From the
The human T-cell leukemia virus type I (HTLV-I) is the causative
agent of an aggressive T-cell malignancy in humans. While the virus
appears to maintain a state of latency in most infected cells, high
level virion production is an essential step in the HTLV-I life cycle.
The virally-encoded Tax protein, a potent activator of gene expression,
is believed to control the switch from latency to replication. Tax
stimulation of HTLV-I transcription is mediated through cellular
activating transcription factor/cAMP response element binding proteins,
which bind the three 21-base pair (bp) repeat viral enhancer elements.
In this report, we show that viral latency may result from a highly
unstable interaction between CREB and the HTLV-I 21-bp repeats,
resulting in rapid dissociation of CREB from the viral promoter. In the
presence Tax, the dissociation rate of CREB from a 21-bp repeat element
is decreased. This stabilization is highly specific, requiring the
amino-terminal region of CREB and appropriate 21-bp repeat sequences.
We suggest that Tax stabilization of CREB binding to the viral promoter
leads to an increase in gene expression, possibly providing the switch
from latency to high level replication of the virus.
Human T-cell leukemia virus type 1 (HTLV-I)
A significant number of studies support a
role for the ATF/CREB proteins in the mediation of transcriptional
stimulation by Tax; however, the precise molecular mechanism of Tax
transactivation is incompletely understood. Several recent studies
provided evidence suggesting that Tax stimulates viral transcription
through enhancement in the DNA binding of cellular activator proteins,
including CREB and ATF-2, to the 21-bp
repeats
(22, 23, 24, 26, 29, 30) .
This Tax-dependent enhancement in DNA binding activity appears to
occur, in part, through an increase in bZIP protein dimerization (26,
30).
To further
characterize the mechanism of Tax transactivation, we used equilibrium
binding and dissociation kinetics to study the interaction of CREB and
ATF-2 with the 21-bp repeat sequences both in the presence and absence
of Tax. We demonstrate in this report that the three 21-bp repeats
represent lower affinity CRE sequences, consistent with a possible role
for these elements in maintaining viral latency. We further demonstrate
that Tax enhances the equilibrium binding affinity of both CREB and
ATF-2 for the 21-bp repeats and a consensus CRE; however, only the
21-bp repeats conferred Tax transactivation to a heterologous promoter
in vivo. Finally, we provide two independent lines of evidence
showing that Tax transactivation may result directly from Tax
stabilization of CREB, and not ATF-2, binding to the 21-bp repeats.
This Tax-dependent stabilization was dependent upon specific nucleotide
sequences located within the 21-bp repeat element and upon amino acids
in the amino-terminal region of CREB.
Together, these data support a
model in which viral latency results from the presence of off-consensus
CRE sequences in the viral 21-bp repeats. These lower affinity
sequences enable rapid dissociation of CREB from the HTLV-I promoter
and therefore fail to support transcriptional activation. In the
presence of Tax, however, the transition from viral latency to high
level expression is achieved via a highly specific interaction between
Tax, CREB, and the 21-bp repeat element. This interaction results in
significant stabilization of CREB binding to the 21-bp repeat sequences
followed by transcriptional activation of the HTLV-I genome.
For the initial experiment, we performed an
equilibrium binding assay to examine CREB binding affinity to a
consensus CRE sequence from the human chorionic gonadotropin gene (hCG)
promoter (Fig. 1A). In this experiment, the amount of
labeled CRE DNA was kept constant, and purified recombinant CREB
protein was varied over a 100-fold concentration range. The binding
reactions were allowed to reach equilibrium, and the protein-DNA
complexes were analyzed by EMSA. Several protein-DNA complexes were
observed. The major complex, observed at lower CREB concentrations,
represented a single CREB homodimer bound to the CRE probe; the slower
migrating complexes observed at high CREB concentrations likely
represented multimeric forms of CREB bound to the 80-bp probe. All
complexes were equally competed by a 100-fold excess of unlabeled
consensus CRE oligonucleotide, whereas an unrelated competitor DNA had
no effect, indicating that CREB bound specifically to the CRE (data not
shown). To determine the apparent binding affinity, the fraction of CRE
probe bound versus the total CREB concentration was plotted
(Fig. 1B). The concentration of protein required for
half-maximal DNA saturation
(K
The DNA binding activities of CREB and ATF-2 (and
other bZIP proteins) are dramatically enhanced in the presence of the
viral Tax protein (22-24, 26). To determine whether enhancement
of DNA binding activity reflects a change in binding affinity,
quantitative equilibrium binding studies were performed with ATF-2 and
the third 21-bp repeat probe in the presence of highly purified
recombinant Tax protein (Fig. 2A). Analysis of a plot of
the binding data obtained in the presence and absence of Tax showed a
large difference in both the concentration range and affinity of ATF-2
binding (Fig. 2B). In the presence of Tax, the apparent
affinity of ATF-2 for the 21-bp repeat was increased, as the
concentration required to bind half of the DNA changed from 12
nM in the absence of Tax to approximately 2.5 nM in
the presence of Tax (Fig. 2B). In addition, ATF-2 bound
over an approximately 10-fold greater range of protein concentration in
the presence of Tax (Fig. 2, A and B).
reports the affinities of CREB and ATF-2 for the third
21-bp repeat and the consensus CRE in the presence of Tax; in each
case, Tax increased the equilibrium binding affinities between 5- and
10-fold.
