they are not activated by TPA
treatment, even though complex formation with the elements was induced
by treatment of Jurkat cells with TPA. This presumably accounts for the
unresponsiveness of the native gp34 promoter to TPA and
further suggests unresponsiveness of the gp34 gene to PHA. Stimulation
with PHA is generally accepted to mimic antigen stimulation. Indeed,
parameters of T cell activation, such as IL-2 production, nuclear
oncogene expression, and cell proliferation, are indistinguishable
between antigen and PHA stimulation. However, gp34 differs in that its
expression is not induced in normal T cells and T cell lines by PHA
stimulation.
We found transcriptionally suppressive activity associated with the
upstream region. This region alone profoundly prevented basic promoter
activity. The suppression was independent of the core promoter. Tax
appeared not to affect the suppressive region by itself. The strictly
regulated gp34 expression may be attributed to the
suppressive activity of the upstream region. Exactly how the upstream
sequence suppresses transcription is presently unknown. Further
experiments are required to precisely identify the suppressive region
and factor(s) associated with such a region.
Kinetics of Tax-induced gp34 expression were far slower than those of
other genes induced by Tax (36). Our results indicate that the
intrinsic gp34 promoter has weak activity even in the presence of Tax (Fig. 1A). Sequences upstream of
823 may
contain some other suppressive activity because the
9000 fragment
exhibited lower promoter activity than the
2700 and
823 fragments.
In addition, a preliminary Northern blot demonstrated that the
half-life of gp34 mRNA was quite long (12-24
h).2 Taken together, it is likely
that gp34 mRNA requires a couple of days to accumulate
to a sufficient level for detection by Northern blotting, and
thereafter, expression is maintained at that level for a certain
period. This may reflect the function of gp34, although the essential
role of the gp34/OX40 system remains obscure. gp34 is induced during
immune activation, and the inducible mechanism of the gp34 gene is
strictly controlled. OX40 is also inducible, unlike other members of
the TNF receptor family (FAS, TNF receptor, and CD40). Thus, production
of gp34 may be important in either maintaining or terminating immune
reactions.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. T. Kanno for
helpful discussion. We thank Drs. M. Yoshida, T. Akagi, and W. Green
for providing Tax mutant plasmids; Y. Matsumura for technical
assistance; and H. Shimizu for assistance in the preparation of the
manuscript.
 |
FOOTNOTES |
*
This work was supported in part by a grant for Core Research
for Evolutional Science and Technology of the Japan Science and Technology Corp., a grant-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of
Japan, a grant from the Inamori Foundation, a grant from the Mochida
Memorial Medical and Pharmaceutical Research Foundation, and a grant
from the Ichiro Kanehara Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been
submitted to DNA Data Bank of Japan, the GenBankTM/EBI Data Bank with
accession number(s) AB007839.

To whom correspondence should be addressed. Tel.:
81-3-5803-5795; Fax: 81-3-5803-0234; E-mail:
naka.gene{at}cmn.tmd.ac.jp.
1
The abbreviations used are: HTLV-I, human T cell
leukemia virus type I; IL-2, interleukin 2; CAT, chloramphenicol
acetyltransferase; TPA,
12-O-tetradecanoylphorbol-13-acetate; PHA,
phytohemagglutinin; CREB/ATF, cyclic AMP-responsive element binding
factor; TNF, tumor necrosis factor; bp, base pair(s); PIPES,
1,4-piperazinediethanesulfonic acid.
2
K. Ohtani and M. Nakamura, unpublished
data.
 |
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