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J. Biol. Chem., Vol. 277, Issue 11, 8775-8789, March 15, 2002
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From the
Received for publication, August 3, 2001, and in revised form, December 5, 2001
The bovine leukemia virus (BLV)
promoter is located in its 5'-long terminal repeat and is composed of
the U3, R, and U5 regions. BLV transcription is regulated by
cis-acting elements located in the U3 region, including
three 21-bp enhancers required for transactivation of the BLV promoter
by the virus-encoded transactivator TaxBLV. In addition to
the U3 cis-acting elements, both the R and U5 regions
contain stimulatory sequences. To date, no transcription factor-binding
site has been identified in the R region. Here sequence analysis of
this region revealed the presence of a potential E box motif
(5'-CACGTG-3'). By competition and supershift gel shift assays, we
demonstrated that the basic helix-loop-helix transcription factors USF1
and USF2 specifically interacted with this R region E box motif.
Mutations abolishing upstream stimulatory factor (USF) binding caused a
reproducible decrease in basal or Tax-activated BLV promoter-driven
gene expression in transient transfection assays of B-lymphoid cell
lines. Cotransfection experiments showed that the USF1 and USF2a
transactivators were able to act through the BLV R region E box. Taken
together, these results physically and functionally characterize a
USF-binding site in the R region of BLV. This E box motif located
downstream of the transcription start site constitutes a new positive
regulatory element involved in the transcriptional activity of the BLV
promoter and could play an important role in virus replication.
Bovine leukemia virus
(BLV)1 is a B-lymphotropic
oncogenic retrovirus that infects cattle and is associated with
enzootic bovine leukosis, a neoplastic proliferation of B-cells (1-6).
BLV is closely related to human T-lymphotropic viruses HTLV-I and -II. The majority of BLV-infected cattle are asymptomatic carriers of the
virus. Only about 30% of BLV-infected animals develop a preneoplastic
condition termed persistent lymphocytosis, with 2-5% developing
B-cell leukemia and/or lymphoma after a long latency period. The virus
can be experimentally transmitted to sheep, in which it causes similar
pathologies, providing a helpful model to understand BLV and
HTLV-induced leukemogenesis. BLV infection is characterized by viral
latency in the large majority of infected cells and by the absence of
viremia. These features are thought to be due to the transcriptional
repression of viral expression in vivo (7-9). The latency
is likely to be a viral strategy to escape the host immune response and
allow tumor development.
BLV transcription initiates at the unique promoter located in the
5'-long terminal repeat (5'-LTR) of the BLV genome. The 5'-LTR is
composed of the U3, R, and U5 regions and transcription initiates at
the U3-R junction. The U3 region contains the main sites that regulate
viral transcription (Fig. 1) as follows:
the promoter CAAT and TATA boxes (10, 11), a glucocorticoid-responsive element (12-15), and a large segment protected in DNase footprinting assays containing NF- In addition to these cis-acting elements all situated in U3,
we and others (10, 22, 23) have previously demonstrated that both the R
and U5 regions of the BLV LTR, located downstream from the
transcription start site, contain additional regulatory sequences
stimulating BLV gene expression. An interferon regulatory factor
(IRF)-binding site in U5 interacts with IRF-1 and IRF-2 and stimulates
viral gene expression in the absence of TaxBLV (22). The
presence of a 64-bp downstream activator sequence (DAS) at the 3' end
of the R region has been reported (10, 23) (Fig. 1). To date however,
no transcription factor-binding site has been identified in the R region.
In this report, the sequence analysis of the BLV R region revealed the
presence of a potential E box site (hereafter referred to as E-box4),
5'-CACGTG-3' (nt +173 to +178), that conforms to the core
hexanucleotide consensus sequence of an E box motif, CANNTG. E box
sites bind proteins that belong to the basic helix-loop-helix (bHLH)
family of transcription factors, including c-Myc, Max, USF, or TFE3
(reviewed in Refs. 24-26). bHLH proteins contain a basic (b) DNA
binding domain and a helix-loop-helix (HLH) dimerization domain; the
latter allows these proteins to interact and to form homo- and/or
heterodimers. In the case of bHLH-ZIP-related proteins, the bHLH domain
is contiguous with a second dimerization domain, a leucine zipper
(ZIP), characterized by heptad repeats of leucines that occur
immediately C-terminal to the bHLH motif. Precise DNA-binding site
selection by individual bHLH family members is determined both by the
central dinucleotide contained in the core hexamer sequence and by the
flanking nucleotides (27-36). Some of these transcription factors can
act as either transactivators or repressors of gene expression,
depending on the gene promoter or on their dimerization partner (33,
37-40).
The objective of the present study was to characterize physically and
functionally the E-box4 motif located in the BLV R region. We performed
competition and supershift gel shift assays with nuclear extracts
prepared either from peripheral blood mononuclear cells (PBMCs) derived
from a BLV-infected sheep or nuclear extracts from the human B-lymphoid
cell line Raji. We demonstrated that the bHLH transcription factors
USF1 and USF2 specifically interacted with the E-box4. A 2-bp mutation
(central CG to TA) and another 2-bp mutation (3' TG to GA) abrogated
USF binding. To assess the transcriptional regulatory function of the
E-box4, we tested the effect of these 2-bp mutations by transient
transfection of B-lymphoid cell lines in the context of an
LTR-luciferase reporter construct in presence or absence of a
TaxBLV expression vector. Both mutations caused a
reproducible 25% decrease in LTR-driven basal gene expression, indicating a positive functional role of the E-box4 motif in R. Ectopically expressed USF1 and USF2a proteins had an
E-box4-dependent stimulatory effect on both the homologous
BLV promoter and a heterologous thymidine kinase (TK) promoter
containing multiple upstream E-box4 motifs. Mutation in the E-box4
impaired the responsiveness of the BLV promoter to TaxBLV
but not to other activators known to up-regulate BLV expression
(overexpression of CREB2, calcium/calmodulin-dependent protein
kinase IV (CaMKIV), or CREB2/CaMKIV and treatment with PMA/ionomycin).
Moreover, mutation in the E-box4 in combination with a mutation in the
IRF site in U5 decreased the LTR basal activity more than 2-fold. The
identification of the E-box4 motif represents the first transcription
factor-binding site reported in the R region of BLV.
PBMC Isolation--
The animal used in this study was a
BLV-seropositive adult sheep (M298) affected with persistent
lymphocytosis, presenting a persistently elevated lymphocyte count and
an inverted B/T-lymphocyte ratio. This sheep was housed at the
Veterinary and Agrochemical Research Center (Uccle, Belgium). Blood
samples were collected by jugular venipuncture, mixed with EDTA as an
anticoagulant, and centrifuged at 1750 × g for 25 min
at room temperature. The PBMCs were then isolated by Percoll gradient
centrifugation (density 1.129 g/ml; Amersham Biosciences) and washed
twice in phosphate-buffered saline containing 0.075% EDTA, with
centrifugation steps at 450 × g for 10 min at room
temperature. The cells were washed with phosphate-buffered saline
(centrifugations at 200 × g for 10 min at room
temperature) until the supernatant became clear.
Cell Lines and Cell Culture--
The Raji and Daudi cell lines
are human B-lymphoid EBV-positive cell lines derived from Burkitt's
lymphomas. The human epithelial HeLa cell line is derived from a
cervical carcinoma and is transformed by human papilloma virus type 18. All media, sera (Myoclone Superplus), and supplements were from
Invitrogen. Raji cells were grown in RPMI 1640-Glutamax I medium
supplemented with 10% fetal bovine serum, 50 units of penicillin/ml,
and 50 µg of streptomycin/ml. Daudi cells were maintained in RPMI
1640-Glutamax I medium with 10% fetal bovine serum, 10 mM
HEPES buffer, 1 mM sodium pyruvate, 50 units of
penicillin/ml, and 50 µg of streptomycin/ml. HeLa cells were cultured
in Dulbecco's modified Eagle's-Glutamax I medium containing 5% fetal
bovine serum, 50 units of penicillin/ml, and 50 µg of
streptomycin/ml. All cells were grown at 37 °C in an atmosphere of
5% CO2.
Plasmid Constructs--
The BLV LTR used in this study is the
344 provirus described by Sagata et al. (41). The first
nucleotide of R and the last nucleotide of U3 are considered +1 and
Mutated constructs were fully resequenced after identification by cycle
sequencing using the Thermosequenase DNA sequencing kit (Amersham
Biosciences). Three mutated plasmids were designated pLTR(E-box4-mutA)-luc, pLTR(E-box4-mutB)-luc, and
pLTR(IRF-mut)-luc. Four constructs containing combinations of mutations
were also generated by site-directed mutagenesis (Stratagene) using
simultaneously two or three of the mutagenic oligonucleotide pairs
described above. These plasmids were generated by combining
CV194/195-CV196/197, CV281/282-CV283/284,
CV192/193-CV194/195-CV196/197, and CV262/265-CV281/282-CV283/284 and
were designated pLTR(E-box1,2,3-mutA)-luc, pLTR(E-box1,2,3-mutB)-luc, pLTR(E-box1,2,3,4-mutA)-luc, and pLTR(E-box1,2,3,4-mutB)-luc, respectively. In addition, pLTR(E-box4-mutA)-luc and
pLTR(E-box4-mutB)-luc were used as substrates for site-directed
mutagenesis using the mutagenic oligonucleotide primers CV311/312,
thereby generating pLTR(E-box4-mutA/IRFmut)-luc and
pLTR(E-box4-mutB/IRFmut)-luc, respectively.
The pTK-luc reporter plasmid contains the herpes simplex virus
thymidine kinase (TK) minimal promoter and was generated by subcloning
the SalI (position 417)-XhoI (position 597)
fragment from pBLCAT2 (42) into the XhoI-restricted
pGL2-BASIC reporter plasmid (Promega).