The three Tax-responsive 21-bp repeats serve as binding sites
for members of the ATF/CREB family of transcription factors, including
CREB and ATF-2. Since Tax does not bind the 21-bp repeats directly, it
is generally believed that members of the ATF/CREB family mediate Tax
transactivation. However, the precise mechanism by which Tax
transactivates through these proteins is not understood. In this
report, we examined the kinetic and equilibrium binding behavior of
CREB and ATF-2 for various DNA sequences in the presence and absence of
Tax. We then compared the in vitro binding data with in
vivo Tax transactivation to correlate the functional significance
of the data.
We demonstrated that, in the absence of Tax, the three
21-bp repeat elements located in the transcriptional control region of
HTLV-I represent binding sites with modest affinity for CREB and ATF-2.
There was a strong correlation between the in vitro binding
affinities of the sites and their ability to confer transcriptional
activation to a heterologous promoter in vivo. The lower
affinity 21-bp repeats conferred essentially no increase in
transcriptional activation, whereas the higher affinity consensus CRE
increased the level of CAT activity approximately 10-fold. We conclude
that the weaker 21-bp repeat elements may be essential to maintain
viral latency, as high affinity CRE recognition elements would likely
promote elevated levels of viral gene expression and prohibit the virus
from escaping immune detection.
In the presence of Tax, both CREB
and ATF-2 were transformed into higher affinity binding forms in
vitro, with each protein demonstrating an approximately 10-fold
higher affinity for the 21-bp repeats and a consensus CRE. It is
interesting to note that the affinity of the third 21-bp repeat in the
presence of Tax approached the affinity of the consensus CRE observed
in the absence of Tax, suggesting that Tax converts the relatively weak
21-bp repeats into higher affinity binding sites in vivo. This
Tax-dependent conversion of the 21-bp repeats into high affinity
recognition elements may play a role in the transition from latency to
transactivation.
Although the equilibrium binding studies indicated
comparable Tax-dependent increases in CREB and ATF-2 binding affinity
for both 21-bp repeats and the consensus CRE, we observed Tax
transactivation only through the 21-bp repeat element. These data
essentially exclude the possibility that Tax transactivation occurs
through direct interaction of Tax with the bound ATF/CREB proteins at
the promoter, as the high affinity consensus CRE should bind more CREB
and thus tether more Tax, resulting in higher levels of transcriptional
activation. These data also suggest that enhancement in DNA binding
activity only partially explains Tax transactivation, as we observed a
significant increase in the affinity of both CREB and ATF-2 for the
consensus CRE sequence, yet the consensus CRE was ineffective in
mediating Tax transactivation. One possible explanation for these data
is that the Tax-dependent increase in ATF/CREB binding affinity for the
consensus CRE sequence does not result in enhanced transcription, as
the high affinity binding sites are fully occupied in the absence of
Tax. The lower affinity 21-bp repeats, however, are unoccupied in
vivo in the absence of Tax, and the Tax-dependent increase in
ATF/CREB binding affinity results in saturation of these sites and
subsequent transcriptional activation from 21-bp repeat containing
promoters.
Although Tax enhanced the binding of both CREB and ATF-2
to the consensus CRE and 21-bp repeat elements, dissociation kinetic
studies revealed a highly specific Tax stabilization of CREB binding to
a 21-bp repeat element. We did not observe Tax stabilization of ATF-2
binding to any of the probes tested. Surprisingly, the 21-bp repeat
sequences responsible for this Tax-dependent stabilization of CREB
binding were not localized to the CRE-like core octanucleotide but
rather to the nucleotide sequences located directly adjacent to the
core. We observed remarkable concordance between our observations
showing the importance of the sequences immediately adjacent to the
octanucleotide core for CREB binding stabilization in vitro and the importance of these sequences in conferring Tax
transactivation in vivo. Our results are consistent with
several other studies demonstrating a functional significance for these
21-bp repeat sequences in Tax transactivation in
vivo(28, 39, 40, 41) . The
correlation between the in vivo significance of the flanking
sequences for Tax transactivation together with our observation of the
importance of these sequences in stabilizing CREB binding in the
presence of Tax in vitro provide strong evidence for a
molecular mechanism of Tax transactivation. It is interesting that the
CRE core sequences, whether consensus or off consensus, were irrelevant
in both Tax-dependent stabilization of CREB binding in vitro and Tax transactivation in vivo. From these data, we
conclude that the core CRE-like sequences are necessary, but not
sufficient, for Tax transactivation. In this manner, the virus has
evolved the most efficient use of the LTR promoter by using low
affinity CRE sequences to maintain viral latency while at the same time
using the sequences flanking the CRE to mediate Tax transactivation.
