From the Division of Molecular Medicine, Tax-1, the transcriptional activation protein of
human T-cell leukemia virus-1, increases transcription from the human
T-cell leukemia virus-1 long terminal repeat and specific cellular
promoters through interactions with cellular DNA-binding proteins. The
Tax response elements (TxREs) of the long terminal repeat resemble cAMP
response elements (CREs), the target of cAMP-responsive element-binding protein (CREB). CREB binds the TxRE with reduced affinity; however, the
interaction is specifically enhanced by Tax. Using a fluorescence quenching method, we determined that CREB dimerizes in the absence of
DNA, and that Tax does not enhance dimerization. DNA footprinting of
the TxRE with 1,10-phenanthroline-copper complex demonstrates that Tax
contacts DNA and extends the footprint of CREB to GC-rich sequences
flanking the core CRE-like element. The minor groove-binding drug
chromomycin A3, but not distamycin A, disrupted
Tax-enhanced CREB binding to the TxRE. Substitution of the guanine-rich
sequences flanking the core of the TxRE with inosine residues also
blocked the Tax effect. Finally, the IC-substituted TxRE binds CREB
with increased affinity, suggesting flanking DNA influences the binding of CREB to the core CRE-like element. These data indicate that Tax does
not regulate DNA binding of CREB by altering dimerization, but rather
enhances DNA binding by additionally interacting with the minor groove
of flanking DNA sequences.
Viruses typically exploit host cellular processes to promote their
own replication. Human T-cell leukemia virus
(HTLV-1),1 a human
retrovirus, is the causative agent of adult T-cell leukemia/lymphoma (reviewed in Ref. 1). Adult T-cell leukemia/lymphoma develops only in a
small percentage of patients, typically many years following infection
with HTLV-1. The virus remains in a state of latency in infected
T-cells, producing low levels of transcripts from the long terminal
repeat (LTR). Activation of viral RNA synthesis depends on cooperation
between virally encoded proteins and cellular factors. The pX region of
the HTLV-1 genome encodes Tax, a 40-kDa protein that is essential for
viral pathogenesis. Tax is a positive transcriptional activator of both
cellular and viral promoters, but Tax is thought to activate
transcription through interactions with cellular proteins rather than
by binding to DNA itself (2-4). Through a positive feedback mechanism,
Tax enhances transcription from the viral LTR through a complex
enhancer containing a series of three 21 base pair repeats, which act
as Tax-responsive elements (or TxREs) (5, 6). The core sequence
(5'-TGACG-3') of the 21-base pair TxRE resembles the cAMP-responsive
element (or CRE), the target of the cellular transcriptional regulatory
protein CREB (7). The central CRE-like core of each 21-base pair repeat is flanked by GC-rich sequences that are required for Tax
transactivation in vivo (2, 8, 9). The TxRE is a low
affinity CREB-binding site in vitro; however, the binding
activity of CREB can be dramatically enhanced by the addition of Tax
(10-19).
We and others have shown that Tax augmentation of CREB DNA binding
activity is sequence-specific; Tax enhances binding of CREB (and
cAMP-responsive element modulator, or CREM) to the TxRE but not to the
perfect palindromic CRE derived from the somatostatin promoter (12-14,
16, 17, 19-21). In addition Tax forms a stable complex with CREB and
DNA, also in a sequence-specific manner, dependent on flanking GC-rich
DNA motifs (12, 14, 16, 17, 19). Although Tax does not associate with
CREB bound to the somatostatin CRE, it will bind to CREB in the absence
of DNA (22-24). The stable complex of Tax, CREB, and the TxRE is
termed the "ternary complex," although the precise stoichiometry is
unknown. This DNA-directed ternary complex recruits the CREB
coactivator CREB-binding protein in a phosphorylation-independent
manner, probably through a direct association with CREB-bound Tax (19,
21, 25). Thus, through the activity of Tax, the TxRE DNA directs the
assembly of a topologically distinct activator-coactivator complex at
the HTLV-1 LTR promoter, permitting transcriptional activation through CREB in a cAMP-independent manner.