The p(E-box4)3TK-luc and p(E-box4)7TK-luc were
generated by inserting three direct repeats in the forward orientation
and seven direct repeats in the reverse orientation, respectively, of
an oligonucleotide with the sequence
5'-ACCGTCTCCACGTGGACTCTCT-3' (the BLV E-box4 motif is
underlined) into SmaI-digested pTK-luc. Similarly, the two
mutated versions, p(E-box4-mutA)3TK-luc and p(E-box4-mutB)3TK-luc, were generated by inserting three
direct repeats in the forward orientation of an oligonucleotide with the sequence 5'-ACCGTCTCCATATGGACTCTCT-3'
and 5'-ACCGTCTCCACGGAGACTCTCT-3' (mutations are highlighted in bold), respectively, into
SmaI-digested pTK-luc. In all these constructs, the
multimerized wild-type or mutated E box motifs are thus positioned
upstream of the TK promoter.
To construct pGEM-LTRBLV used in RNase protection analysis,
a 201-bp fragment containing the BLV 5'-LTR from position
To construct the expression plasmid for ovine c-Myc, we used PCR to
amplify the coding region of the ovine c-myc cDNA
described in Kiermer et al. (43). HindIII and
BamHI restriction sites were introduced into the 5' and 3'
PCR primers, respectively, and the
HindIII-BamHI-restricted PCR fragment was cloned
in pcDNA3.1 (+/
The eukaryotic expression vectors pSG-TAXBLV and pSG-CREB2
(44) were gifts from Drs. Luc Willems and Richard Kettmann. The expression plasmid pCaMKIV was a gift from Dr. Anthony Means (45).
The USF1 and USF2a expression vectors (kindly provided by Drs. A. Kahn
and M. Raymondjean) contained the human USF1 and USF2a cDNAs cloned
in the pCR3 expression vector parent plasmid (Invitrogen) and were
described previously (46).
Transient Transfection and Luciferase Assays--
Raji cells
were transfected by using the DEAE-dextran procedure as described
previously (47). Briefly, cells were harvested at density of
106/ml, washed with STBS (25 mM Tris-HCl (pH
7.5), 137 mM NaCl, 5 mM KCl, 700 µM CaCl2, 500 µM
MgCl2, 600 µM
Na2PO4), and resuspended at a concentration of
6 × 106 in 600 µl of a mixture containing 500 ng of
the pGL3-BASIC-derived constructs (with or without cotransfected DNAs)
and 450 µg of DEAE-dextran (Amersham Biosciences)/ml in STBS. Cells
were incubated for 1 h at 37 °C, washed twice with STBS and
once with culture medium, and grown in 3 ml of supplemented medium for
40-44 h. Cells were then lysed and assayed for luciferase activity
(Promega). Luciferase activities derived from BLV LTRs were normalized
with respect to protein concentrations using the Detergent-compatible Protein Assay (Bio-Rad).
Daudi cells were transfected by electroporation. Cells were harvested
in exponential growth phase and resuspended in supplemented medium at a
concentration of 5 × 106 per 400 µl. The 400 µl
of cells were mixed with 8 µg of pGL3-BASIC-derived constructs and 50 ng of pRL-TK (Promega) and incubated for 15 min at room temperature,
transferred to electroporation vials, and electroporated at 250 V with
a capacitance of 960 microfarads (by using a Bio-Rad gene pulser).
Transfected cells were collected, plated out immediately in 4 ml of
preheated medium (a 1:1 mixture of fresh culture medium and supernatant
of a 24-h culture), and grown for 48 h at 37 °C. All
transfection mixtures contained the pRL-TK, in which a cDNA
encoding Renilla luciferase is under the control of the
herpes simplex virus thymidine kinase promoter region and is used as an
internal control for transfection efficiency. At 48 h
post-transfection, luciferase activities (firefly and Renilla) were measured in cell lysates, and firefly
luciferase activities derived from the BLV promoters were normalized
with respect to the Renilla luciferase activity by using the
dual luciferase reporter assay system (Promega).
HeLa cells were transfected using FuGENETM-6 (Roche
Molecular Biochemicals) according to the manufacturer's protocol.
Briefly, cells were seeded at a density of 2 × 105
cells/well in 6-well plates. FuGENETM-6 was added directly
to serum-free medium 5 min prior to addition to the DNA. Hundred
microliters of this FuGENETM-6/serum-free medium mix was
added to the DNA mixture in microcentrifuge tubes. The mixture was
incubated for 15 min at room temperature and, finally, was added to
each well of a 6-well plate. Transfected cells were grown in 2 ml of
supplemented medium for 40-44 h. Cells were then lysed and assayed for
luciferase activity (Promega). Luciferase activities derived from BLV
or TK promoters were normalized with respect to protein concentrations
using the Detergent-Compatible Protein Assay (Bio-Rad).
Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts were prepared by a rapid method described by Osborn et
al. (48). All buffers contained the protease inhibitors antipain
(10 µg/ml), aprotinin (2 µg/ml), chymostatin (10 µg/ml), leupeptin (1 µg/ml), and pepstatin (1 µg/ml). Protein
concentrations were determined by the method of Bradford (49). The DNA
sequences of the coding strand of the double-stranded oligonucleotides
used for this study are listed in Fig. 3A or in the figure
legends. EMSAs were performed as described previously (47). Briefly, nuclear extract (4 µg of protein) was first incubated at room temperature for 10 min in the absence of probe and specific competitor DNA in a 16-µl reaction mixture containing 10 µg of DNase-free bovine serum albumin (Amersham Biosciences), 6 µg of poly(dI-dC) (Amersham Biosciences) as nonspecific competitor DNA, 1 mM
dithiothreitol, 20 mM Tris-HCl (pH 7.5), 60 mM
KCl, 1 mM MgCl2, 0.1 mM EDTA, and 10% (v/v) glycerol. Fifteen thousand cpm of probe (10-40 fmol) was
then added to the mixture with or without a molar excess of an
unlabeled specific DNA competitor, and the mixture was incubated for 20 min at room temperature. Samples were subjected to electrophoresis at
room temperature on 6% polyacrylamide gels at 150 V for 2-3 h in 1×
TGE buffer (25 mM Tris acetate (pH 8.3), 190 mM
glycine, 1 mM EDTA). Gels were dried and autoradiographed
for 24-48 h at
In vitro translated Ebox-binding proteins Max, Mad1, and
c-Myc were used in control EMSAs. These proteins were generated by using the TNT-coupled Reticulocyte Lysate System (Promega) with the
pGEM expression plasmid encoding mouse Max (p22 long form), the pRC/CMV
expression vector for human Mad1 (both kindly provided by Dr. S. Segal), and the pcDNA3 expression vector for ovine c-Myc, pcDNA3-cMyc. Eight µl of in vitro translated Mad1 or 4 µl of in vitro translated c-Myc were preincubated with 1 µl of in vitro translated Max for 10 min at 42 °C and
then for an additional 20-min period at room temperature to promote
formation of Mad1-Max or c-Myc-Max dimers, respectively. These
complexes were then incubated for 30 min at room temperature in the
presence of 30,000 cpm of a 32P-labeled CMD (specific E
box) probe (an oligonucleotide with a c-Myc consensus binding site,
5'-AGCTTCAGACCACGTGGTCGGG-3' (50)) in a buffer containing
10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.5 mM dithiothreitol, 0.05% Nonidet P-40, 1% glycerol, and 1 µg of poly(dI-dC) in a total volume of 20 µl. For supershift
assays, 2 µl of either anti-Max (sc-197), either anti-Mad-1 (sc-222), or anti-c-Myc (sc-764) antibodies (Santa Cruz Biochemicals, Santa Cruz,
CA) were added to the reaction mixture and further incubated overnight
at 4 °C. The protein-DNA complexes were then separated from the free
probe by electrophoresis on 4% polyacrylamide gels in 0.5× Tris
borate/EDTA at 150 V. The free probe was run out of the gel for better
separation of the complexes.
RNase Protection Assays--
Raji cells were harvested by
centrifugation 44 h post-transfection, washed in
phosphate-buffered saline, pelleted, and kept on ice. Total RNA samples
were prepared using the commercial RNAqueous Phenol-free Total RNA
Isolation Kit (Ambion) and treated with 2 units/µg of RNase-free
DNase I (10 units/µl, Roche Molecular Biochemicals) for 30 min at
37 °C in a total volume of 240 µl containing 1× NEB2 buffer (New
England Biolabs) and 1.6 units/µg RNasin (40 units/µl, Promega).
RNA was then extracted with phenol/chloroform/isoamyl alcohol,
ethanol-precipitated, and resuspended in sterile and RNase-free water
(United States Biochemical Corp.).
A BLV-specific 32P-labeled antisense riboprobe was
synthesized in vitro by transcription of
XbaI-restricted pGEM-LTRBLV with SP6 polymerase
according to the protocol provided with the Riboprobe in
vitro Transcription Systems (Promega). A luciferase antisense riboprobe was similarly synthesized by transcription of
SgrAI-restricted pSP-luc+ vector (Promega) with
T7 polymerase. As control, a glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)-specific antisense probe was synthesized by the same method and
used on the same RNA samples.
The RNase protection assays were performed with the RPA II Kit (Ambion)
according to the manufacturer's recommendations. Briefly, hybridization reactions (20 µl) containing 120 µg of total cellular RNA and 200,000 cpm of probe in hybridization buffer were heated to
95 °C for 4 min to denature the RNA and then incubated at 42 °C
for 16 h. The reaction mixtures were diluted by the addition of
200 µl of digestion buffer, and the single-stranded sequences were
digested with RNase T1 and RNase A for 1 h at 37 °C. Following addition of 300 µl of inactivation buffer and ethanol precipitation, the protected RNA fragments were analyzed by electrophoresis through 6% urea polyacrylamide gels.