The effect of Tax on the dissociation kinetics of CREB from the
21-bp repeat strongly suggests that Tax directly interacts with the
CREB
Although we provide strong
evidence supporting a direct interaction between Tax and the
CREB
It is of interest that Tax
enhances the DNA binding activity of a wide range of transcriptional
activator proteins to a wide range of DNA sequences, yet the change in
dissociation rate kinetics was very specific for CREB and the 21-bp
repeats. We (data not shown) and others (26) have demonstrated that the
pleiotropic enhancement in DNA binding activity results from an
increase in the association rate of bZIP protein binding to DNA. The
increase in association rate reflects a decrease in binding energy
resulting from an interaction between Tax and the basic-spacer region
of the bZIP proteins.
In summary, we provide both DNA binding and functional
evidence to indicate that the interaction between the ATF/CREB proteins
and the HTLV-I 21-bp repeats is highly unstable, resulting in rapid
dissociation of the transcription factors from the promoter DNA. This
unstable protein-DNA interaction does not support efficient
transcriptional initiation and, therefore, likely contributes to viral
latency. The HTLV-I Tax protein enters into a transient or metastable
complex with CREB and the 21-bp repeat elements. This complex is highly
specific, requiring amino acids in the amino-terminal region of CREB
and appropriate nucleotide sequences flanking the CRE octanucleotide
core sequence. The formation of this complex results in the
stabilization of CREB binding to a 21-bp repeat, thus slowing
dissociation of CREB from the viral promoter. We suggest that this
interaction provides the switch from viral latency to Tax
transactivation, with subsequent high level replication of the virus
in vivo.
The mismatches
in the 21-bp repeats, relative to the consensus sequence, are
underlined. The third 21-bp repeat is the most promoter proximal.
The CRE and CRE-like
octanucleotide core sequences are shown in boldface.
We thank members of the department, especially Drs.
Robert and A. Young Woody, for generous advice and support.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is the retrovirus found to be the causative agent of adult
T-cell leukemia, an aggressive and fatal malignancy of T-helper cells
(for review, see Ref. 1). It is estimated that less than 4% of
HTLV-I-infected individuals develop adult T-cell leukemia, with disease
onset occurring several decades after infection (2-4). The virus
is believed to establish and maintain a state of latency in the
infected T-cell, with very low levels of viral gene
expression
(5, 6) . A critical step in the life cycle of
the virus is the transition from the latent infection to high levels of
viral gene expression, resulting in virion production. The key viral
regulatory protein, which may control this switch, is the HTLV-I
oncoprotein Tax. Tax stimulates transcription of HTLV-I through
interaction with host cell transcription factors rather than through
direct binding to the viral promoter
DNA
(7, 8, 9, 10) . These cellular
transcription factors mediate Tax transcriptional activation through
three 21-bp repeat enhancer elements located in the transcriptional
control region of the
virus
(11, 12, 13, 14) . Each 21-bp
repeat contains a core nucleotide sequence with similarity to the
palindromic cAMP response element (CRE). The CRE's are
recognition sites for members of the ATF/CREB family of basic leucine
zipper (bZIP) transcription factors
(15) . The ATF/CREB proteins
have been widely studied as mediators of both viral gene expression and
Tax
transactivation
(16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28) .
We have previously shown that two members of this family, CREB and
ATF-2, are the principal T-cell proteins that directly bind the HTLV-I
21-bp repeats, and activate transcription in
vitro(24) .
(
)
Other studies provide evidence for a
distinct mechanism of Tax transactivation in which Tax stimulates
transcription through direct tethering to the HTLV-I 21-bp repeats. In
this model, Tax is anchored to the promoter via protein-protein
interactions with the bound ATF/CREB proteins. This hypothesis is
supported primarily by data demonstrating transcriptional activation
properties intrinsic to the Tax
protein
(32, 33, 34, 35) .
Recombinant Plasmids, Cell Culture, and
Transfections
The reporter plasmid pminCAT (formerly called
pGLCAT, see Ref. 36) was used as the parent vector for cloning
double-stranded oligonucleotides representing the CRE and CRE-like
elements given in the text (see I). Three copies of each
element were cloned into pminCAT immediately upstream of the
herpesvirus thymidine kinase promoter TATA box, at position -35
relative to the start site of transcription. The final constructs were
confirmed by DNA sequencing. The reporter plasmids were tested in the
presence and absence of the Tax expression plasmid, HTLV-I
Tax
(14) . Transient co-transfection assays were performed in
CV-1 cells, grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mML-glutamine, and antibiotics, with 12 µg of DNA total.
CV-1 cells were used in the transfection assay, as they have been
widely used to demonstrate Tax transactivation. The percent of
acetylated C-labeled chloramphenicol was determined by
PhosphorImager analysis.