The proposed molecular mechanisms of Tax-enhanced binding of CREB to
DNA and ternary complex formation are controversial. Wagner and Green
(26) have proposed that Tax augments the dimerization of CREB as well
as other B/ZIP factors. Although Tax interacts specifically with the
basic region of B/ZIP proteins (18, 24, 27), the contribution of Tax to
dimerization, mediated by the leucine zipper motif, is unclear. Tax
increases the DNA binding of chimeric peptides consisting of the GCN4
basic region and unrelated dimerization motifs (27) and, in the context
of CREB, specific residues within the leucine zipper may be essential
for the Tax effect on DNA binding (17, 20). Nevertheless, by using
nonspecific cross-linking, Wagner and Green (26), and Perini et
al. (27), suggest that Tax increases dimeric association of the
GCN4 B/ZIP through interactions with the basic segment. The DNA
sequence specificity of the Tax effect, however, argues against the
idea that enhanced dimerization accounts for the increased DNA binding activity. Indeed, the ability of Tax to stimulate the DNA binding activity of cross-linked basic region peptides independently of dimerization (15) suggests that Tax might associate with specific DNA
sequences, despite the lack of evidence to date for direct Tax-DNA
interactions (12, 26).
In this report, we directly measure CREB dimerization by using a
solution-binding assay based on fluorescence quenching and demonstrate
that Tax does not affect this aspect of CREB function. By DNA
footprinting with 1,10-phenanthroline-copper complex, a minor
groove-selective cleavage reagent, we demonstrate that Tax interacts
with the minor groove of the DNA flanking the core CRE-like motif of
the TxRE, in the context of the CREB-Tax-DNA ternary complex. The
GC-selective minor groove-binding drug chromomycin A3
selectively disrupts the Tax effect on CREB recruitment. Furthermore, alteration of minor groove structure by substitution of GC with IC base
pairs in the flanking region disrupts Tax-enhanced DNA binding by CREB.
These findings support a model of Tax acting through minor groove DNA
contacts, specifically increasing the association of CREB with the
HTLV-1 LTR TxRE.
Plasmids and Expression Vectors--
The bacterial expression
vector for HTLV-1 Tax has been described previously (28). A cDNA
encoding a single cysteine form of CREB327 was prepared by
site directed mutagenesis of cysteines at codons 286 and 323 to serines
using the Altered Sites system (Promega) (29). Wild type
CREB327 and the single cysteine mutant of
CREB327 (cysteine Protein Purification--
E. coli BL21(DE3) cells
harboring the CREB expression vectors were grown as described
previously (29). Cells were harvested after 3 h at 37 °C,
washed in ice-cold phosphate-buffered saline, resuspended in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM DTT (TED) and lysed by two passages through a French
pressure cell at 14,000 p.s.i. (Aminco). Lysates were cleared of
insoluble cellular debris by centrifugation at 30,000 × g for 30 min. The lysate was then heated to 70 °C for 15 min, and recentrifuged at 12,000 × g for 10 min.
Nucleic acid was precipitated from the supernatant by addition 0.2%
polyethyleneimine following adjustment of the supernatant to 0.3 M NaCl. CREB was then precipitated from the supernatant with 20% (w/v) ammonium sulfate. Pellets were solubilized in TED and
dialyzed, and then soluble protein was applied to a Q-Sepharose column
(Amersham Pharmacia Biotech). Proteins were then eluted with a
continuous concentration gradient from 0 to 1 M NaCl in TED. Fractions containing CREB were then adjusted to 2 M
NaCl and applied to a phenyl-Sepharose column, washed, and eluted with low salt. Fractions containing CREB were pooled and fractionated by
heparin-agarose chromatography. CREB was dialyzed into TED+100 mM NaCl and 5% glycerol, aliquoted, and stored at
Department
of Medicine, ** Department of Biochemistry and Molecular Biology,
Division of Hematology/Oncology,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
serine 286, cysteine 296, cysteine
serine 323) were each subcloned into the prokaryotic expression
vector pET11d as a NcoI-BamHI fragment (Novagen)
and expressed as full-length proteins without polyhistidine tags in
Escherichia coli BL21(DE3) by identical procedures.