The BLV R Region E-box4 Motif Specifically Binds USF1 and USF2 in
Vitro--
A data base search for potential transcription
factor-binding sites in the R region of the BLV LTR revealed the
presence of a putative E box site (nt +173/+178 according to the
sequence reported by Sagata et al. (41)), 5'-CACGTG-3'. In
order to identify cellular factors that bind to this potential E box
motif (designated E-box4), EMSAs were performed by using as probe a
22-bp E-box4-containing oligonucleotide corresponding to positions +165
to +186 of the 5'-LTR. This probe (referred to as E-box4-wt) was
incubated with nuclear extracts from PBMCs derived from a BLV-infected
sheep (BLV-infected sheep M298 presenting a persistently elevated
lymphocyte count and an inverted B/T-lymphocyte ratio). A major
retarded protein-DNA complex was detected with a major band indicated
by an arrow and the fainter band indicated by an asterisk
(Fig. 2A, lane
1). To evaluate the specificity of these interactions, unlabeled double-stranded oligonucleotides were prepared and used as competitors in the EMSAs. Binding of proteins in the major complex was shown to be
sequence-specific by competition EMSAs. Indeed, the formation of this
complex was competed for by an excess of the unlabeled homologous
E-box4-wt oligonucleotide (Fig. 2A, lanes 2-5)
but not by the same molar excess of a heterologous oligonucleotide of
unrelated sequence containing the HIV-1 NF-
To identify directly the factors present in the specific major retarded
complex, we performed supershift assays using antibodies directed
against individual members of the bHLH family of transcription factors
(Fig. 2B, left panel). Labeled E-box4-wt
oligonucleotide probe was incubated with nuclear extracts from
BLV-infected ovine PBMCs. Polyclonal antibodies directed against USF1,
USF2, Max, Mad-1, Mad-2, Mad-3, Mad-4, c-Myc, and Mnt were added to the
binding reaction mixture. Addition of either the anti-USF1 or the
anti-USF2 antibody (Fig. 2B, left panel, lanes 2 or 3, respectively) interfered with the formation of the
major complex and generated supershifted complexes of decreased
mobility (Fig. 2B, left panel). Although the
anti-USF1 antibody eliminated almost completely the major complex, the
anti-USF2 antibody only slightly decreased the intensity of this
complex. When both anti-USF1 and anti-USF2 antibodies were included in
the binding reaction, the entire complex was eliminated (Fig.
2B, left panel, lane 4). In contrast,
the binding pattern was not affected by the addition of the antibodies
directed against the other bHLH proteins (Fig. 2B,
left panel, lanes 5-11), showing that the major
retarded complex did not seem to involve these other proteins. No
supershifted complex was observed with a control purified rabbit IgG
(Fig. 2B, left panel, lane 1) or when
the antibodies were added to the probe alone (data not shown), thus
indicating the specificity of the protein-antibody interactions. Similar results were observed with Raji nuclear extracts (data not shown).
In order to establish the validity and/or the specificity of some of
the antibodies other than anti-USF1/2 used in the supershift assays,
control EMSAs were performed by incubating in vitro
translated E box-binding proteins (Max, Mad1, and c-Myc) with a labeled
CMD (specific E box) probe (50) (Fig. 2B, right
panel). Addition of the anti-Mad1 or anti-c-Myc antibody
interfered with formation of the Mad1-Max or c-Myc-Max complex (Fig.
2B, right panel, lanes 9 or
12, respectively). The anti-Max antibody supershifted the Max homodimers (Fig. 2B, right panel, lane 6) as
well as the Mad1-Max (Fig. 2B, right panel,
lane 8) and c-Myc-Max (Fig. 2B, right
panel, lane 11) heterodimers.
Overall, our results demonstrate that both the USF1 and USF2
transcription factors interact with the E-box4 motif located in the BLV
R region. Moreover, they suggest that the predominant USF species that
bind to this motif are the heterodimer USF1/USF2 and the homodimer
USF1/USF1 and that a small amount of homodimer USF2/USF2 might also
bind. These data are consistent with several reports (53-56)
demonstrating that the major USF species present in most tissues and
cell lines is the heterodimer between USF1 and USF2. USF1 homodimers
are less abundant, and USF2 homodimers are usually quite scarce.
Identification of Point Mutations Abolishing USF Binding to the
E-box4 Motif in the 5'-LTR BLV R Region--
To further characterize
physically the E-box4 motif located in the R region of the BLV 5'-LTR,
we studied by EMSA the effect of selected mutations on binding
affinity. Two mutations were designed to abolish binding of factors to
the E-box4 motif. The first mutation, designated E-box4-mutA, consisted
of the substitution of the two central nucleotides CG with the
dinucleotide TA (57). The second mutation, designated E-box4-mutB,
consisted of the substitution of the 3'-TG with the dinucleotide GA
(58) (Fig. 3A).
The effects of these 2-bp mutations on binding affinity were analyzed
by competition EMSAs with the E-box4-wt oligonucleotide as a probe and
nuclear extracts from sheep PBMCs (Fig. 3B). The E
box-specific retarded complex was inhibited by competition with an
excess of the homologous oligonucleotide (Fig. 3B,
lanes 2-5). In contrast, the complex was not competed by the
E-box4-mutA and E-box4-mutB oligonucleotides (Fig. 3B, lanes
6-9 and lanes 10-13, respectively), demonstrating
that both mutations abolished USF binding to the E-box4. Moreover, we
confirmed the lack of USFs binding to probes corresponding to the
mutated E-box4 motifs (data not shown). Similar results were observed
with Raji nuclear extracts (data not shown).
Taken together, our results identify an E box motif in the R region of
the BLV 5'-LTR (nt +173 to +178). This E box motif specifically binds
the bHLH proteins USF1 and USF2 in vitro. We report a couple
of 2-bp mutations, referred to as E-box4-mutA and E-box4-mutB, that
abrogate USF binding to this motif.
The E-box4 Motif in the R Region of the BLV 5'-LTR Is Required for
Optimal Basal Gene Expression from the BLV Promoter--
In order to
examine the functional role of the E-box4 motif in the basal
transcriptional activity of the BLV promoter, the same 2-bp mutations
described above (E-box4-mutA and E-box4-mutB) were introduced by
site-directed mutagenesis in the context of a
pLTRBLV-luciferase reporter construct, called pLTRwt-luc
and containing the firefly luciferase (luc) gene under the control of
the complete 5'-LTR (nt
To assess the transcriptional regulatory function of the E-box4
motif, the constructs pLTRwt-luc, pLTR(E-box4-mutA)-luc, and pLTR(E-box4-mutB)-luc were transiently transfected into Raji cells. At
44 h post-transfection, cells were lysed and assayed for
luciferase activity. Results presented in Fig.
4A show that both mutations, E-box4-mutA and E-box4-mutB, reproducibly reduced the basal activity of
the BLV promoter by 25%. These results are consistent with the binding
of a transcriptional activator, such as USF1 and/or USF2, to the E-box4
motif.
To confirm these results in another B-lymphoid cell line, the same
reporter constructs (pLTRwt-luc, pLTR(E-box4-mutA)-luc, and
pLTR(E-box4-mutB)-luc) were transiently transfected into the human cell
line Daudi. As shown in Fig. 4B, the E-box4-mutA mutation caused a 15% decrease in basal LTR-directed gene expression.
Interestingly, the E-box4-mutB mutation more strongly decreased LTR
activity because it caused a 56% decrease in BLV 5'-LTR-driven basal
gene expression. These functional results confirmed in another B-cell line the results observed in Raji cells. However, the E-box4-mutA and
E-box4-mutB mutations decreased LTR basal activity with less strength
and more strength, respectively, in Daudi cells than observed in Raji
cells. These differential effects of both mutations between the two
cell lines may result from differences in the expression, binding,
and/or transcriptional activity of USF proteins, transcriptional
cofactors, and/or other DNA-binding proteins.
Our functional results thus demonstrate a positive regulatory role of
the E-box4 motif located in the R region of the BLV 5'-LTR in
Tax-independent BLV promoter-driven gene expression.
Ectopic Expression of USF1 and USF2a Transcription Factors
Up-regulates BLV Promoter Activity in Part through the E-box4
Motif--
To examine the role of USF in regulation of the BLV
promoter, we studied the effect of overexpression of USF on luciferase activity. Cotransfection of expression constructs for USF1 and/or USF2a
with the pLTRwt-luc reporter construct had no significant effect on
luciferase activity in the transformed Raji cell line (data not shown).
Considering that USF is abundantly expressed in most cell types, one
possible explanation is that USFs would be non-limiting factors for the
BLV promoter activity in Raji cells. Moreover, recent studies comparing
USF function in different normal and cancerous breast cell lines have
demonstrated that a partial or complete loss of USF transcriptional
activity is a common event in transformed cells as compared with normal
cells, even though there is no difference both in expression and
DNA-binding activity of USF proteins (56, 59). These latter studies
suggest a correlation between the loss of USF function and
tumorigenesis. Therefore, the potential inactivation of USF in the
transformed Raji cells we used is another possible explanation.
Furthermore, USF was originally identified and characterized in HeLa
cells (30, 60-64), and it has been demonstrated that USF proteins are transcriptionally active in this cell line (59).
Together, these observations prompted us to examine in HeLa cells
whether USF1 or USF2a overexpression had an effect mediated by the
E-box4 on BLV promoter activity. We performed cotransfection experiments using USF1 or USF2a expression vectors and the pLTRwt-luc reporter construct as well as the mutated derivative
pLTR(E-box4-mutB)-luc. As shown in Fig.
5, USF1 and USF2a transactivated the BLV
promoter in a dose-dependent manner up to 4- and 3.2-fold,
respectively. Maximal activation was seen at concentrations of 2000 ng
of USF1 (Fig. 5A) and 250 ng of USF2a (Fig. 5B).
When the amounts of USF2a were further increased, the stimulation was
reduced. When expressed at very high levels, many transcriptional
activators squelch activated transcription (65). This squelching
phenomenon is thought to be due to the sequestration in solution of
transcriptional components, preventing their interaction at gene
promoters. Moreover, cotransfection of both USF1 and USF2a together in
equal amounts resulted in transactivation levels of the BLV promoter
similar to those obtained with USF1 and USF2a cotransfected separately
(data not shown). This observation has also been reported in numerous
other studies (66-71) and is consistent with the idea that USF
homodimers can function as well as heterodimers, although in
purified nuclear extracts USFs were generally found as heterodimers
(72).