Expression and Purification of CREB, ATF-2, and
Tax
CREB (amino acids 1-327), truncated CREB (amino acids
254-327), and ATF-2 (amino acids 1-505) were expressed in
Escherichia coli and purified by the methods described
previously
(37, 24) followed by heparin agarose
chromatography. All proteins were purified to greater than 95%
homogeneity. We have previously demonstrated sequence-specific DNA
binding of each protein
(24) . Tax was expressed in E. coli from the pTaxH expression vector (20) and purified to
apparent homogeneity by Ni
chelate chromatography
followed by gel filtration (see Ref. 23). The Tax concentration ranged
between 40 and 80 ng/µl. Mock Tax was prepared from E. coli cells transformed with a control plasmid not encoding the Tax
gene. This mock preparation of Tax was expressed, purified, and
analyzed exactly as described for wild-type Tax (see Ref. 24).
Electrophoretic Mobility Shift Assays
(EMSA)
Protein-DNA interactions were studied by incubation of
the purified proteins with the appropriate P-end labeled
DNA probes. The binding reactions contained the DNA probe, 12.5
mM HEPES, pH 7.9, 6.25 mM MgCl
, 5
µM ZnS0
, 75 mM KCl, 2 mM
-mercaptoethanol, 10% (v/v) glycerol, 0.05% (v/v) Nonidet P-40,
and the indicated concentrations of purified CREB, truncated CREB, or
ATF-2, and/or Tax in a 20-µl sample. Reactions were incubated at
room temperature and analyzed on 5.2% nondenaturing polyacrylamide gels
(49:1, acrylamide/N,N-methylenebisacrylamide). The
electrophoresis buffer contained 0.025 M Tris, 0.189
M glycine, pH 8.5, and 0.1% Nonidet P-40. Gels were dried,
autoradiographed, and quantitated by PhosphorImager analysis.
Equilibrium and Kinetic Binding Studies
For the
equilibrium binding studies, we ensured that the concentration of free
protein approximated the total protein concentration by keeping the
amount of labeled DNA probe constant at a level below 0.05 nM.
Time course studies, performed for both CREB and ATF-2, were used to
establish when DNA binding reactions reached equilibrium (data not
shown). Both bound and free probe were quantitated to determine the
percent of DNA complexed with protein. The concentration of active
protein was determined by the method of DNA titration
(38) and
adapted for the electrophoretic mobility shift assay. The apparent
K values were determined as described
previously
(38) . Computer analysis of the data was performed
with the KaleidaGraph software program. Comparison of equilibrium
binding curves produced by parallel EMSA and DNase I footprinting
assays showed essentially identical results (data not shown). Inclusion
of 100-fold excess of nonspecific competitor DNA or 1 µg of BSA in
the binding reactions had no effect on the binding curve in the
presence or absence of Tax (data not shown). For the dissociation
kinetic studies, the third 21-bp repeat or consensus CRE probes were
incubated with a concentration of CREB to produce approximately 50% of
the probe bound in complex. The binding reactions were allowed to reach
equilibrium and then challenged with a 1000-fold molar excess of
unlabeled homologous binding site. The samples were loaded onto a
running gel at appropriate times following challenge and resolved by
EMSA, and the percent bound was determined as described above.
DNA Probes
Complimentary double-stranded
oligonucleotides were cloned into the BamHI site in the
polylinker of pUC19, and the 80-bp EcoRI-HindIII
fragments were purified and
P-5`-end labeled for use as
probes in the EMSA reactions. These larger cloned fragments were more
stable than synthetic oligonucleotide probes, enabling accurate
quantitation. DNA fragment concentrations were determined by UV
absorbance at 260 nm. The nucleotide sequence of the binding sites used
in this study are reported (see I).
The Determination of CREB and ATF-2 DNA Binding
Affinities in the Absence and Presence of Tax
We have previously
identified CREB and ATF-2 as the principal T-cell proteins that bind
the three 21-bp repeats in the HTLV-I promoter and stimulate HTLV-I
transcription in a cell-free extract
(24) . The CRE-like
sequences present within each 21-bp repeat share similarity, but not
identity, with the consensus palindromic CRE sequence, TGACGTCA.
Because the initiation of transcription depends upon the stable binding
of transcription factors to specific DNA elements, we were interested
in determining whether the binding affinities of CREB and ATF-2 for the
HTLV-I 21-bp repeat elements were significantly lower than for a
consensus CRE.
) was used to determine
the apparent affinity of CREB for the CRE. The affinities are reported
as K
as the shape of the
curve suggests multistep binding. Analysis of the midpoint of binding
indicated an apparent K
of CREB binding
to the hCG consensus CRE of approximately 0.6 nM.
Figure 1:
Equilibrium binding of
CREB to a consensus CRE sequence. A, protein titration
experiment analyzed by EMSA. Equilibrium binding reactions contained a
constant amount of CRE probe and the indicated concentration of CREB
monomer. The positions of protein-DNA complex and free probe are
indicated. B, a graph showing the fraction of CRE probe bound,
from EMSA shown in A, plotted as a function of CREB
concentration. The dashedline is the best fit to the
data.
Equilibrium binding titrations were performed as above to determine
the relative affinity of both ATF-2 and CREB for the three 21-bp
repeats. The result of these studies are summarized in .
