(Received for publication, September 18, 1996, and in revised form, October 18, 1996)
From the the Vollum Institute and the Departments of Cell and Developmental Biology, Biochemistry and Molecular Biology, Pediatrics, and Internal Medicine, Oregon Health Sciences University, Portland, Oregon 97201
We have previously proposed that cAMP-responsive
element-binding protein (CREB) activity is stimulated by human T-cell
lymphotropic virus-1 (HTLV-1) Tax through two mechanisms that are
differentially dependent upon CREB phosphorylation. We have tested this
model by examining how Tax affects transcriptional activation mediated by the cAMP-responsive element (CRE) modulator (CREM). The CREM proteins are highly homologous to CREB, particularly in their DNA-binding domains and the kinase-inducible domain (KID), a region that interacts with the coactivator CREB-binding protein (CBP) in a
phosphorylation-dependent manner. Despite this similarity, most CREM isoforms are transcriptional repressors. CREM lacks the
glutamine-rich domains found in CREB that are essential for transcriptional activation. We show that the normally repressive CREM
activates the HTLV-1 and cellular CREs in the presence of Tax;
activation of the viral element is phosphorylation-independent, and
activation of the cellular CRE is
phosphorylation-dependent. CREM
(C-G) lacks both the KID
and the glutamine-rich regions. This isoform activates the HTLV-1 long
terminal repeat in a phosphorylation-independent manner, but does not
activate the cellular CRE. This study suggests that Tax, interacting
with the basic/zipper region of CREM, recruits CBP to the viral
promoter. Tax activation of the cellular CRE depends on the KID and its
ability to interact with CBP in a phosphorylation-dependent manner.
Human T-cell lymphotropic virus-1 (HTLV-1)1 is the causative agent in adult T-cell leukemia (1). The HTLV-1 genome encodes several regulatory proteins that are involved in controlling viral gene expression and pathogenesis. One such protein is the 40-kDa transactivator Tax (2). While Tax contains a transcriptional activation domain, the native Tax protein is unable to bind DNA directly (3, 4). Thus, it has been proposed that the transactivating functions of Tax require its interaction with cellular DNA-binding proteins. Indeed, several basic/leucine zipper transcription factors have been shown to interact with the Tax-responsive elements of the HTLV-1 promoter in vitro, including CREB, CREM, and ATF-1 (5-7). Binding of these factors to the Tax-responsive elements, which resemble the cAMP-responsive elements (CREs) of cellular genes, is markedly enhanced by the Tax protein (8-11). Furthermore, studies have shown that the HTLV-1 long terminal repeat (LTR) can be activated by protein kinase A (PKA) and that the level of activation is augmented by Tax (12). However, the precise molecular mechanisms underlying Tax transactivation are not clear.
Recently, we proposed that Tax potentiates transactivation of cellular CRE-containing genes through a direct interaction with the transcriptional coactivator CREB-binding protein (CBP) (8). We additionally showed that the ability of Tax to function in this manner depends upon CREB phosphorylation, as the augmentation is prevented when PKA is omitted and is significantly decreased when the consensus PKA site in the kinase-inducible domain (KID) of CREB is mutated (8). In contrast, Tax activates expression of the HTLV-1 LTR even in the absence of PKA (8). These results suggest that Tax activates cellular and viral CREs through mechanisms that are differentially dependent on phosphorylation.
The structurally related transcription factors CREB, CREM, and ATF-1
are key mediators of cAMP-regulated gene transcription. While the
regulatory properties of ATF-1 remain poorly defined, isoforms of CREB
and CREM have well characterized functions as activators and repressors
of transcription, respectively. The CREB and CREM genes both encode an
amino-terminal phosphorylation domain (KID) flanked by glutamine-rich
transcriptional activation domains (13). The KID of CREB has been shown
to interact with the transcriptional coactivator CBP in a
phosphorylation-dependent manner (14-16). Alternative
splicing and selective translational initiation of CREB and CREM
transcripts generate a family of proteins ranging from full-length
transcriptional activators to truncated transcriptional repressors
(17). Specifically, CREM, which lacks the glutamine-rich domains
found in activator isoforms such as CREB and CREM
, has been shown to
be a potent repressor of cAMP-activated transcription (18).