However, when analyzing in the same cotransfection assays the effect of
USF1 and USF2a overexpression on the mutated construct pLTR(E-box4-mutB)-luc, we observed that mutation of the E-box4 did not
reduce the USF stimulatory effect. Indeed, USF1 and USF2a transactivated pLTR(E-box4-mutB)-luc as efficiently as the wild-type construct pLTRwt-luc (Fig. 5, A and B),
indicating that other LTR region(s) could mediated partially or totally
transactivation by USF. These other regions could mask the potential
transactivation mediated by the E-box4 motif. Because three E boxes are
located in the BLV LTR U3 region (E-box1, -2, and
-3, Fig. 1), we evaluated the potential contribution of
these motifs in the LTR response to USF1 and USF2a. Mutant reporter
constructs were generated, in which the E-box1, -2, and -3 were mutated
and in which all four E boxes were mutated. The plasmids were
designated pLTR(E-box1,2,3-mutB)-luc and pLTR(E-box1,2,3,4-mutB)-luc,
respectively, and were used in cotransfection experiments of HeLa cells
with the USF1 and USF2a expression vectors. As shown in Fig. 5,
A and B, mutation of the E boxes 1- 3 in
combination attenuated the stimulatory effect of USF1 and USF2a. There
was a 28 (USF1) and 39% (USF2a) reduction of stimulation compared with
the USF stimulations observed with pLTRwt-luc. Interestingly, when all
four E boxes were mutated, we observed a 53 (USF1) and 57% (USF2a)
reduction of stimulation, suggesting that USF proteins act in part
through the E-box4 motif to stimulate BLV promoter activity. It is
noteworthy that, although a mutated LTR in which all four E boxes were
mutated was strongly impaired in terms of stimulation by USF, this
mutated LTR was still responsive to ectopic USF. This residual response
may result either from unidentified E boxes present in the LTR and/or
from USF action directly or indirectly through other
cis-regulatory elements.
These results thus indicate that the E-box4 plays a role in the
transactivation of the BLV promoter by USF but that the three other E
boxes located in U3 are also responsible for part of the USF response.
Moreover, besides these four E boxes, other cis-elements that are directly or indirectly affected by USF seem to contribute to
the residual USF induction observed with the total mutant construct pLTR(E-box1,2,3,4-mutB)-luc.
Multimerized Copies of the BLV E-box4 Motif Confer USF Inducibility
to a Heterologous Minimal Promoter--
To assess better the
contribution of the E-box4 to the ectopic USF response, we constructed
three luciferase reporter plasmids driven by the herpes simplex virus
TK minimal promoter with or without three or seven tandem repeats of
the E-box4 motif inserted upstream of the TK promoter. These three
plasmids were referred to as pTK-luc, p(E-box4)3TK-luc, and
p(E-box4)7TK-luc, respectively. Moreover, three copies of
the E-box4-mutA and E-box4-mutB oligonucleotide were also cloned into
pTK-luc and called p(E-box4-mutA)3TK-luc and
p(E-box4-mutB)3TK-luc, respectively. To examine the USF
inducibility of these reporter constructs, HeLa cells were transiently
cotransfected with each of them and increasing amounts of either the
USF1 or USF2a expression vector (Fig. 6,
A or B, respectively) and then assayed for
luciferase activity. The control pTK-luc construct was moderately
transactivated by USF1 (up to 3.5-fold) and by USF2a (up to 7.7-fold).
Cotransfection of USF1 or USF2a expression vectors with the reporter
constructs containing the wild-type E-box4 motifs resulted in a
stimulation of luciferase activity that was dependent on the number of
E boxes [p(E-box4)3TK-luc versus
p(E-box4)7TK-luc]. Addition of three copies of the E-box4 motif upstream of the TK promoter resulted in a
dose-dependent increase in luciferase activity by
ectopically expressed USF1 (up to 8.2-fold) and USF2a (up to
21.1-fold), thus representing a 2.3- and 2.7-fold up-regulation when
compared with the USF response of the control pTK-luc devoid of
upstream E-box4 motifs. Seven copies of the E-box4 further increased
the USF stimulation: 4.5- (USF1) and 4-fold (USF2a) up-regulation
compared with the pTK-luc. This effect required intact E-box4 motifs,
because mutations in these motifs [p(E-box4-mutB)3TK-luc
(Fig. 6, A and B) and
p(E-box4-mutA)3TK-luc (data not shown)] resulted in levels
of USF-mediated transactivation similar to those obtained with the
control pTK-luc. Moreover, using each TK reporter construct, the
combination of USF1 and USF2a transfected together in equal amounts had
no more effect than either factor transfected alone (data not
shown).
We conclude from these experiments that ectopic USF1 and USF2a proteins
have an E-box4-dependent stimulatory effect on the heterologous TK promoter containing multiple upstream E-box4 motifs. These results thus establish the functional significance of USF through
the BLV E-box4 motif.
The E-box4 Motif in the R Region of the BLV 5'-LTR Regulates
Tax-dependent BLV Promoter-driven Gene
Expression--
Efficient transcription and replication of the BLV
genome require both the viral LTR and the virus-encoded transcriptional activator TaxBLV, which functions through the TxREs in U3.
In the next experiments, we studied the effect of the E-box4-mutA and
E-box4-mutB mutations on the responsiveness of the BLV promoter to
TaxBLV. Toward this end, cotransfections of Raji cells were performed using either pLTRwt-luc, pLTR(E-box4-mutA)-luc, or
pLTR(E-box4-mutB)-luc and increasing amounts of the TaxBLV
expression vector, pSG-TAXBLV. At 44 h
post-transfection, luciferase activity was measured in cell lysates. As
shown in Fig. 7A,
TaxBLV activation of the wild-type LTR ranged from 44.3- to
214-fold. In comparison, TaxBLV activation of the
pLTR(E-box4-mutA)-luc and of the pLTR(E-box4-mutB)-luc ranged from
32.6- to 160-fold and from 24.7- to 126-fold, respectively. Thus, the
E-box4-mutA and E-box4-mutB mutations reproducibly reduced TaxBLV -mediated transactivation by 21-26 and by 29-44%,
respectively. These results demonstrate a positive regulatory role of
the E-box4 motif in Tax-dependent BLV promoter-driven gene
expression.
We next wanted to test the effect of the E-box4-mutA and E-box4-mutB
mutations on the activation of the BLV LTR by other stimuli. It has
been reported previously (19, 44) by transient transfections of D17
osteosarcoma cells that the bovine CREB2 protein is able to
transactivate the BLV LTR in the absence of the TaxBLV
protein and that the cAMP-dependent protein kinase A or the
calcium/calmodulin-dependent protein kinase IV (CaMKIV)
substantially increases the ability of CREB2 to stimulate gene
expression. Moreover, ex vivo, BLV expression can be
up-regulated by several lymphocyte activators, including phorbol ester
PMA (73) and calcium ionophore ionomycin (74).
We performed transient cotransfections of Raji cells with either
pLTRwt-luc, pLTR(E-box4-mutA)-luc, or pLTR(E-box4-mutB)-luc and
increasing amounts of both the CREB2 expression vector, pSG-CREB2, and
the CaMKIV expression vector pCaMKIV. At 44 h post-transfection, luciferase activity was measured in cell lysates. As shown in Fig.
7B, CREB2/CaMKIV activation of the wild-type LTR ranged from 61.7- to 2201-fold. In comparison, CREB2/CaMKIV activation of the
pLTR(E-box4-mutA)-luc and of the pLTR(E-box4-mutB)-luc ranged from
76.4- to 2656-fold, and from 95.1- to 2836-fold, respectively. Thus, in
contrast to what we observed with TaxBLV, the E-box4-mutA and E-box4-mutB mutations did not reduce CREB2/CaMKIV responsiveness of
the BLV LTR compared with the wild-type construct pLTRwt-luc. Similar
results were observed in cotransfection experiments using either
pSG-CREB2 alone, pCaMKIV alone, or after treatment with PMA/ionomycin
(data not shown).
Thus, our results demonstrate that mutations of the E-box4 motif
located in the BLV R region impaired the responsiveness of the viral
promoter to the viral protein TaxBLV but not to other activators known to up-regulate BLV expression.
The Effects of the Mutations in the E-box4 Motif Occur at the
Transcriptional Level--
In order to demonstrate that the amount of
transcription (i.e. RNA levels) has been affected by the
point mutations in the E-box4 motif, transcript levels in transiently
transfected Raji cells were measured by RNase protection assays using
probes proximal and distal to the BLV promoter (Fig.
8). The proximal probe, which overlaps
the start of transcription in the BLV reporter plasmids, stretches from
nt
We thus demonstrate that the mutations A and B introduced in the E-box4
motif decrease the amount of transcription directed by the BLV
promoter. These results are consistent with those of the LTR-luciferase
assays and show that the effects of the E-box4 mutations occur at the
level of transcription.
Mutations of Both the R Region E-box4 Site and the U5 Region IRF
Site Decrease Basal Gene Expression from the BLV Promoter to a Greater
Extent Than the Individual Mutations--
Previous studies (22) from
our laboratory have identified a binding site for the interferon
regulatory factors IRF-1 and IRF-2 in the first half of the BLV U5
region and have shown that mutation in this site causes a decrease in
Tax-independent BLV LTR gene expression. Together with the E-box4 site
described in this report, the IRF site in U5 constitutes the only two
characterized transcription factor-binding sites located downstream of
the BLV transcription start site. Therefore, double mutant constructs were generated in which both the E-box4 and the IRF site were mutated.
The constructs pLTRwt-luc, pLTR(E-box4-mutA)-luc, and pLTR(E-box4-mutB)-luc were used as substrates to introduce a 3-bp mutation in the IRF motif. The three mutated plasmids were
designated pLTR(IRFmut)-luc,
pLTR(E-box4-mutA/IRFmut)-luc, and
pLTR(E-box4-mutB/IRFmut)-luc, respectively. We tested
the functional effects of the E-box4-mutA and E-box4-mutB mutations in
combination with the mutation of the IRF-binding site by transient
transfection of these plasmids into Raji cells. As shown in Fig.