For comparison, the affinity for the consensus CRE is also reported. As
indicated in , the CRE-like sequences present in the 21-bp
repeats of HTLV-I each contain one or more base changes from the
consensus CRE sequence. These base changes likely contribute to the
approximately 10-fold lower affinity observed for each of the three
21-bp repeats, relative to a consensus CRE site. Interestingly, both
CREB and ATF-2 demonstrated parallel reduced affinity for the three
21-bp repeats.
Figure 2:
Equilibrium binding of ATF-2 to the third
21-bp repeat in the presence and absence of Tax. A, protein
titration analyzed by EMSA. Equilibrium binding reactions contained a
constant amount of the third 21-bp repeat probe and the indicated
amount of ATF-2 monomer. 100 nM Tax was added to the indicated
reactions. The positions of protein-DNA complex and free probe are
indicated. B, a graph showing the fraction of the third 21-bp
repeat probe bound, from EMSA shown in A, plotted as a
function of ATF-2 concentration. ATF-2 binding in the absence of Tax
(diamonds) and in the presence of Tax (triangles) is
shown.
To characterize the concentration dependence of Tax on bZIP
protein binding, we measured the affinity of CREB to a 21-bp repeat in
the presence of incremental changes in Tax protein concentration.
Increasing concentrations of Tax produced a corresponding incremental
decrease in apparent K of CREB for the
third 21-bp repeat element (Fig. 3). Together, these data
indicate that the mechanism of Tax enhancement of CREB and ATF-2 DNA
binding involves a Tax concentration-dependent increase in the affinity
of the bZIP proteins for their CRE and CRE-like recognition elements.
Figure 3:
The
change in affinity of CREB for a 21-bp repeat is directly proportional
to the concentration of Tax. Equilibrium binding reactions, containing
a constant amount of the third 21-bp repeat probe, varying amounts of
Tax, and the indicated amount of CREB monomer, were assayed by EMSA,
and the results were plotted as described in Fig. 1B. CREB
binding in the absence of Tax (diamonds) and in the presence
of 200 ng (circles) and 400 ng (triangles) of Tax is
shown.
Correlation between DNA Binding Affinities and
Transcriptional Activation in the Absence and Presence of
Tax
Since enhancement in DNA binding activity may represent a
mechanism of Tax transactivation, we were interested in determining
whether a correlation existed between the in vitro binding
affinities, obtained in both the presence and absence of Tax, and basal
and Tax-induced transcriptional activation in vivo. To test
this, we constructed heterologous promoters containing three copies of
either the third 21-bp repeat or the consensus CRE, cloned immediately
upstream of the herpesvirus thymidine kinase minimal promoter linked to
the CAT gene. We focused our study on the third, most
promoter-proximal, 21-bp repeat, as comparable binding affinities were
obtained for all three 21-bp repeat elements. The chimeric constructs
were transfected into CV-1 cells in the presence and absence of a Tax
expression vector. The HTLV-I promoter, LTR-CAT, and the parent
construct, pminCAT, were tested in parallel as positive and negative
controls, respectively. The results of the CAT assay are presented in
Fig. 4
. In the absence of Tax, the consensus CRE-containing
construct produced an approximately 10-fold higher CAT activity
relative to the third 21-bp repeat construct (compare lanes9 and 10 with lanes13 and
14). The third 21-bp repeat construct conferred no increase in
CAT activity over the parent vector (compare lanes5 and 6 with lanes 13and 14).
This difference between the 21-bp repeat and the consensus CRE
constructs correlated nicely with the approximately 10-fold difference
in CREB and ATF-2 binding affinities observed between these two sites
in the in vitro binding assays. This strong correlation
between the in vitro binding data and in vivo functional data suggest that CREB and/or ATF-2 play a role in
basal expression of the virus in the cell. In the presence of Tax,
however, we observed over a 40-fold increase in CAT activity with the
third 21-bp repeat construct (Fig. 4, lanes13-16), with no effect of Tax on the consensus
CRE-CAT construct (Fig. 4, lanes9-12).
Tax transactivation mediated through the 21-bp repeat was dependent
upon wild-type Tax, as transfection of a mutant Tax construct had no
effect (data not shown). These in vivo results, obtained in
the presence of Tax, contrast with our equilibrium binding data, as we
observed similar Tax-dependent increases in DNA binding activity in
vitro with both the third 21-bp repeats and the consensus CRE.
Figure 4:
Comparison of Tax transactivation mediated
through the third 21-bp repeat and consensus CRE. Transient
co-transfection assays were performed in CV-1 cells with 10 µg of
the indicated CAT reporter plasmids in the absence or presence of 2
µg of the HTLV-I-Tax expression plasmid (14) as indicated
(lanes5-16). As a positive control, pU3RCAT (2
µg), the HTLV-I LTR CAT reporter plasmid, was assayed in the
presence and absence of Tax (lanes1-4). The
percent conversion to acetylated
[C]chloramphenicol together with the -fold
activation in the presence of Tax are shown in the figure. The -fold
activation is based on the average of the duplicates. Although the
figure represents the results of a single experiment, similar Tax
activation was observed in four independent experiments. A schematic
representation of the CAT reporter plasmid (pminCAT), carrying
three copies of the third 21-bp repeat (pminCAT-21-bp repeat)
or consensus CRE (pminCAT-CRE) cloned upstream of the
thymidine kinase proximal promoter is shown.