Sequences were confirmed by the dideoxy chain termination method
(Sequenase, U. S. Biochemical Corp.).
70 °C. CREB protein concentrations were determined as described
previously (31).
Fluorescence Labeling-- Single cysteine CREB327 (approximately 20 µM) was dialyzed extensively into 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 0.5 mM DTT, and then incubated with 2 mM fluorescein-maleimide or tetramethylrhodamine-5-maleimide (Molecular Probes, Eugene, OR) for 2 h at 20 °C and then 4 °C overnight. Reactions were quenched with 5 mM DTT and modified protein separated from unreacted fluor by gel filtration (HiTrap Desalting column, Amersham Pharmacia Biotech). Modified proteins were then dialyzed extensively against 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 mM DTT.
Oligodeoxyribonucleotides--
Oligonucleotides corresponding to
the somatostatin CRE have been described previously (32). A duplex
oligonucleotide corresponding to the promoter proximal
Tax-responsive element (107 to
81; Ref. 1) was formed by annealing
5'-GATCTCCTCAGGCGTTGACGACAACCCCTCAC-3'(upper strand), to the
complementary oligonucleotide, 5'-GATCGTGAGGGGTTGTCGTCAACGCCTGAGGA-3'. Oligonucleotides with inosine substitutions flanking the CRE-like sequence of the TxRE (IC-TxRE) were synthesized commercially (Oligos Etc., Wilsonville, OR) with the sequences
5'-GATCTCCTCAIICITTGACGACAACCCCTCAC-3' (upper
strand), and
5'-GATCGTGAIIIITTGTCGTCAACICCTGAGGA-3'.
Fluorescence Measurements-- Steady state fluorescence measurements of labeled proteins were made using a SPEX Fluoromax spectrofluorimeter. All binding measurements were determined in 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 100 mM NaCl, 1 mM DTT, 5% glycerol, 50 µg/ml BSA, at 20 °C in a 1-ml binding reaction. For dimerization reactions, efficiency of fluorescence quenching was determined from fluorescein emission at 515 nm, following excitation at 485 nm. Data were fitted to a simple bimolecular binding model by nonlinear regression (33).
DNA Footprinting--
1,10-Phenanthroline-copper DNA
footprinting was performed as described previously (34) using a DNA
fragment corresponding to the 1 to
300 region of the HTLV-1 LTR
end-labeled at a single terminus with [
-32P]ATP and T4
polynucleotide kinase (1). Footprinting ladders were resolved on 6%
acrylamide, 8 M urea sequencing gel and exposed to a
storage phosphor screen (Molecular Dynamics). A DNA sequencing ladder
(30) was run next to the cleavage reactions to identify the location of
the TxRE 21 base pair repeats.
DNA Binding Assays--
DNA binding was determined by
electrophoretic mobility shift assay (32). Duplex
oligodeoxyribonucleotides were radiolabeled with T4 polynucleotide
kinase and [-32P]ATP (>6000 Ci/mmol; NEN Life Science
Products). Binding reactions consisted of a buffer containing 25 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA,
1 mM DTT, 5 mM MgCl2, 50 µg/ml
poly(dI·dC), 200 µg/ml BSA (New England Biolabs), 1 nM
32P-labeled duplex oligodeoxyribonucleotide, and Tax and
CREB concentrations as indicated in the figure legends. Free and
protein-bound DNA were electrophoretically separated by 5% native
polyacrylamide gel electrophoresis in 0.5× Tris-borate-EDTA, and
exposed to a storage phosphor screen. Minor groove-binding drugs
distamycin A and chromomycin A3 were obtained from Sigma.