9, mutation of the E-box4 motif resulted
in a 22% reduction and a 35% reduction (depending on the mutation) of
LTR-directed luciferase expression. Mutation of the IRF site resulted
in a 42% reduction of LTR-directed luciferase expression. This result
confirmed, in the context of the 344 BLV strain, the previous
observation of our laboratory (22) showing that the IRF mutation
induces a 2-fold reduction in basal transcription of an LTR isolated
from the T15 BLV strain. Importantly, for the double mutants
pLTR(E-box4-mutA/ IRFmut)-luc and pLTR(E-box4-mutB/IRFmut)-luc, we
observed in a highly reproducible manner a 64 and 54% reduction, respectively, of LTR-directed luciferase expression (Fig. 9).
Thus, we showed that mutation of the only two transcription
factor-binding sites identified to date downstream of the transcription initiation site decreased the LTR basal activity more than 2-fold. These results reinforce the positive regulatory role played by the R
region E-box4 motif and indicate that transcription factor-binding sites at the R/U5 junction are critical for optimal basal BLV promoter activity.
In this report, we have physically and functionally characterized
an E box motif (referred to as E-box4) located in the R region of the
BLV 5'-LTR. We have demonstrated by competition and supershift EMSAs
that the bHLH transcription factors USF1 and USF2 bound in a
sequence-specific manner to the E-box4-containing sequence. Mutations
abolishing USF binding caused a reproducible decrease in BLV
promoter-driven gene expression in transient transfection assays of
B-lymphoid cell lines. Cotransfection experiments showed that the USF1
and USF2a transactivators were able to act through the BLV R region
E-box4. Moreover, combined mutation of both this E-box4 motif and the
IRF site in U5 decreased the LTR basal activity to a greater degree
than the individual mutations. This E box motif represents the first
transcription factor-binding site reported in the BLV R region.
USF proteins belong to the bHLH-ZIP family of transcription factors,
which includes c-Myc, Max (75), Mad (76), Mxi1 (77), AP4 (78), TFEB
(29), TFE3 (27), MiTF (79, 80), and ADD1 (81). USF was initially
identified from HeLa cell nuclei and was shown to be necessary to
stimulate transcription from the adenovirus major late promoter through
the core sequence CACGTG (30, 60-62). USF factors are encoded by two
distinct genes (the USF1 and USF2 genes)
(53, 54, 63, 64, 82). The USF1 gene encodes a single 43-kDa
protein. The USF2 gene, due to alternative splicing, encodes
two proteins, USF2a and USF2b of 44 and 38 kDa, respectively. The three
USF isoforms differ in their N-terminal moieties, whereas they have
highly conserved bHLH-ZIP domains (53, 54). USF proteins form homo- and
heterodimers both in vitro and in vivo (53, 82).
Dimerization with other bHLH proteins has not been observed (27, 33,
75, 76, 83). Although expression of the USF1 and USF2 species is
ubiquitous, different ratios of USF homo- and heterodimers are found in
different cell types (53). The major USF species present in most
tissues and cell lines is the USF1-USF2 heterodimer. USF1 homodimers
are less abundant, and USF2 homodimers are usually quite scarce (53, 54). USF proteins are ubiquitous transcription factors that were
initially considered to play a role in housekeeping functions (61).
Furthermore, these USF proteins have also been recognized as important
players in the transcriptional regulation of tissue-specific genes (26,
84-88) and in the specific response of genes to external modulators
(89), as glucose (46, 90, 91). The mechanisms by which USF proteins
contribute to these specific functions are yet unclear and may overlay
several phenomena. USF-binding sites have been found, and the
involvement of USF in transcriptional regulation has been studied in a
number of cellular and viral genes (46, 90, 92-95).
In this report, we clearly demonstrated a significant decrease in BLV
promoter activity accompanying mutation in the E-box4, indicating a
positive functional role of this motif. Moreover, cotransfection
experiments using USF1 and USF2a expression vectors demonstrated the
ability of a DNA fragment corresponding to multiple E-box4 copies to
confer USF inducibility to a minimal TK promoter in an
E-box4-dependent manner. We also showed that ectopic USF1 and USF2a transcription factors up-regulated BLV promoter activity. This USF responsiveness of the BLV promoter was mediated in part through the E-box4 motif. However, the three other E boxes located in
U3 were also responsible for part of the USF response. We are currently
investigating the identity of the transcription factors binding to
these three E boxes. Interestingly, we observed that mutation of all
four E boxes did not completely attenuate the stimulatory effect of
USF1 and USF2a in cotransfection experiments. Rather, there was a
reduction of stimulation to ~50% that observed with the wild-type
BLV promoter reporter construct. The presence of other yet unidentified
E boxes in the BLV LTR could be a first explanation. However, computer
analysis searches of the complete BLV LTR did not reveal any other
CANNTG consensus sequence (data not shown), suggesting that non-E box
cis-elements within the BLV promoter are directly or
indirectly responsive to USF. A report by Desbarats et al.
(96) documented a similar situation with the rat prothymosin- Studies by other investigators (60) have reported that USF is able to
bind sequences other than CACGTG such as the initiator element in the
adenovirus major late promoter. Moreover, some studies have shown that
USF factors can stimulate transcription by direct interaction with
members of the basal initiation complex as follows: the basal factor
TFIID (60, 63), the initiator-binding protein TFII-I (99, 100), and
TBP-associated factor TAFII55 (101). USF proteins have also been shown
to interact with other transcription factors, such as Fra1 (102), c-Maf
(103), Ets1 (104), and Sp1/Sp3 (105). Thus, substantial evidence
indicates that USF is involved in regulating gene expression by direct
binding to E boxes and/or initiator elements and by functionally
interacting with basal and/or specific transcription factors other than
USF. The present study showed that USF regulated the expression of BLV
in part through four E boxes (the E-box-1-3 in U3 and the E-box4 in
R). The residual USF induction observed with the LTR construct mutated
in all four E boxes could result from USF action directly or indirectly
through other cis-regulatory elements. Additional
experiments will be necessary to test this hypothesis.
Our in vitro binding studies demonstrated that both USF1 and
USF2 specifically interacted with the E-box4 motif. Our ex
vivo functional studies showed that ectopic USF1 and USF2a
proteins activated transcription of a reporter gene driven by either
the homologous BLV promoter or a minimal heterologous promoter
containing multiple upstream E-box4 motifs. In these latter studies,
both USF1 and USF2a exerted their stimulatory effect with similar
efficiency. Although from different genes, USF1 and USF2 display a high
degree of homology in their C-terminal DNA binding domain and
USF-specific region that is thought to play a role in transcriptional
activation of promoters (53, 106). Moreover, studies with USF null mice (55) and specific genes such as L-type pyruvate kinase (54), liver
fatty-acid synthase (66), and human dipeptidyl peptidase IV (107) have
shown that USF1 and USF2 have overlapping, redundant activities and can
transactivate some promoters with similar efficiency. Therefore, our
results are consistent with these previous observations, because they
demonstrate that the BLV LTR is another promoter activated equally well
by USF1 or USF2a, suggesting that both USF genes are involved in
BLV transcriptional activation.
The members of the bHLH transcription factor family have been divided
into four classes (A-D) depending on the sequence of the canonical
bHLH-binding site CANNTG (25). Class B proteins, which include c-Myc,
Max, MyoD, myogenin, and USF, bind to CACGTG or CATGTG. The E-box4
motif identified here in the BLV R region is an E box class B consensus
motif and accordingly was found to bind the class B proteins USF1 and
USF2. We could not observe the binding of c-Myc to this motif by
supershift experiments, even though Myc/Max heterodimers share the same
binding site requirements as USF dimers. Interestingly, in
vivo, the myc oncogene is overexpressed in
B-lymphocytes from tumors (108) and PBMCs isolated from BLV-infected animals with persistent lymphocytosis (109). Moreover, it has been
reported that levels of Myc/Max proteins in nuclear extracts are very
difficult to detect, whereas USF is the major binding activity detected
by in vitro assays with crude nuclear extracts from several
cell types and species (98, 110-114), even from cells that are
transformed by c-Myc (112). For all these reasons, although we
did not observe Myc binding under our in vitro conditions, we considered the possibility that, under physiological conditions, the
E-box4 motif would be nevertheless permissive for Myc interactions. Toward this end, we decided to analyze the consequences of c-Myc overexpression on the activity of the BLV promoter. We cloned the ovine
c-Myc cDNA (43) in the eukaryotic expression vector pCDNA3, and
we performed transient cotransfection experiments into both B-cell
lines (Raji and Daudi) and non-B-cell lines (HeLa) of this Myc
expression construct with the LTRBLV-luciferase vector in
the absence or presence of a Max expression vector. Our results did not
reveal any stimulatory effect of Myc/Max on the BLV
promoter.2 Therefore, our
data strongly suggest that USF proteins but not Myc-Max complexes are
the functional transcription factors that can activate the E-box4 in
the R region of BLV. However, it should be stressed that we did not
investigate the factors that bind to the E-box4 motif in
vivo, and therefore the binding of factors different from both USF
and Myc cannot be excluded at this time.
Our data showed that the E-box4 positively regulated BLV LTR-directed
gene expression both in the absence and presence of TaxBLV ex vivo and could therefore be involved
in the early and late stages of viral infection, respectively. USF
proteins, in conjunction with CREB, ATF-1, ATF-2 (18, 19), IRF-1, and
IRF2 (22), could thus be important transcription factors involved in
the initiation of BLV transcription. Indeed, the E-box4 motif may with
others initiate a low level of transcription from the BLV LTR promoter
and lead to the synthesis of small amounts of the TaxBLV
transactivator, which could then amplify transcription of the viral
genome. Although a 64-bp DAS at the 3' end of the R region (+147 to
+211) was reported previously (23), no transcription factor-binding
site had been identified in or close to the DAS. The E-box4 motif
described in this report is located right in the center of DAS and
could therefore be, at least in part, responsible for the positive
regulatory activity of DAS. Moreover, we have demonstrated that
mutation of both the R region E-box4 site and the U5 region IRF site
decreased TaxBLV-independent LTR-driven gene expression to
a greater degree than mutation of either site separately. These results
indicate that the two transcription factor-binding sites identified to
date downstream of the transcription start site play a critical role in
the basal transcriptional regulation of the BLV promoter. Downstream
regulatory sequences have also been identified in the HTLV-I LTR.