Dissociation Kinetics Reveal a Highly Specific Tax
Stabilization of CREB Binding to a 21-bp Repeat
To investigate
the molecular basis for the dramatic difference between Tax
transactivation mediated by the third 21-bp repeat and the consensus
CRE, we analyzed the dissociation kinetics of CREB binding to these
sites in vitro. Probes representing either the third 21-bp
repeat or the consensus CRE were incubated with CREB in the presence or
absence of Tax. Binding reactions were then challenged with a large
excess of unlabeled DNA binding site, and the kinetics of dissociation
were determined by quantitative analysis of the EMSA. A plot of CREB
dissociation from each DNA sequence is shown in Fig. 5. In the
absence of Tax, the dissociation of CREB from the third 21-bp repeat
was very rapid (t 30 s), whereas CREB binding to the
consensus CRE was significantly more stable (t = 17
min). The presence of Tax in the reaction significantly stabilized the
binding of CREB to the third 21-bp repeat, whereas Tax had little or no
effect on CREB binding to the consensus CRE. Surprisingly, we did not
observe Tax stabilization of the binding of ATF-2, or a truncated form
of CREB, containing principally the DNA binding and dimerization domain
of the protein on either the consensus CRE or the 21-bp repeat sequence
(data not shown). These data indicate that the effect of Tax on
dissociation kinetics is specific for full-length CREB binding to a
21-bp repeat. Interestingly, the Tax-dependent stabilization of CREB on
the 21-bp repeat, and not the consensus CRE, correlates precisely with
the in vivo Tax transactivation results presented above. Tax
has also been shown to stimulate the rate of bZIP protein association
for DNA
(26) . Together, these data suggest that the effect of
Tax on the dissociation rate, and not the association rate, contributes
to Tax transactivation of HTLV-I in vivo.
Figure 5:
Kinetics of CREB dissociation in the
presence and absence of Tax. Tax prevents dissociation of CREB from a
21-bp repeat but not to the consensus CRE. Purified, recombinant CREB
was incubated with either the consensus CRE or the third 21-bp repeat
probe in the absence or presence of purified Tax. Equilibrium binding
reactions were then challenged with a 1000-fold molar excess of the
unlabeled binding site, and the kinetics of dissociation were analyzed
by EMSA (see ``Experimental Procedures''). CREB dissociation
was quantitated, and the concentration of CREB, remaining bound
relative to the concentration bound at time zero (no added competitor)
(B/B), was plotted as a function of time
following challenge. CREB dissociation from either the third 21-bp
repeat probe in the absence (closedtriangles) and
presence (closedcircles) of Tax or the consensus CRE
probe in the absence (opentriangles) and presence
(opencircles) of Tax is
shown.
The strong
correlation showing a dependence upon the 21-bp repeat element for both
Tax stabilization of CREB binding and Tax transactivation in vivo prompted us to test whether the critical nucleotide sequences lie
within the 21-bp CRE-like octanucleotide core or within the DNA
sequence adjacent to the core. To test this hypothesis, we prepared the
two hybrid binding sites shown in I. The first contains
the off-consensus CRE-like core sequence (TGACGACA) from the
third 21-bp repeat, with adjacent flanking sequences derived from the
hCG consensus CRE site (site 3 core/CRE). The second contains the
consensus CRE core sequence (TGACGTCA), with adjacent flanking
sequences derived from the third 21-bp repeat (CRE core/site 3). We
measured the dissociation rate of CREB binding to these hybrid
sequences in the presence and absence of Tax (Fig. 6, A and B). Examination of the kinetic data indicates that
the core CRE octanucleotide, whether consensus or off-consensus, had no
effect on Tax-dependent stabilization. The sequences that directly
flank the core, however, had a dramatic effect on CREB binding
stability in the presence of Tax.
Figure 6:
Tax
stabilization of CREB binding is dependent upon 21-bp sequences
adjacent to the CRE octanucleotide core. Plot of the dissociation
kinetic data was performed and analyzed exactly as described for Fig.
5, except CREB was assayed in the absence (closedcircles) and presence (opentriangles)
of Tax with the following probes: A, the off-consensus
CRE-like core sequence from the third 21-bp repeat, with adjacent
flanking sequences derived from the hCG consensus CRE site (site 3
core/CRE) (see Table III for sequence); B, the consensus CRE
core sequence, with adjacent flanking sequences derived from the third
21-bp repeat (CRE core/site 3) (see Table III for
sequence).