The different CREM isoforms provide valuable reagents for testing our
model of Tax-activated transcription because the various functional
domains in these isoforms are clearly delineated. Our model suggested
that Tax activates transcription by interacting with distinct domains
of the CREB transcription factor, depending on the promoter context and
the presence or absence of PKA. Thus, we would predict that Tax should
convert the repressor CREM into an activator of the cellular CRE
because the KID and DNA-binding domain of CREM
are very similar to
those of CREB. Moreover, CREM
-mediated activation of the HTLV-1 LTR
should occur in the absence of PKA, in a manner similar to that
reported previously for CREB. The CREM
(C-G) isoform lacks both the
glutamine-rich regions and the KID (19). This form is predicted to be
incapable of mediating Tax activation of the cellular CRE, but because
it contains a CREB-like basic/zipper region, it should still be able to
contribute to activation of the viral LTR. By using a combination of
in vivo transfection and in vitro fluorescence
polarization binding assays, we show that the Tax activation of
cellular and viral CRE-containing genes is indeed mediated through
different functional domains of the bZIP proteins in a manner that is
differentially dependent upon phosphorylation. Our studies suggest that
activation of the viral promoter occurs through the recruitment of CBP
and requires, in addition to Tax, only the bZIP domain that is
conserved among CREB, CREM
, and CREM
(C-G). In contrast,
activation of the cellular CRE by Tax occurs only in the presence of
PKA and requires isoforms of CREB or CREM that contain the KID. Thus,
recruitment of Tax to the cellular CRE depends upon a phosphorylated
KID and its ability to bind to the coactivator CBP.
F9 teratocarcinoma
cells were grown on 0.7% gelatin-coated plates in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum, penicillin,
and streptomycin. Cells were seeded at 3.5 × 105
cells/plate 18 h prior to transfection. DNA was prepared by
alkaline lysis, purified twice in CsCl gradients, phenol-extracted, and ethanol-precipitated prior to use. F9 cells were transfected by calcium
precipitation (20) with 4 µg of RSV-PKA, 2 µg of RSV-luciferase, 0-8 µg of RSV-Tax, 0-8 µg of RSV-CREM, 5 µg of p(
71)
somatostatin-CAT (21), 0-8 µg of CMV-CREM
(C-G), and 2 µg of
pU3R-CAT (22), as indicated. CREM
was a gift from P. Sassone-Corsi;
CREM
(C-G) was a gift from J. F. Habener. All other plasmids used in
this study have been described previously. The total amount of DNA in
each transfection was 30 µg, the balance being made up with Rc/RSV.
Cells were washed, refed, and allowed to grow for 24 h before
harvesting. CAT activity was determined as described previously (23),
and values were normalized for luciferase activity as a control for
transfection efficiency. RSV-luciferase expression was not affected by
introduction of CMV-CREM
(C-G).
Recombinant CREM protein was
generated using a pET15b expression vector (Novagen). BL21(DE3) cells
transformed with a plasmid containing the entire coding sequence of
CREM
were grown to A600 = 0.6 in Luria broth
containing 2 g/liter glucose and 100 µg/ml ampicillin. Cultures were
induced to express the recombinant protein by the addition of
isopropyl-
-D-thiogalactopyranoside to a final concentration of 1 mM. After 3 h, cells were harvested
and lysed by two passages through a French press. Bacterial debris was
cleared by centrifugation, and the cleared supernatant was heated to
72 °C for 10 min. Precipitated proteins were cleared by
centrifugation. CREM
was purified from the supernatant over an FPLC
HiTrap Q anion-exchange column (Pharmacia Biotech Inc.). The minimal
bZIP cDNA was constructed from CREB/SER (24) by engineering a
transcription start site and an NcoI cleavage site at
residue 282 using site-directed mutagenesis. This construct, in which
the cysteine residues in the basic/zipper region have been changed to
serine, was subcloned into the pET15b expression vector using the
NcoI/BamHI sites. An oligonucleotide encoding a
histidine tag extension (MHHHHHHSSG) and compatible NcoI
ends was ligated to the bZIP/SER vector at the NcoI site to
aid in purification. BL21(DE3) cells were transformed with the bZIP/SER
plasmid as described above, and cells were induced with 0.8 mM isopropyl-
-D-thiogalactopyranoside. Cells
were harvested by centrifugation and resuspended in 50 mM
potassium phosphate buffer, pH 7.5, containing 300 mM KCl,
5% glycerol, and 0.1% Triton X-100. Extracts were prepared as
described above, and the supernatants were heated to 80 °C for 10 min. The peptide was purified over a nickel-nitrilotriacetic acid resin
(QIAGEN Inc.); dialyzed against 25 mM Tris-HCl buffer, pH
8.0, containing 1 mM EDTA; and further purified over an
FPLC Q-Sepharose anion-exchange column (Pharmacia Biotech Inc.).
Generation of histidine-tagged Tax and CBP fusion proteins was as
described (25, 26).