Indeed, a 45-bp element, which is located at the boundary of R-U5 and
binds the YB-1 transcription factor, is required for
TaxHTLV-I-independent transcription (115, 116). On the
other hand, binding of the Sp1 and Sp3 transcription factors to the
HTLV-1 U5 region has been associated with transcriptional repression of
the LTR (117, 118). Furthermore, it has been suggested that the
interaction of CREB and ATF-2 with the R region of the HTLV-1 LTR is
associated with viral latency (119, 120).
We have shown that the E-box4 motif also positively regulates BLV
LTR-directed gene expression in the presence of TaxBLV and could therefore be involved in the late stage of viral replication. How
USF affects the responsiveness of the BLV LTR to TaxBLV and whether this effect is mediated by direct or indirect interactions of
USF with TaxBLV remains to be established. Interestingly,
the cellular transcription factor USF has been reported to cooperate with other viral transactivators, such as the E2 proteins of human papillomavirus type 16 and bovine papillomavirus type 1 (121), and the
immediate-early protein 62 of varicella-zoster virus (93). USF has been
demonstrated to interact with the adenovirus E1A protein, which
stimulates transcription of adenovirus genes as well as a wide variety
of other viral and cellular genes (122). Moreover, a few studies show
that the Tax protein of HTLV-1 represses the expression of cellular
genes through the bHLH transcription factors. First, Uittenbogaard
et al. (123, 124) have demonstrated that members of the bHLH
protein family mediate repression by the HTLV-1 Tax protein, including
the transcriptional repression of the p53 gene. They show that this
repression of p53 by TaxHTLV-1 is dependent upon the p53
promoter E box element and is mediated by the class B bHLH proteins
c-Myc and USF (124). Second, TaxHTLV-1 has been shown to
mask c-Myc function through a c-AMP-dependent pathway
(125). The region in c-Myc perturbed by TaxHTLV-1 is contained within a highly conserved transformation/activation domain.
Their data provide evidence that the N-terminal portion of c-Myc is
conformationally altered by TaxHTLV-1, without additional effects on overall protein stability. A conformational disruption of
this protein could account for an abrogation of the transactivation and
transformation properties of Myc (125). Third, the biological behavior
of p56lck is modulated by the expression of the viral
regulatory tax gene in HTLV-1-infected T-cells through a
mechanism of repression that involves the E box DNA recognition
sequence encountered in the lck gene distal promoter (126).
Fourth, a molecular mechanism for TaxHTLV1-mediated
repression of the transcriptional activity of the bHLH myogenic MyoD
protein has been proposed recently (127). In this study, the authors
show that TaxHTLV-1 binding to the KIX domain of the
cellular coactivator p300 prevents MyoD from contacting this N-terminal
domain of p300, thus resulting in repression of
MyoD-dependent transcription (127). Regarding the results reported in the present study, it would be interesting to study the
possibility that TaxBLV may inhibit the
transcriptional activation function of bHLH proteins,
thereby interfering with cellular gene expression and/or BLV expression.
We have characterized a novel E box motif in the R region of the BLV
LTR. Functionally important E box motifs have been identified in the
regulatory regions of other viruses. Latency-associated promoter 1 (LAP1) of herpes simplex virus type 1 is required to generate a series
of latency-associated transcripts in sensory neurons of latently
infected animals, and a USF element and a CRE site contribute to LAP1
function during latency (128). The transcriptionally regulatory regions
of the lymphomagenic Akv and SL3-3 murine leukemia retroviruses
contain E box motifs that are important determinants for murine
leukemia retrovirus transcriptional activity in lymphocytic cell lines
(129). The murine sarcoma virus enhancer has six E box targets for MyoD
family proteins (130). Activation of the Epstein-Barr virus (EBV) DNA
polymerase promoter by the BRLF1 immediate-early viral transactivator
is mediated through USF and E2F (131). The latent membrane protein 1 (LMP1) gene promoter in the EBV genome contains a silencing activity overlapping with a transcriptional enhancer in a region that
contains an E box motif (71). The latter study shows that bHLH
transcription factors Max, Mad1, USF, E12, and E47 and the corepressor
mSin3A bind in vitro to this E box (71). The silencing and
enhancer activities correlate with the binding of Max-Mad1 and of USF,
respectively (71). Finally, four E boxes are present in the promoter
region of human immunodeficiency virus type 1 (HIV-1) (51, 52,
132-135), and cooperative interaction of USF1 with Ets-1 is required
for full transcriptional activity of the HIV-1 LTR in T-cells
(104).
In summary, we provided evidence that the BLV R region E-box4 is
required, and USFs are components for BLV promoter regulation by
correlating functional assays and USF binding activities to the E-box4.
Our findings contribute toward a clearer understanding of the
transcriptional regulation of BLV. Further studies will focus on
characterizing the physiological role of the R region E-box4 site in
the context of the complete infectious BLV provirus. A BLV provirus
mutated in this site will be constructed. This mutated provirus will be
injected into sheep in order to test for infectivity and ability to
induce pathogenesis. These in vivo experiments should
provide new insight into the molecular mechanisms of BLV
transcriptional activation.
We thank Séverine Nizet for excellent
technical assistance. We are grateful to Richard Kettmann, Luc Willems,
and members of the Van Lint laboratory for critical reading of the
manuscript. We thank Drs. Luc Willems and Richard Kettmann (Faculty of
Agronomy, Gembloux, Belgium), Dr. Axel Kahn (Institut Cochin de
Génétique Moléculaire, Paris, France), Dr. Shoshana
Segal (National Naval Medical Center, NCI, National Institutes of
Health, Bethesda), and Dr. Anthony Means (Duke University Medical
Center, Durham, NC) for reagents used in this study.
*
This work was supported by grants from the
FNRS-Télévie, Free University of Brussels (ARC),
Internationale Brachet Stiftung, CGRI-INSERM Cooperation, Région
Wallonne-Commission Européenne FEDER, and Theyskens-Mineur
Foundation (to C. V. L.).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.
§
Fellow of the FNRS-Télévie.
¶
Fellow of the Belgian "Fonds pour la Recherche dans
l'Industrie et l'Agriculture (FRIA)."
¶¶
"Chercheurs Qualifiés" of the "Fonds
National de la Recherche Scientifique" (Belgium).
Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.M107441200
2
C. Calomme and C. van Lint, unpublished results.
The abbreviations used are:
BLV, bovine leukemia
virus;
EMSA, electrophoretic mobility shift assay;
CRE, cAMP-responsive
element;
CREB, CRE-binding protein;
ATF-1 and ATF-2, activating
transcription factor-1 and -2;
USF, upstream stimulatory factor;
NF-
Upstream Stimulatory Factors Binding to an E Box Motif in the
R Region of the Bovine Leukemia Virus Long Terminal Repeat Stimulates
Viral Gene Expression*
§,¶,
**,
,, and¶¶
Université Libre de Bruxelles,
Institut de Biologie et de Médecine Moléculaires, Service
de Chimie Biologique, Laboratoire de Virologie Moléculaire, Rue
des Profs Jeener et Brachet 12, 6041 Gosselies, Belgium,
Université Libre de Bruxelles, Faculté de
Médecine, Laboratoire de Microbiologie, 808 Route de Lennik,
1070 Bruxelles, Belgium, ** Institut Pasteur de Lille,
Institut de Biologie de Lille, UMR 8526 CNRS, 1 Rue Calmette BP 447, 59021 Lille Cedex, France, and
§§ Université Libre de Bruxelles,
Laboratoire de Microbiologie, Institut de Recherche du CERIA, 1 Avenue
Emile Gryson, 1070 Bruxelles, Belgium
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B-related sites (16, 17). Among the most
important sites are three copies of an imperfectly conserved 21-bp
sequence harboring in the middle a common 8-bp core sequence known as
the cAMP-responsive element (CRE). At least three proteins, CRE-binding
protein (CREB) and activating transcription factors-1 and -2 (ATF-1 and
ATF-2), bind to these 21-bp enhancers in bovine B-lymphocytes, and the
amount of generated complex correlates with the level of viral
expression (18, 19). The 21-bp enhancers are also called Tax-responsive
elements (TxREs) because transactivation of the BLV LTR by the
virus-encoded transcriptional activator TaxBLV requires
these enhancers. Because there is no evidence for direct binding of
TaxBLV to DNA (20), it has been proposed that
TaxBLV activation of transcription could be mediated, as reported for the HTLV system, through increased binding of the cellular
proteins CREB, ATF-1, and ATF-2 (and possibly other factors yet to be
identified) to the TxREs. Each of the 21-bp enhancers also contains an
E box-homologous motif overlapping the CRE (Fig. 1). From 5' to 3',
these E boxes are referred to as E-box1, E-box2 and E-box3,
respectively (5, 21).
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Fig. 1.
Transcription factor-binding sites in the
5'-LTR of the BLV genome. The 5'-LTR contains the "CAAT" (nt
97/92) and "TATA" (nt 43/37) boxes upstream of the
transcription initiation site at the U3-R junction (mRNA start site
at +1 is indicated by an arrow). The TxREs are three
imperfectly repeated sequences of 21 bp. These are major
transcriptional enhancers, which interact with the cellular
transcription factors CREB, ATF-1, and ATF-2. Transcriptional
activation of the BLV LTR by the viral encoded TaxBLV
transactivator requires these enhancers. Moreover, the activity of the
factors CREB, ATF-1, and ATF-2 is increased by protein kinases A and
CaMKIV. Each of the 21-bp enhancers contains a sequence homologous to
the consensus E box-binding motif (referred to as E-box1,
E-box2, and E-box3) overlapping an imperfect CRE
(referred to as CRE1, CRE2, and CRE3). The U3
region also contains a glucocorticoid-responsive element
(GRE) and a large segment protected in DNase footprinting
assays (nt 118/70) containing NF-B-related sites. Outside of U3,
a DAS (nt +147/+210) in the R region and an IRF-binding site (nt
+246/+269) in the U5 region, interacting with IRF-1 and IRF-2, are
regulatory sequences important for BLV gene expression. The E box motif
identified in the present report and located in the R region is also
indicated and is referred to as E-box4.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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1, respectively. To construct the luciferase reporter plasmid
pLTRwt-luc, we used PCR to amplify the BLV promoter region from a 344 wild-type (wt) plasmid (a kind gift from Dr. Luc Willems).