Distamycin A (100 mM) was prepared in distilled water and
stored at
20 °C. Chromomycin A3 (100 mM)
was prepared in dimethyl sulfoxide and stored at
20 °C.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Measurement of CREB Dimerization by Fluorescence Quenching-- We utilized an approach based on fluorescence resonance energy transfer to detect self-association of CREB in the absence of DNA binding and to determine whether Tax enhances the DNA binding activity of CREB through enhancement of dimerization. Fluorescence resonance energy transfer measurements have been used previously to study dimerization of Fos and Jun, the components of the AP-1 complex (35). The specificity of this approach relies on the strict distance dependence of nonradiative energy transfer (36).
For this assay, we used a single cysteine form of CREB327 in which cysteines at positions 286 and 323 were changed to serine residues by site-directed mutagenesis, preserving a single cysteine residue at amino acid 296 (at the first c position within the coiled-coil heptad repeat, Fig. 1A). Previous studies have demonstrated that these cysteines are dispensable for high affinity DNA binding. For example, the B/ZIP of GCN4 lacks these cysteine residues, yet binds CRE sequences with high affinity (37, 38). Santiago-Rivera et al. (39) demonstrated that iodoacetamide carboxymethylation of CREB B/ZIP peptides at these cysteines did not alter DNA binding activity, but rather diminished aggregation and enhanced solubility of these peptides in solution. Mutation of a cysteine to a serine in the basic regions of Fos and Jun had a similar stabilizing effect (40). Finally, Richards et al. (29) demonstrated that DNA binding of CREB341 lacking these three cysteine residues was not altered. The structure of the CREB B/ZIP is not known, but may be modeled on the structure of the GCN4 B/ZIP bound to DNA (41, 42). Based on GCN4, the cysteine at position 296 is predicted to lie on the outer face of the dimeric coiled-coil leucine zipper motif, away from the dimerization interface. The sulfur-sulfur distance between subunits of a homodimer of single cysteine CREB is predicted to be approximately 18 Å.
|
|
|
Tax Extends the Footprint of the CREB-DNA Complex--
Previous
studies from several laboratories have demonstrated that Tax forms a
stable complex with CREB both in the absence (22, 24) and presence of
DNA (16, 17, 19). We and others demonstrated previously that the
association of Tax with CREB depends on the DNA context (16, 17, 19).
Tax and CREB form a ternary complex with the proximal TxRE, but not
with the palindromic somatostatin CRE, correlating with the ability of
Tax to enhance the affinity of CREB only for the former element. The
DNA sequence dependence of the Tax effect suggests that Tax itself
participates in DNA recognition in the context of a complex with CREB
and DNA, even though Tax alone does not bind DNA. Previous studies of
the Tax-DNA interaction by DNase I footprinting and dimethyl sulfate methylation interference failed to detect differences between the
footprint formed by CREB alone bound to the TxRE and the footprint formed by the complex of CREB-Tax (12). We also failed to demonstrate differences between CREB alone and CREB-Tax in methylation protection or methylation interference assays (data not shown). These results are
surprising given the dependence of the Tax effect on flanking CG-rich
sequences (8, 12); however, the approaches utilized address only a
limited portion of the DNA available for interaction with DNA-binding
proteins. For example, the primary site of modification of DNA by
dimethyl sulfate under neutral conditions is N-7 of guanine, in the
major groove (30). Because of the possibility that Tax might interact
with the minor groove of DNA, we employed the 2:1 complex of
1,10-phenanthroline-copper ((OP)2Cu+), a
chemical nuclease that cleaves selectively in the minor groove (34,
43). (OP)2Cu+ cleaves the phosphodiester bond
in the floor of the minor groove through oxidative attack of the C-1
hydrogen of deoxyribose, a cleavage reaction sensitive to protein
binding in the minor groove, or major groove binding that disrupts the
structure of the minor groove (43). We consequently asked whether Tax
extended or altered the footprint of the CREB-binding site on the TxRE,
using (OP)2Cu+, reasoning that Tax might make
minor groove contacts with DNA in the GC-rich sequences flanking the
central CRE-like element of the 21 base pair repeats. Fig.