CREM was phosphorylated using the
purified catalytic subunit of PKA (a gift from R. A. Maurer). The
phosphorylation reaction was carried out in 50 mM MOPS
buffer, pH 6.8, containing 50 mM NaCl, 5 mM
MgCl2, 1 mM dithiothreitol, and 1 mM ATP at 30 °C for 30 min. To measure the incorporation
of phosphate, [
-32P]ATP was added to the reaction at a
final concentration of 0.6 nM. The extent of
phosphorylation was determined by trichloroacetic acid-precipitable
counts. For binding studies, excess ATP was removed by extensive
dialysis against binding buffer.
Fluorescence polarization measurements were
performed as described (25, 27). Briefly, a 5-fluorescein-labeled
28-base oligonucleotide representing the sense strand of the promoter proximal HTLV-1 Tax-responsive element
(5
F-TCCTCAGGCGTTGACGACAACCCCTCAC-3
) and a 23-base 5
-fluoresceinated
oligonucleotide representing the somatostatin CRE
(5
F-CCTTGGCTGACGTCAGAGAGAGC-3) were annealed to antisense
oligonucleotides. An N-terminal fragment of CBP (amino acids 1-682)
was titrated in 1 ml of reaction buffer (25 mM, Tris pH
7.6, 50 mM NaCl, 1 mM dithiothreitol 0.1%
Triton X-100, 5% glycerol, 6 µg of bovine serum albumin, 10 µg of
poly[d(I-C)], and 5 mM MgCl2) in borosilicate
glass tubes containing 5 nM fluoresceinated HTLV-1 or
somatostatin CRE saturated with either 30 nM CREM
(phosphorylated or non-phosphorylated) or 50 nM bZIP
peptide and, in selected experiments, 1 µM Tax. The
fluorescence anisotropy of each titration point was determined at room
temperature using a PanVera Beacon fluorescence polarization system.
Four measurements of each binding condition were averaged for each
determination. The apparent Kd for CBP binding was
determined from a plot of the anisotropy versus CBP
concentration. Binding isotherms were fitted to a simple binding model
for the interaction of CBP with CREM
or bZIP peptide, including a
linear nonspecific binding component, by nonlinear regression. Protein
concentrations were determined by measurement of extinction coefficients and by amino acid analysis.
CREM, which lacks the glutamine-rich domains present in
the activators CREB and CREM
(Fig. 1), generally
represses CRE-mediated transcription (18, 28). Our model for Tax
activation of CRE-containing genes (8) suggested that CREM
should be
converted into a PKA-dependent activator by the addition of
Tax. We tested this possibility by measuring expression of a
somatostatin-CAT reporter gene in F9 teratocarcinoma cells in the
presence or absence of PKA and Tax. F9 cells were chosen for these
studies because they lack functional levels of CREB and PKA (29). As
shown in Fig. 2, Tax activated expression of the
reporter in a dose-dependent manner in the presence of
CREM
and PKA (lanes 6-10) by >40-fold. More important,
this activation was eliminated in the absence of PKA (lanes
1-5) or in the absence of exogenous CREM
(data not shown).
These results demonstrate that CREM
becomes an activator of the
cellular CRE in the context of the viral transactivator Tax and that
this activation is PKA-dependent.
Activation of the HTLV-1 Promoter through CREM
Our model additionally predicted that CREM
should be able to mediate the Tax activation of the HTLV-1 LTR and that
this activation should not require PKA. To test this possibility, F9
cells were cotransfected with Tax and increasing amounts of CREM
in
the absence of PKA. The results in Fig. 3 indicate that,
in the presence of Tax, CREM
activates the HTLV-1 LTR reporter by
11-fold over basal levels (lanes 1-6). Control
transfections indicate that this activation does not occur in the
presence of exogenous CREM
alone (lanes 7-11). These
results show that the glutamine-rich regions (lacking in CREM
) are
not essential for Tax activation of either the viral or cellular CREs.
In addition, these data confirm the differential requirement for
phosphorylation in Tax-mediated activation of cellular and viral
promoters.
Activation of gene expression by PKA-phosphorylated CREB has been
proposed to involve the transcriptional coactivator CBP (14-16).
Moreover, we have proposed that CBP may participate in the
PKA-independent activation of the HTLV-1 LTR mediated by Tax and CREB
(8). The evidence that somatostatin and HTLV-1 CRE activation by the
repressor CREM is differentially dependent on phosphorylation
suggests that CBP might be involved in these processes as well.
The conservation of the KIDs of
CREB and CREM suggested that both factors might interact with CBP in
a PKA-dependent manner. Binding of CBP to CREM
associated with a somatostatin CRE oligonucleotide was shown by
fluorescence anisotropy analysis (Fig. 4A).