SmaI sites were introduced into the 5' and 3' PCR primers,
and the SmaI-restricted PCR fragment was cloned in
pGL3-BASIC (Promega) digested with SmaI. The 5' primer oligonucleotide corresponding to nt 211 to 182 contained an added
SmaI site (in bold) at the 5' end
(5'-TCCCCCGGGTnt
211GTATGAAAGATCATGCCGGCCTAGGCGCC-3').
The 3' primer oligonucleotide corresponding to nt +291 to +320
contained an added SmaI site (in bold) at the
5'end
(5'-TCCCCCGGGTnt+320GTTTGCCGGTCTCTCCTGGCCGCTAGAGG-3').
Amplification reaction was conducted with 100 ng of template
plasmid DNA as specified in the protocol provided with the high
fidelity Pfu DNA polymerase (Stratagene, La Jolla, CA) with
a DNA thermal cycler 480 (PerkinElmer Life Sciences). The resulting
plasmid was designated pLTRwt-luc. This construct was used as a
substrate for mutagenesis by the QuickChange Site-directed Mutagenesis
method (Stratagene). Different mutations were generated with the
following pairs of mutagenic oligonucleotide primers (mutations are
highlighted in bold and E box or IRF motifs are underlined on the
coding strand primer): CV192/193 (E-box4-mutA),
5'-CCTCTGACCGTCTCCATATGGACTCTCTCTC-3'; CV262/265 (E-box4-mutB),
5'-ACCGTCTCCACGGAGACTCTCT-3'; CV311/312
(IRFmut),
5'-GTTTCCTGTCTTACAGTCTGTGTCTCGCGGCCCGCG-3'; CV194/195 (Eboxes1,2-mutA),
5'-GACAGAGACGTCATATGCCAGAAAAGCTGGTGACGGCATATGGTGGCTAGAATCC-3'; CV281/282 (Eboxes1,2mutB),
5'-GACAGAGACGTCAGCGACCAGAAAAGCTGGTGACGGCAGCGAGTGGCTAGAATCC-3'; CV196/197 (E-box3-mutA),
5'-GAGCTGCTGACCTCATATGCTGATAAAACAATAA-3'; and
CV283/284 (E-box3-mutB),
5'-GAGCTGCTGACCTCACCGACTGATAAAACAATAA-3.
118 to +83
was generated by PCR amplification of pLTRwt-luc. XbaI and
PstI restriction sites were introduced into the 5' and 3' PCR primers, respectively, and the
XbaI-PstI-restricted PCR fragment was cloned in
pGEM-3Zf(+) vector (Promega) digested with XbaI and
PstI. The 5' primer oligonucleotide corresponding to nt
118 to 87 contained an added XbaI site (in
bold) at the 5' end
(5'-CGCTCTAGAGnt-118GCTAGAATCCCCGTACCTCCCCAACTTCCCC-3').
The 3' primer oligonucleotide corresponding to nt +55 to +83 contained
an added PstI site (in bold) at the 5' end
(5'-GGTTTTCTGCAGCnt+83GGATAGCCGACCAGAAGGTCTCGGGAGC-3').
) vector (Invitrogen) digested with
HindIII and BamHI. The 5' primer oligonucleotide
corresponding to nt 203-226 (according to the ovine c-Myc cDNA
sequence, GenBankTM accession number Z68501)
contained an added HindIII site (in bold) at the 5' end
(5'-GCCCAAGCTTAnt203CCGCGATGCCCCTCAACGTCAGC-3').
The 3' primer oligonucleotide corresponding to nt 1571-1598 contained
an added BamHI site (in bold) at the 5' end
(5'-GCCGGATCCTnt1598CCTCACTTTCCCTTAGTAACAAATGAG-3').
The resulting plasmid was designated pcDNA3-cMyc.
70 °C. For supershift assays, polyclonal
antibodies against USF1 (sc-229), USF2 (sc-862), Max (sc-197), Mad-1
(sc-222), Mad-2 (sc-1720), Mad-3 (sc-933), Mad-4 (sc-771), c-Myc
(sc-764), and Mnt (sc-769) (Santa Cruz Biochemicals, Santa Cruz, CA)
were added at a final concentration of 2 µg/reaction to the binding
reaction mixture at the end of the binding reaction for an additional
30 min incubation at room temperature before electrophoresis.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B sites (Fig.
2A, lanes 10-13). A well characterized E box
motif from the HIV-1 promoter (51, 52) was also used as a competitor
and inhibited the formation of the major complex as efficiently as the
homologous oligonucleotide (Fig. 2A, lanes 6-9),
demonstrating that the major complex was specific to the E box motif.
In contrast, the specificity of the fainter and slower migrating band
was uncertain because this band was competed by all oligonucleotides
tested (the homologous and the heterologous oligonucleotides, Fig.
2A, and the two mutated E-box4 oligonucleotides, see below
Fig. 3B), except by the HIV-1 E box oligonucleotide (Fig. 2A, lanes 6-9).
Moreover, this fainter band was not affected by any of the E
box-specific antibodies we used in supershift assays (Fig.
2B, see below) and was not further characterized. Similar
results were obtained with nuclear extracts from the Raji cell line, a
human B-lymphoid cell line (data not shown).
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Fig. 2.
Characterization of the factors binding to
the E-box4 motif located in the BLV R region. A, EMSA
analysis of nuclear factors interacting specifically with the E-box4
motif. The E-box4-wt oligonucleotide
(5'-ACCGTCTCCACGTGGACTCTCT-3') was used as probe and
incubated with 4 µg of nuclear extracts from BLV-infected ovine PBMCs
in the absence of competitor (lane 1) or in the presence of
increasing concentrations (5-, 10-, -20, and 80-fold molar excess) of
the homologous E-box4-wt oligonucleotide (lanes 2-5), of
the HIV-1 E box oligonucleotide (lanes 6-9), or of the
heterologous HIV-1 NF- B oligonucleotide (lanes 10-13).
The sequence of the coding strand of the HIV-1 E box oligonucleotide is
5'-ATTTCATCACGTGGCCCGAG-3' (nt +281 to +300 according to
the numbering of the HIV-1 BRU genome where nt +1 is the start of U3 in
the 5'-LTR; the E box motif is underlined) (52). The HIV-1
NF-B oligonucleotide contains the two NF-B-binding sites of the
HIV-1 enhancer (nt +350 to +373) and has been described previously
(136). The major DNA-protein complex and the fainter band are indicated
by an arrow and an asterisk, respectively. The
free probe (FP) is also indicated. B, supershift
assays. Left panel, the E-box4-wt oligonucleotide probe was
incubated with 4 µg of nuclear extracts from BLV-infected ovine
PBMCs. Next, antibodies directed against different members of the bHLH
family of transcriptional regulatory proteins (lanes 2-11)
or purified rabbit IgG as a negative control (lane 1) were
added to the binding reaction. The polyclonal antibodies used are
indicated at the top of each lane. The major DNA-protein
complex and free probe are indicated by arrows. The fainter
band is indicated by an asterisk. The supershifted complexes
are also indicated. Right panel, the CMD (specific E box)
oligonucleotide probe was incubated with either in vitro
translated Max (lanes 5 and 6), in
vitro translated Mad1-Max complexes (lanes 7-9),
in vitro-translated c-Myc-Max complexes (lanes
10-12), or unprogrammed reticulocyte lysates (lanes
1-4) as a negative control. The anti-Max, anti-Mad1, and
anti-c-Myc polyclonal antibodies added to the binding reaction are
indicated at the top of each lane. The major DNA-protein
complexes are indicated by arrows. An E box-binding protein
constitutively present in the reticulocyte lysate is indicated by an
asterisk. The supershifted complexes are also indicated. The
free probe was run out of the gel for better separation of the
complexes.
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Fig. 3.
Mutagenesis of the E-box4 motif located in
the BLV R region. A, nucleotide sequence of the
E-box4-wt oligonucleotide. The underlined bases indicate the
E box motif, which is aligned with the E box consensus sequence. For
the E-box4-mutA and E-box4-mutB oligonucleotides, only the bases that
are changed compared with the wt sequence are indicated. B,
E-box4-wt and mutated oligonucleotides were tested in competition
EMSAs. The E-box4-wt oligonucleotide was 5'-end-labeled and used as
probe. This probe was incubated with 4 µg of nuclear extracts from
BLV-infected ovine PBMCs either in the absence of competitor
(lane 1) or in the presence of increasing concentrations
(5-, 10-, 20-, and 80-fold molar excess) of the homologous E-box4-wt
oligonucleotide (lanes 2-5), of the E-box4-mutA
oligonucleotide (lanes 6-9), or of the E-box4-mutB
oligonucleotide (lanes 10-13). The major DNA-protein
complex and the fainter band are indicated by an arrow and
an asterisk, respectively. The free probe (FP) is
also indicated.
211 to +320) of the 344 BLV provirus. Strain
344 is an infectious and pathogenic molecular clone. The two mutated
plasmids were designated pLTR(E-box4-mutA)-luc and pLTR(E-box4-mutB)-luc, respectively.
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Fig. 4.
Effects of the E-box4 mutations on BLV basal
gene expression. A, Raji cells were transiently
transfected with 500 ng of either pLTRwt-luc, pLTR(E-box4-mutA)-luc, or
pLTR(E-box4-mutB)-luc using the DEAE-dextran procedure. Luciferase
activity was measured in cell lysates 44 h after transfection. The
results are presented as histograms indicating luciferase activities
(arbitrary units) normalized to protein concentrations. Means and
standard errors of the means from 10 independent transfections
performed with at least two different DNA preparations are shown.