4 shows the results of the footprinting of CREB alone or CREB+Tax on the HTLV-1 LTR, demonstrating a protection of cleavage around the promoter-proximal TxRE (113 to
77). The protection of CREB-bound DNA from (OP)2Cu+
cleavage is modest in the absence of Tax. Nevertheless, the cleavage pattern is diminished within the immediate 5' and 3' GC-rich regions flanking the CRE-like core (
97 to
90) of the TxRE in the presence of CREB+Tax relative to CREB alone. Tax M47 (L319R, L320S), a mutant
that fails to transcriptionally activate the HTLV-1 LTR promoter (59),
also fails to augment the footprint (data not shown).
|
Minor Groove-binding Drugs Prevent Tax Enhancement of CREB DNA Binding Activity and Ternary Complex Formation-- Distamycin A and chromomycin A3 are reversible minor groove-binding drugs with sequence preference for AT- or GC-rich regions, respectively. This relative specificity in DNA interaction has proven useful for selective disruption of the DNA binding activity of minor groove-binding proteins (44, 45). The Tax-dependent interaction of CREB with the TxRE depends on flanking GC-rich sequences (8, 16, 17); thus, we expected that minor groove-binding drugs with GC preference might specifically disrupt Tax activity. Because CREB, like other B/ZIP factors, interacts with the major groove of DNA, we reasoned that these drugs would not disturb the interaction of CREB to DNA, but rather specifically disrupt the Tax enhancement of DNA binding.
Gel mobility shift assay was used to measure the association of CREB with either the consensus somatostatin CRE or the TxRE derived from the promoter proximal 21 base pair repeat of the HTLV-1 LTR (21) in the absence or presence of 250 nM bacterially expressed Tax. As we demonstrated previously by using fluorescence polarization, CREB binds with high affinity to the somatostatin CRE (Fig. 5B, upper panel). In contrast, CREB binds poorly to the promoter proximal TxRE, although the core CRE-like sequence differs from the somatostatin CRE by only one base pair (-TGACGTCA- versus -TGACGACA-) (Fig. 5A, upper panel). Tax enhances the DNA binding activity of CREB to the TxRE by greater than 10-fold. In addition, we reproducibly observe a modest supershift of the complex consistent with the findings of other laboratories (indicated by * complex, Fig. 5A, upper panel) (14, 25, 46), reflecting either the increased size of the CREB-Tax-DNA complex, or some alteration in the shape of the complex, such as DNA bending (47).
|
|
Disruption of Minor Groove Interactions with Inosine-Cytosine-substituted Oligonucleotides-- The disruption of Tax-enhanced DNA binding by the GC-selective minor groove-binding drug chromomycin A3 suggested that the effect of Tax on DNA binding of CREB was mediated through the minor groove of the GC-rich DNA flanking the core CRE-like sequence. We therefore asked whether components of the minor groove of the GC-rich flanking sequences of the promoter proximal TxRE were required for Tax action. We synthesized a synthetic duplex oligonucleotide corresponding to the DNA sequence of the promoter proximal TxRE, except that guanine:cytosine base pairs in the 5' and 3' flanking domains were replaced with inosine:cytosine base pairs (IC-TxRE). Inosine differs from guanine only in the absence of an exocyclic amino group at the 2-position of the purine ring. Thus, the IC base pair has the appearance of an AT base pair in the minor groove, while preserving the stereochemistry of a GC base pair in the major groove (49).
CREB binding to the IC-TxRE is not enhanced by Tax (Fig. 7A) and, in fact, in the absence of Tax is nearly identical to CREB binding to the native TxRE in the presence of Tax (see Fig. 6, upper panel). In addition, Tax fails to supershift the CREB-IC-TxRE, suggesting Tax is unable to form a ternary complex in this context. An oligonucleotide with mutation of the CRE-like sequence of the proximal TxRE to match the consensus palindromic somatostatin CRE (to 5'-TGACGTCA-3', from 5'-TGACGACA-3'), has an increased affinity for CREB in the absence of Tax, and retains Tax-enhanced CREB binding (Fig. 7B). The palindromic site in the context of flanking GC-rich sequences also recruits Tax, indicated by a modest supershift of the protein-DNA complex.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We proposed previously that Tax activates transcription of viral and cellular CRE-containing promoters through distinct mechanisms depending on the DNA context and the cellular CRE-binding protein that Tax targets (19, 21). In this model, the DNA sequence of the CRE plays a critical role in determining the topology of transcription factors in the activator complex. The finding that Tax enhances DNA binding of CREB via interactions through the minor groove of DNA flanking the core CRE-like sequences of the TxRE is consistent with this model. We suspect that Tax does not merely enhance CREB dimerization, but rather contributes to the recognition of specific DNA sequences and directs the assembly of a specific multiprotein complex that includes CRE-binding proteins and the transcriptional coactivator CREB-binding protein (19, 21, 25).