CBP binds to phosphorylated CREM
with an apparent
Kd of 500 nM. In the presence of Tax,
the affinity of this interaction is only minimally higher
(Kd = 430 nM). As expected, the
interaction of CBP with CREM
depends on phosphorylation, as there is
no binding of CBP to non-phosphorylated CREM
in the presence or
absence of Tax (Fig. 4B). These data indicate that the
phosphorylation-dependent activation of the somatostatin
CRE reporter by Tax in the presence of CREM
is probably not due to a
change in the affinity of the coactivator CBP, but rather to the
recruitment of Tax to the promoter via CBP.
Tax Increases the Affinity of CBP for CREM
Our model suggests that CREM and CBP allow activation of
the HTLV-1 LTR by Tax in a PKA-independent manner. We characterized the
assembly of this complex by fluorescence polarization binding assays.
In the presence of Tax, CBP has a higher affinity for phosphorylated
CREM
bound to the HTLV-1 CRE (Kd = 130 nM) (Fig. 5A) than to the
somatostatin CRE (Kd = 430 nM). In
addition, Tax facilitates a high affinity interaction between CBP and
the non-phosphorylated form of CREM
(Kd = 140 nM) (Fig. 5B, closed triangles).
Recruitment of CBP to the HTLV-1 CRE·non-phosphorylated CREM
complex completely depends on Tax, as there is no specific binding of
CBP in the absence of Tax (Fig. 5B, open
triangles). These data suggest that the PKA-independent activation
seen in the context of the HTLV-1 LTR reporter may be due, in part, to
recruitment of CBP. The phosphorylation-independent nature of this
interaction suggests that it might involve a region of CREM
distinct
from the KID.
Tax Activates CREM
To elucidate the mechanism underlying this
differential dependence on phosphorylation, we utilized a CREM isoform,
CREM(C-G), which contains neither the KID nor the glutamine-rich
domains, but has a bZIP domain that is nearly identical to that of CREB (19). This factor, like CREM
, is normally a transcriptional repressor. Adya et al. (30) have proposed that Tax interacts directly with a region of CREB shared by the CREM proteins that is
amino-terminal to the basic region. Because this region is conserved in
CREM
(C-G), we predicted that CBP should be recruited to the viral
promoter through a direct interaction between Tax and the conserved
bZIP domain of this isoform. Conversely, CREM
(C-G) should bind to
the somatostatin CRE, but should be unable to recruit CBP, and
therefore Tax, to the cellular promoter. As a result, even though it
lacks the glutamine-rich domains and the KID, we predicted that
CREM
(C-G) would activate the viral CRE, but not the cellular CRE. We
utilized an expression vector encoding CREM
(C-G) in functional
assays to test this prediction. F9 cells were cotransfected with
CREM
(C-G), Tax, and either the somatostatin or the HTLV-1 LTR
reporter. As shown in Fig. 6 (left panel,
lanes 1-6), CREM
(C-G) activates the HTLV-1 reporter by
25-fold in the presence of Tax. CREM
(C-G) is unable to activate
expression of the somatostatin CRE, however, regardless of Tax
(right panel, lanes 1-6). These data support a
mechanism of activation in which CBP is recruited to the promoter
through different domains of the CREB/CREM factors, depending on the
particular promoter context.
Tax Facilitates High Affinity Binding of CBP to the CREB bZIP Peptide in an Enhancer-dependent Manner
Our model
predicts that a minimal CREB bZIP peptide should allow Tax to
facilitate an interaction between CBP and the HTLV-1 CRE, but should
not promote CBP binding to the somatostatin CRE. Indeed, as
seen in fluorescence polarization binding assays (Fig. 7, closed circles), CBP binds to the HTLV-1
CRE saturated with bZIP peptide in the presence of Tax
(Kd = 306 nM). There was no specific
interaction between CBP and bZIP peptide in the absence of Tax (data
not shown). In addition, no specific binding of CBP to the somatostatin
CRE saturated with bZIP peptide was detected, regardless of the
presence of Tax (Fig. 7, open circles). These data support
our model for differential activation of viral and cellular
CRE-containing genes. Specifically, CBP is recruited to the
Tax-responsive viral CRE through direct interaction between Tax and the
basic domain conserved among CREB, CREM, and CREM
(C-G). Recruitment of CBP, and therefore Tax, to the cellular CRE depends on
the phosphorylation of the KID conserved in CREB and CREM
.