Correlation between Tax-dependent Changes in Dissociation
Kinetics and Tax Transactivation in Vivo
Our observation that
the DNA sequences immediately adjacent to the core octanucleotide
promoted specific stabilization of CREB binding prompted us to test
whether these same DNA sequences may confer increased Tax
transactivation in vivo. To test this hypothesis, we
constructed heterologous promoters containing three copies of the two
hybrid binding sites (I). The binding sites were cloned
into pminCAT as described above. The chimeric constructs were
transfected into CV-1 cells in the presence and absence of a Tax
expression vector. The results of the CAT assay are presented in
Fig. 7
. In surprising agreement with the in vitro dissociation rate data, we found that the construct carrying the
consensus CRE core and the 21-bp repeat flanking sequence (CRE
core/site 3) conferred Tax transactivation, whereas the construct
carrying the 21-bp repeat core and CRE flanking sequence was
unresponsive (Fig. 7). These data confirm that the sequences that
immediately flank the 21-bp repeat CRE-like core were critical in
conferring Tax transactivation. This CAT assay data correlates
precisely with the dissociation kinetic data presented above and
provides strong support for a model of Tax transactivation occurring
through the specific stabilization of CREB binding a 21-bp repeat.
Figure 7:
Identification of 21-bp repeat nucleotide
sequence elements required for Tax transactivation. Transient
co-transfection assays were performed in CV-1 cells using 10 µg
each of the pminCAT reporter plasmids with three copies of the
indicated cloned hybrid binding sites (see Table III for sequence).
Transfections were performed in the absence or presence of 2 µg of
the HTLV-I-Tax expression plasmid, as indicated. The percent conversion
to acetylated [C]chloramphenicol together with
the -fold activation in the presence of Tax are shown in the figure.
The -fold activation is based on the average of the duplicates. This
experiment is a continuation of the data shown in Fig. 4 (see Fig. 4
for controls).
Alterations in the Mobility of EMSA Complexes Further
Support a Role for CREB and a 21-bp Repeat in Mediating Tax
Transactivation
Our demonstration that Tax stabilized
full-length CREB binding in a DNA sequence-dependent manner prompted us
to directly compare CREBDNA complexes formed with each probe by
EMSA. Labeled probes representing the third 21-bp repeat, a consensus
CRE, and the two hybrid sites (see I) were compared in
binding assays with CREB in the presence or absence of Tax
(Fig. 8A). In all cases, Tax enhanced the DNA binding
activity of CREB to the labeled probes as expected. However,
examination of the CREB
DNA interaction in the presence of Tax
revealed differences in complex mobility in a probe-dependent manner.
Tax significantly reduced the mobility of the CREB
DNA complex
obtained with the third 21-bp repeat probe, but had only a modest
effect on the mobility of the complex formed with the consensus CRE
(Fig. 8A, lanes1-8). In
addition, comparison of the two hybrid sites shows that flanking
sequences derived from the third 21-bp repeat were responsible for this
reduced mobility complex (Fig. 8A, lanes9-16). A mock preparation of Tax, which was
expressed, purified, and analyzed in parallel with wild-type Tax, did
not produce enhancement in CREB DNA binding activity or alterations in
the mobility of the CREB
DNA complex (Fig. 8B). We
also did not observe Tax-dependent changes in the EMSA migration of the
ATF-2-DNA complex (data not shown).
Figure 8:
A, Tax alters the mobility of
CREBDNA complexes in a sequence-dependent manner. 10 ng of
purified, recombinant CREB was incubated with probes representing
sequences from the consensus CRE (lanes1-4)
and the third 21-bp repeat (lanes5-8). The
hybrid binding sites (see Table III) representing the consensus CRE
core/third 21-bp repeat flank (CRE core/site 3) (lanes9-12), and the third 21-bp repeat CRE-like
core/consensus CRE flank (site 3 core/CRE) (lanes13-16) were tested in parallel. Binding reactions
were performed in the absence or presence of the indicated volume of
purified Tax and analyzed by EMSA. The position of the free probe is
indicated. B, mock Tax, expressed and purified exactly as
wild-type Tax from E. coli, does not alter the mobility of the
CREB
DNA complex. Reaction mixtures contained the third 21-bp
repeat probe, 10 ng of purified recombinant CREB, and the indicated
amount of mock Tax or authentic Tax, as indicated. Binding reactions
were analyzed by EMSA. The position of the free probe is
indicated.
To delineate specific regions of
CREB that might contribute to complex formation, we also tested the
truncated form of CREB containing the DNA binding/dimerization domain
(carboxyl-terminal 73 amino acids; positions 254-327).
Fig. 9
shows that under conditions where full-length CREB binding
to the third 21-bp repeat in the presence of Tax produced a complex
with reduced mobility, truncated CREB was unaffected (compare lanes3-5 with lanes 6-8). The migration
of both full-length and truncated CREB were unaffected by Tax on the
consensus CRE probe (Fig. 9, lanes9-16).
This observation was further supported by the absence of an effect of
Tax on the dissociation rate of truncated CREB from the third 21-bp
repeat probe (data not shown). Furthermore, the gel shift migration of
complexes formed with the labeled 21-bp repeat probe and additional
bZIP proteins (including ATF-1, truncated, and full-length forms of
ATF-2, and Jun) were unaffected by Tax (data not shown). These data
indicate that the association between Tax and the CREBDNA complex
occurs only with appropriate DNA sequences and full-length CREB
protein. Furthermore, the data implicate amino acids amino-terminal to
the DNA binding-dimerization domain in the interaction.