For B/ZIP factors, dimerization is a prerequisite for DNA binding. Dimerization through the leucine zipper is complex, depending on hydrophobic interactions within the core coiled-coil interface (a and d positions) as well as interhelical electrostatic interactions between e and g positions between peptide strands (50, 51). Although peptides corresponding to the basic regions of B/ZIP proteins may specifically interact with DNA with low affinity (52, 53), mutations in the leucine zipper motif have deleterious effects on DNA binding affinity (32). Despite the importance of dimerization on DNA binding activity, and the potential of this step for regulation of DNA binding, few studies quantitatively address this interaction. In fact, most arguments that favor an effect of Tax on dimerization use the measurement of DNA binding as a surrogate for dimerization potential (15). Studies of the yeast B/ZIP factor GCN4 suggest that Tax enhances DNA binding through enhancement of dimerization (26, 27); however, the GCN4 B/ZIP may not be an appropriate model for all B/ZIP factors. Weiss et al. (38) demonstrated that the affinity of GCN4 B/ZIP for the CRE is at least 10-20-fold lower than the affinity of CREB for the same site (29). Also, unlike CREB, GCN4 binds both CRE and AP-1 elements (37, 38).
The dimerization affinity that we measured for CREB in the absence of
DNA is higher than expected based on comparison to the GCN4 B/ZIP, and
represents the first determination of the dimerization affinity of an
intact B/ZIP transcription factor. Weiss et al. (38)
indirectly measured GCN4 B/ZIP association by following changes in
-helical content by circular dichroism at varying peptide
concentrations. In the absence of DNA, the GCN4 B/ZIP underwent a
helical transition at micromolar concentrations of peptide, consistent
with folding of the leucine zipper into a coiled-coil conformation. The
peptide corresponding to the isolated GCN4 leucine zipper may have a
lower relative affinity of association (54), suggesting that protein
sequences outside of the leucine zipper per se may contribute to the
folding of the dimerization domain. Santiago-Rivera et al.
(39) likewise examined the behavior of the CREB B/ZIP module by
circular dichroism, and suggested that CREB dimerizes with micromolar
affinity. The B/ZIP peptide used for that study bound DNA with 200-fold
lower affinity than either native, full-length CREB (29, 55) or
bacterially expressed CREB
B/ZIP,2 however. Estimations
of the dimerization affinities of Fos and Jun are also widely
divergent, with measured values over nearly a 100-fold range (35, 56,
57). This variation perhaps also reflects differences in the size of
the peptides used, or in the methods of determination.
The observation that Tax does not enhance the dimerization of CREB is
not surprising, given the DNA sequence specificity of the Tax effect.
Factors that strengthen dimerization of B/ZIP proteins would be
expected to enhance the binding to all target DNA sequences because of
the coupled equilibrium for dimerization and the subsequent dimer-DNA
interaction in the DNA-binding pathway. For Tax and CREB at least, this
seems not to be the case, although several investigators have
demonstrated that Tax enhances the general affinity of the GCN4 B/ZIP
peptide for DNA (15, 26, 27). The discrepancy between the results with
CREB and the studies of GCN4 may relate to a lower affinity of
dimerization for the yeast DNA-binding protein. In addition, Tax itself
may dimerize (27, 46), and appears to interact with specific residues
within the basic region of B/ZIP factors (24, 27). The enhancement of
the DNA binding activity of CREB occurs at a half-maximal effective concentration of 107 M Tax (data not shown),
although dimerization of Tax is proposed to occur at low nanomolar
concentrations (46). Consequently, dimerized Tax may contribute to
dimerization of B/ZIP factors that have low affinity leucine zippers,
such as GCN4, through symmetric intersubunit contacts between the basic
regions.