The finding that CREB participates in Tax-mediated activation of
the HTLV-1 and cellular CREs, along with the high degree of
conservation among regions of CREB and CREM, suggested that Tax might
also interact with some CREM isoforms. Indeed, several lines of
evidence have supported this possibility (31, 32). Whether the
interaction of Tax and CREM would result in activation or repression of
transcription of viral and cellular CREs was unclear, however. Many of
the CREM isoforms, including CREM and CREM
(C-G), are known to be
inhibitors of transcription (18, 19). One model suggests that Tax
functions by increasing dimerization of bZIP transcription factors,
thereby enhancing binding of these factors to CREs (9, 11). If this
were the primary mode of Tax-mediated gene activation, we would expect
that Tax would enhance repression by CREM
, rather than cause
activation. The fact that CREM
activates both viral and cellular
CRE-containing genes in the presence of Tax and that activation of the
two types of promoters is differentially dependent on phosphorylation
argues for an alternative mode of action for the Tax
transactivator.
The CREB transcription factor has provided a good model for
understanding the role of phosphorylation in gene activation. We (14,
15) and others (16) have shown that phosphorylation of CREB by PKA
facilitates a high affinity interaction between the KID and the
coactivator CBP. The KID is highly conserved between CREB and CREM,
suggesting that CBP could interact with the repressor upon
phosphorylation by PKA. Here, we show that the binding affinity of CBP
for phosphorylated CREM
is equivalent to that of CREB. Although
phosphorylated CREM
interacts with the coactivator CBP in
vitro, the repression of cellular CRE-containing genes by CREM
in vivo suggests that this interaction is not sufficient for
gene activation. The lack of the glutamine-rich domains in CREM
may contribute to this repression by failing to recruit other activators to
the CREM
·CBP complex or by failing to provide additional
interactions with components of the basal transcriptional machinery.
This report shows that phosphorylated CREM
becomes an activator of
the somatostatin promoter in the presence of Tax. Presumably,
recruitment of the activation domain in the Tax C terminus (33-35) by
CBP (8) allows CREM
to participate in activating the cellular
promoter. The idea that CBP is an essential component of this
activation process is supported by our finding that the activation
domain of Tax cannot induce expression from the somatostatin CRE in the
presence of CREB or CREM isoforms that are incapable of interacting
with the coactivator CBP, i.e. CREB-M1 (8) or CREM
(C-G).
In contrast, CREM isoforms lacking only the glutamine-rich domains can
still participate in Tax-mediated activation of cellular CREs if they are capable of interacting with CBP.
The PKA-independent activation of the HTLV-1 CRE by Tax and CREM may
provide further insights into the mechanism of Tax action. Adya
et al. (30) have suggested that a conserved Ala-Ala-Arg motif flanking the CREB basic region is necessary for a direct Tax-CREB
interaction. This motif is conserved in CREM
, suggesting that a
direct Tax-bZIP interaction may be sufficient for activation of the
HTLV-1 CRE. By utilizing another repressor isoform of CREM, CREM
(C-G), which contains the conserved bZIP domain but not the KID,
we demonstrated that the KID is not essential for PKA-independent viral
promoter activation. Binding assays suggest that this activation is due
to the ability of Tax, interacting with the minimal bZIP domain, to
recruit the coactivator CBP to the viral promoter. Thus, Tax may
provide a bridge between the HTLV-1 promoter and the coactivator CBP
through a mechanism that does not depend on phosphorylation. The
different modes of Tax action emphasize that while Tax cannot interact
directly with DNA, the sequence of viral and cellular Tax-responsive
elements has a strong influence on the behavior of this
transactivator.
The influence of promoter sequence on the behavior of specific
transcription factors is well established (36). Indeed, while CREM
is primarily believed to be a repressor of cAMP-mediated transcription,
there is some evidence that its ability to stimulate or repress gene
transcription depends upon the promoter context of the CRE sequence
(37). How promoter context influences CREM
activity is unclear,
however. In this paper, we provide evidence that CREM
can activate
transcription of viral and cellular promoters in the presence of Tax in
a manner that is differentially dependent on phosphorylation.
Specifically, we demonstrate that while phosphorylated CREM
can
interact with the coactivator CBP in a manner similar to CREB, this
interaction does not result in activation of a cellular CRE. However,
additional recruitment of the viral transactivator Tax through CBP
results in a complex that promotes transcription. It remains unclear,
however, whether activation domains in CBP are essential for activation
of the HTLV-1 CRE or whether the activation domain of Tax functions
through other coactivators or components of the basal transcriptional
machinery (38).
We thank R. Brennan for advice on the fluorescence polarization measurements and P. Sassone-Corsi, J. Habener, and R. Maurer for reagents.