Figure 9:
Amino acids amino-terminal to the bZIP
region of CREB are required to support interaction with Tax. 2 ng of
either full-length CREB (amino acids 1-327) or truncated CREB
(CREB BR) (amino acids 254-327) were incubated in the presence of
the indicated amount of Tax protein. Identical reactions were performed
with the third 21-bp repeat (lanes1-8) or
consensus CRE probes (lanes9-16), as
indicated. The protein-DNA complexes were analyzed by EMSA. The
position of free probe is indicated.
To further
examine the Tax-dependent alteration in complex mobility, we performed
a Tax titration experiment. We reasoned that if the reduced mobility
complex represented a stable stoichiometric association of Tax with
CREB and DNA, as has been reported previously (27), a single
``supershifted'' gel shift complex of a discrete size should
be observed. Furthermore, at lower Tax concentrations, two bands should
appear; one representing the ternary complex and the second
representing the CREBDNA complex without Tax. Fig. 10shows
the result of this titration experiment. Surprisingly, the inclusion of
increasing amounts of Tax in the gel shift reaction produced an
incremental change in the mobility of the CREB
DNA complex, with
progression to a slower migrating, supershifted complex at higher Tax
concentrations. These data support a weak or transient interaction
between Tax and the CREB
DNA complex, resulting in a
dose-dependent reduction in complex mobility.
Figure 10:
The degree of supershift is dependent
upon the Tax concentration in the reaction. 2 ng of CREB were incubated
in the presence of the indicated amount of Tax protein. Binding
reactions were performed with the third 21-bp repeat probe, and the
protein-DNA complexes were analyzed by EMSA. The positions of the
protein-DNA complex (arrow) and free probe are
indicated.
DNA complex to stabilize CREB binding. Further support for
this hypothesis was provided by the Tax-dependent formation of
CREB
21-bp repeat complexes with reduced mobility in an EMSA. We
observed these reduced mobility complexes with full-length CREB
protein, but not with a truncated form of CREB containing only its
DNA-binding/dimerization domain, again implicating amino acids in the
amino-terminal region of the protein as critical in the interaction.
Furthermore, the same 21-bp repeat nucleotide sequences were required
for this interaction as were required for Tax transactivation in
vivo and Tax stabilization of CREB binding in vitro,
lending further support for the significance of these sequences in
Tax-mediated transcriptional activation.
21-bp repeat complex, our evidence also suggests that the
association of Tax is weak and/or transient. This conclusion is based
primarily on our observation of Tax concentration-dependent changes in
the position of the supershifted EMSA complex (Fig. 10). Our
detection of Tax concentration-dependent incremental changes in the
mobility of the CREB
DNA complex is consistent with a rapid
association-dissociation of Tax with the CREB
21-bp repeat
complex. This observation does not exclude the possibility that a
stable ternary complex forms but dissociates under the conditions of
our EMSA. We continue, however, to favor a model in which Tax forms a
weak or transient interaction with the CREB
21-bp repeat complex,
as several additional lines of evidence support this interpretation.
These include the absence of a Tax-specific protein-DNA interaction
using either DNase I or methidium propyl EDTA:Fe(II) footprinting (data
not shown and Ref. 28) or methylation interference
(28) and,
finally, failure to detect Tax-CREB interactions by affinity
chromatography (data not shown and Ref. 31). While we conclude that the
interaction is weak or transient, the relative strength of the Tax
interaction remains very controversial, as evidence supporting a more
stable ternary complex between Tax, a 21-bp repeat element, and 3
specific amino acids in the basic DNA binding region of CREB has
previously been reported
(27) .
This increase in association rate
likely accounts for a portion of the Tax-dependent increase in DNA
binding affinity that we observe for CREB, with an additional
contribution in binding stability derived from the decrease in
dissociation rate. Taken together, these data indicate that Tax
interacts with the basic-spacer segment of CREB to increase the
association rate of CREB to the DNA sequence. This reaction is
concurrent with, or immediately followed by, a second reaction in which
Tax interacts with the amino-terminal region of CREB and nucleotides
within the 21-bp repeat element to decrease the dissociation rate of
CREB from the DNA. In the context of the HTLV-I promoter, both
reactions appear necessary for efficient Tax transactivation. It is
tempting to speculate, however, that the effect of Tax on the DNA
association rate of a wide range of cellular transcription factors is
responsible for the highly pleiotropic deregulation of cellular gene
expression.
Table:
Apparent affinities for CREB and ATF-2 binding
to a consensus CRE and the three HTLV-I 21-bp repeats
Table:
Apparent affinities for CREB and ATF-2 DNA
binding in the presence of Tax
Table:
Nucleotide sequences of the consensus CRE,
third 21-bp repeat, and hybrid binding sites
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.