Our experiments suggest that Tax enhances the DNA binding activity of CREB for the TxRE through minor groove interactions with the GC-rich flanking sequences. However, the observation that Tax extends the CREB footprint within the context of the TxRE contrasts with previous studies. For example, Pacca-Uccaralertkun et al. (12) did not observe an extension of the CREB footprint on the Tax-responsive element by using either DNase I footprinting or methylation interference assays. However, these methods lack the resolution to detect subtle differences in protein-DNA interactions. For example, dimethyl sulfate modification of guanine occurs primarily at the N-7 of the purine ring in the major groove and would not interfere with minor groove interactions.
The GC-rich sequences flanking the core CRE-like element are essential for Tax enhanced binding of CREB to the TxRE (8, 12, 16). Although the direct interaction of Tax with DNA cannot be demonstrated with the techniques described, our conclusion that Tax interacts with the GC-rich portion of flanking DNA is based on three lines of experimental evidence. First, Tax extends the footprint of CREB produced by the chemical nuclease, 1,10-phenanthroline-copper complex ((OP)2Cu+), a minor groove-selective cleavage agent. (OP)2Cu+cleavage is, however, partially sequence-dependent and cleavage could potentially be altered by distortion of the minor groove by a major groove protein binding (43). The inability to detect major groove interactions by methylation interference makes this possibility unlikely. Second, chromomycin A3, a reversible minor groove-binding drug with GC selectivity, specifically blocks the Tax-enhanced DNA binding activity of CREB to the TxRE and prevents formation of the Tax-CREB-TxRE ternary complex. Finally, the specificity of contact in the minor groove is confirmed by substitution of deoxyinosine for deoxyguanosine in the sequences immediately flanking the core CRE-like portion of the TxRE. The dI-dC base pair has the appearance of a dA-dT base pair in the minor groove, while preserving major groove stereochemistry (49). The promoter proximal TxRE has the core sequence 5'-TGACGACA-3', differing from the canonical somatostatin CRE (5'-TGACGTCA-3') by the inversion of a single base pair (1), yet has more than a 20-fold lower affinity for CREB (19). Surprisingly, CREB binds the dI-dC-substituted TxRE with increased affinity relative to the native TxRE, and Tax fails to enhance binding further or associate with the CREB-DNA complex. The hierarchies of affinities of CREB for various CRE sequences have not yet been systematically determined, although functional studies suggest that flanking sequences contribute to activity (58). In addition, the affinity of CREB for the even more divergent tyrosine aminotransferase (TAT) promoter-derived CRE (5'-TGACGCAG-3') differs by less than 5-fold from that of the somatostatin CRE, as measured by fluorescence polarization (29). The most plausible explanation for our observations is that the efficacy of the CRE-like sequence within the viral LTR is determined in part by the context of flanking DNA.
The TxREs of the HTLV-1 LTR vary considerably (see Ref. 1), not only in the extent of divergence of the core sequence from the canonical CRE, but also in the specific sequence of the flanking GC-rich DNA, although core elements are conserved. Oligonucleotide binding site selection experiments using the CREB-Tax complex demonstrate the imprecise nature of DNA sequence recognition by Tax and suggest that Tax targets elements with similar sequence composition but non-identical sequences (12). Features other than primary DNA sequence per se, such as DNA deformability, or conformational microheterogeneity, may define which CREs are responsive to Tax. For example, optimal binding of CREB to the TxRE may require alteration of the DNA structure (47). Structural characterization of different GCN4-B/ZIP-DNA complexes demonstrates how this class of proteins might interact with dissimilar sequences. The GCN4 B/ZIP binds both the AP-1 site (-TGACTCA-) and the CRE site, which contains an additional central GC base pair. Comparison of the structures of these GCN4 B/ZIP-DNA complexes demonstrates that this is accomplished primarily by local distortion of the DNA helix (41, 42). Thus, Tax may interact with the minor groove to cause a local DNA unwinding or melting and therefore allow CREB to overcome an unfavorable binding energy by altering the DNA structure.