B, Daudi cells were transiently cotransfected by
electroporation with 8 µg of either pLTRwt-luc,
pLTR(E-box4-mutA)-luc, or pLTR(E-box4mutB)-luc and with 50 ng of the
internal control plasmid pRL-TK. Luciferase activities (firefly and
Renilla) were measured in cell lysates 44 h after
transfection. Results are expressed as
LuciferaseFirefly/LuciferaseRenilla. Means and
standard errors of the means from six independent transfections
performed with at least two different DNA preparations are shown.
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Fig. 5.
Response of the BLV promoter to ectopically
expressed USF transcription factors. HeLa cells were transiently
cotransfected with 500 ng of an LTR luciferase reporter construct wild
type or mutated in the different E boxes (as indicated) and increasing
amounts (0, 100, 250, 500, 1000, and 2000 ng) of either the USF1 or the
USF2a expression vector, pCR3-USF1 or pCR3-USF2a (A or
B, respectively). To maintain the same amount of transfected
DNA and to avoid squelching artifacts, the different amounts of USF
expression vector cotransfected were complemented to 2000 ng of DNA by
using the empty pCR3 vector. Luciferase activities were measured in
cell lysates 44 h after transfection and normalized to protein
concentrations. Results are presented as histograms indicating the
induction by USF1 or USF2a (in fold) with respect to the activity of
each LTR reporter construct in the absence of USF, which was assigned a
value of 1. Means and standard errors of the means are shown. A
representative experiment of four independent transfections is
shown.
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Fig. 6.
Ability of multimerized BLV E-box4 motifs to
confer USF stimulation to a TK minimal promoter. HeLa cells were
transiently cotransfected with 500 ng of a TK luciferase reporter
construct devoid or not of upstream wild-type or mutated E-box4 motifs
(as indicated) and increasing amounts (0, 100, 250, 500, 1000, and 2000 ng) of either the USF1 or the USF2a expression vector, pCR3-USF1 or
pCR3-USF2a (A or B, respectively). To maintain
the same amount of transfected DNA and to avoid squelching artifacts,
the different amounts of USF expression vector cotransfected were
complemented to 2000 ng of DNA by using the empty pCR3 vector.
Luciferase activities were measured in cell lysates 44 h after
transfection and normalized to protein concentrations. Results are
presented as histograms indicating the induction by USF1 or USF2a (in
fold) with respect to the activity of each TK reporter construct in the
absence of USF, which was assigned a value of 1. Means and standard
errors of the means are shown. A representative experiment of six
independent transfections is shown.
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Fig. 7.
Effects of the E-box4 mutations on
Tax-dependent BLV promoter-driven gene expression.
A, Raji cells were transiently cotransfected with 500 ng of
either pLTRwt-luc, pLTR(E-box4-mutA)-luc, or pLTR(E-box4-mutB)-luc and
increasing amounts of the TAXBLV expression vector
pSG-TAXBLV (0, 1, 2, 4, and 8 ng of plasmid DNA). To
maintain the same amount of transfected DNA and to avoid squelching
artifacts, the different amounts of pSG-TAXBLV
cotransfected were complemented to 8 ng of DNA by using the pSG5 empty
plasmid. Luciferase activity was measured in cell lysates 44 h
after transfection. The results are presented as histograms indicating
luciferase activities (arbitrary units) normalized to protein
concentrations (top panel) or indicating the induction by
TaxBLV (in fold) with respect to the activity of the same
reporter construct in the absence of TaxBLV, which was
assigned a value of 1 (bottom panel). Means and standard
errors of the means from six independent transfections performed with
at least two different DNA preparations are shown. B, Raji
cells were transiently cotransfected with 500 ng of either pLTRwt-luc,
pLTR(E-box4-mutA)-luc, or pLTR(E-box4-mutB)-luc and increasing amounts
of both pSG-CREB2 (0, 50, 150, 300, and 600 ng) and pCaMKIV (0, 50, 150, 300, and 600 ng). To maintain the same amount of transfected DNA
and to avoid squelching artifacts, the different amounts of
pSG-CREB2-pCaMKIV cotransfected were complemented to 1200 ng of DNA by
using the empty plasmids pSG5 and a pCMV-based vector, respectively.
Luciferase activity was measured in cell lysates 44 h after
transfection. The results are presented as histograms indicating
luciferase activities (arbitrary units) normalized to protein
concentrations (top panel) or indicating the induction by
CREB2/CaMKIV (in fold) with respect to the activity of the same
reporter construct in the absence of cotransfected expression vectors,
which was assigned a value of 1 (bottom panel). Means and
standard errors of the means from four independent transfections
performed with at least two different DNA preparations are shown.
118 to +83 and therefore hybridizes to all transcripts that
initiate at the BLV LTR to produce a protected species of 83 nt. The
distal probe, producing a 225-nt protected luciferase product, can only
detect transcripts that have extended into the luciferase gene and
therefore provides a measure of elongation. We failed to observe any
reporter transcripts in the absence of the transactivator
TaxBLV with both the BLV promoter-specific probe and the
luciferase gene-specific probe (data not shown), probably as a
consequence of the weak BLV promoter activity in absence of
TaxBLV and of the weak transfection efficiency of the DEAE-dextran procedure. Because TaxBLV is known to increase
considerably the BLV LTR transcriptional activity and because the
E-box4-mutA and E-box4-mutB mutations reduced both the basal and
TaxBLV-activated BLV-driven gene expression (see
top of Figs. 4 and 7A, respectively), we decided
to analyze the RNA synthesis in the presence of TaxBLV to
facilitate the evaluation of the luciferase gene expression directed by
the wild-type and mutated LTRs. To this end, we performed RNase
protection assays using RNAs extracted from Raji cells transiently cotransfected with either pLTRwt-luc, pLTR(E-box4-mutA)-luc, or pLTR(E-box4-mutB)-luc and 25 ng of the TaxBLV expression
vector, pSG-TAXBLV. As expected, the E-box4-mutA and
E-box4-mutB mutations reduced the luciferase activity by approximately
2-fold (Fig. 8A). In the same experiment, analysis of the
steady-state mRNA level showed that these mutations also reduced
transcript production, as detected with the proximal BLV
promoter-specific probe or with the distal luciferase gene-specific
probe (Fig. 8B, lanes 2 and 3). As an
internal control, RNase protection analysis of the same RNA samples
using an antisense probe corresponding to the GAPDH gene showed no
change in the level of mRNA.
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Fig. 8.
Effects of the E-box4 mutations at the
mRNA level. A, Raji cells were transiently
cotransfected with 750 ng of either pLTRwt-luc, pLTR(E-box4-mutA)-luc,
or pLTR(E-box4-mutB)-luc and 25 ng of the TaxBLV expression
vector, pSG-TAXBLV. Luciferase activity was measured in
cell lysates 44 h after transfection. The results are presented as
histograms indicating luciferase activities (arbitrary units)
normalized to protein concentrations. Means and standard errors of the
means are shown. B, total RNA samples were prepared from
Raji cells cotransfected with either pLTRwt-luc (lane 1),
pLTR(E-box4-mutA)-luc (lane 2), or pLTR(E-box4-mutB)-luc
(lane 3) and with 25 ng of the TaxBLV expression
vector, pSG-TAXBLV. The RNA samples were incubated either
with a BLV-specific antisense riboprobe corresponding to the LTR or
with a luciferase-specific riboprobe. The figure shows the 83-nt
LTRBLV protected band (top panel) and the 225-nt
luciferase protected band (middle panel). As control, the
same RNA samples were incubated with a specific probe corresponding to
the GAPDH gene (bottom panel).
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Fig. 9.
Combined mutation of both the E-box4 site and
the IRF site decreases basal BLV promoter activity to a greater degree
than the individual mutations. Raji cells were transiently
transfected with 500 ng of either pLTRwt-luc, the single mutants
pLTR(E-box4-mutA)-luc, pLTR(E-box4-mutB)-luc, and pLTR(IRFmut)-luc, or
the double mutants pLTR(E-box4-mutA/IRFmut)-luc and
pLTR(E-box4-mutB/IRFmut)-luc. Luciferase activity was measured in cell
lysates 44 h after transfection. The results are presented as
histograms indicating luciferase activities (arbitrary units)
normalized to protein concentrations. Means and standard errors of the
means from four independent transfections performed with at least two
different DNA preparations are shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
intron
enhancer that contains a critical E box; ectopic expression of USF was
shown to equally transactivate reporter plasmids carrying this sequence
with the E box either intact or mutated, indicating that activation did
not occur entirely through the E box element. Similarly, activation by
USF has been reported for a number of other promoters either lacking an
E box or containing a mutated E box (93, 97, 98). Our results are
consistent with these previous studies and suggest that USF may
interact directly and indirectly with non-E box cis-elements in the BLV promoter region. Alternatively, overexpression of USF may be
modulating the level or activity of other proteins or basal transcription factors that mediate the activity of the BLV promoter.
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: The Gladstone Institute of Virology and
Immunology, University of California, San Francisco, CA 94141.
To whom correspondence should be addressed:
Université Libre de Bruxelles, Institut de Biologie et de
Médecine Moléculaires, Service de Chimie Biologique,
Laboratoire de Virologie Moléculaire, Rue des Profs Jeener et
Brachet, 12, 6041 Gosselies, Belgium. Tel.: 32-2-650-9807; Fax:
32-2-650-9800; E-mail: cvlint@dbm.ulb.ac.be.
ABBREVIATIONS
B, nuclear factor B;
IRF, interferon regulatory factor;
nt, nucleotide;
LTR, long terminal repeat;
HTLV, human T-lymphotropic
virus;
HIV-1, human immunodeficiency virus, type 1;
TxRE, Tax-responsive element;
HLH, helix-loop-helix;
bHLH, basic HLH;
ZIP, leucine zipper;
PMA, phorbol myristate acetate;
DAS, downstream
activator sequence;
PBMCs, peripheral blood mononuclear cells;
EBV, Epstein-Barr virus;
CaMKIV, calcium/calmodulin-dependent
protein kinase IV;
TK, thymidine kinase;
wt, wild-type;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
luc, luciferase.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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