The conversion of the TxRE from a low affinity to a relatively high affinity CREB-binding site by substitution of inosine for guanine bases in flanking sequences favors an architectural role for Tax in DNA binding. DNA with inosine substitution will have altered thermal stability due to the loss of the third hydrogen bond of a dG-dC pair, and altered base pair stacking. In this model, Tax would act through DNA bending, melting or otherwise altering the structure of an unfavorable CREB-binding site to reduce the free energy of CREB binding. CREB provides the specificity, targeting the Tax-CREB complex to the core CRE-like motif, augmented by Tax interactions with GC-rich flanking DNA sequences. The Tax interaction may require the exocyclic 2-amino group of guanine as a hydrogen bond donor. Alternatively, the narrow minor groove in IC-substituted DNA might preclude other minor groove-Tax interactions with, for example, backbone sugars or bases. A model suggesting that Tax facilitates "prefolding" of the basic segment of the B/ZIP domain (15) cannot account for the dependence of the Tax effect on GC-flanking sequences.
Fig. 8 shows a model for the interaction of CREB with DNA, indicating the positions of postulated flanking sequence minor groove interactions. In this model, Tax interacts with the outer face of the CREB basic region segment as it lies in the major groove of the core CRE-like DNA sequence, and additionally interacts with flanking GC-rich DNA on the same face of the DNA helix through minor groove contacts. The model suggests two molecules of Tax per CREB dimer bound to DNA; however, the stoichiometry of the components within this complex is unknown.
|
Are these processes unique to the Tax-responsive sequences of the viral promoter? The hepatitis B virus X protein may employ a similar mechanism for enhancement of the DNA binding activity of CREB and other B/ZIP factors to variant CREs within the hepatitis B virus enhancer (60-62). In addition, subsets of cellular genes responsive to Tax may also have elements that are specifically recognized by the Tax-CREB complex. Low et al. (63) showed that the enkephalin CRE might be a target of Tax, through recruitment of the CRE-binding protein, ATF-3.
This model also raises the possibility that cellular CREs divergent from the canonical somatostatin CRE may also utilize accessory cellular factors to specifically recruit CREB in vivo. Other examples of complexes in which the DNA binding specificity of individual components is expanded by protein-protein interactions include the p65-C/EBP protein complex (64), and AP-1/glucocorticoid receptor interactions (65). The model suggests a greatly increased DNA binding specificity for CREB, and provides a basis for the selectivity of the transcriptional response to this ubiquitous factor.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Madeleine Pham and Carolyn Sousa for technical assistance and Drs. C.-Z. Giam and J. N. Brady for plasmid DNAs.
![]() |
Note Added in Proof |
---|
While this manuscript was under review, Lenzmeier et al. (66) reported that recruitment of CREB to the Tax-responsive element depends on an interaction of Tax with the DNA minor groove.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R29 DK51732 (to J. R. 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.
§ To whom correspondence should be addressed: Div. of Molecular Medicine, NRC-5, Dept. of Medicine, Oregon Health Sciences University, Portland, OR 97201. Tel.: 503-494-4392; Fax: 503-494-1933; E-mail: lundblad{at}ohsu.edu.
1 The abbreviations used are: HTLV-1, human T-cell leukemia virus-1; LTR, long terminal repeat; TxRE, Tax-responsive element; CRE, cAMP-responsive element; CREB, cAMP-responsive element-binding protein; B/ZIP, basic region-leucine zipper; BSA, bovine serum albumin; DTT, dithiothreitol; F-CREB, thiol-reactive fluorescein-maleimide-CREB; Rh-CREB, tetramethylrhodamine-maleimide-CREB.
2 J. Richards, unpublished observation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|