Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene
Promoter Activity by 2'-Aminoethoxy-modified Triplex-forming
Oligonucleotides*
Adrian M.
Stütz
,
Jutta
Hoeck
,
Francois
Natt§,
Bernard
Cuenoud¶, and
Maximilian
Woisetschläger
From the
Department of Allergic Diseases, Novartis
Research Institute, Brunnerstrasse 59, A-1230 Vienna, Austria,
§ Novartis Pharma Research, 4002 Basel, Switzerland, and
¶ Respiratory Diseases, Novartis Horsham Research Centre,
Horsham RH12 5AB, United Kingdom
Received for publication, November 10, 2000, and in revised form, January 18, 2001
 |
ABSTRACT |
Elevated levels of IgE are intimately
associated with a number of allergic diseases, such as allergic
rhinitis or asthma. Therefore, prevention of IgE production in human
B-cells represents an attractive therapeutic target. IL-4-induced IgE
germline gene transcription represents a crucial early step during IgE
isotype switch differentiation. Gene induction is orchestrated by the coordinated action of the transcription factors STAT6 (signal transducer and activator of transcription), NF-
B, PU.1, and C/EBP. This study shows that 2'-aminoethoxy-modified oligonucleotides, which
partially overlap with the STAT6 and the adjacent PU.1/NF-
B binding
site, inhibit DNA binding of all three proteins with high affinity in a
dose- and time-dependent fashion in vitro. Loss of protein binding correlated strongly with increasing DNA triplex formation. Importantly, the oligomers also effectively displaced pre-bound recombinant NF-
B p50 from double-stranded DNA in
vitro. Functionally, the oligonucleotides led to a selective
inhibition of IL-4-induced reporter gene activity from a construct
driven by the IgE germline gene promoter in human B-cells. These data confirm the critical role of this cytokine-responsive regulatory region
in IgE germline gene induction and further support the concept of
specific modulation of gene expression by DNA triplex formation induced
with chemically modified oligonucleotides.
 |
INTRODUCTION |
Interleukin-4 (IL-4)1 is
a multifunctional cytokine that plays a critical role in the regulation
of immune responses. It is intimately associated with the
differentiation of antigen-stimulated naive T-cells into specialized
helper T-cells (Th2 cells) (1, 2). In addition, IL-4 increases the
expression of genes involved in cell activation and inflammation, such
as class II major histocompatibility complex molecules on B cells (3),
CD23 (4), the IL-4 receptor (5) and, together with tumor necrosis
factor-
, eotaxin (6) and VCAM-1 (7). IL-4 also controls the
specificity of immunoglobulin class switching toward the IgE isotype
(8-11) by inducing the expression of IgE germline transcripts during
the early phase of the switch cascade (12). CD40-mediated signaling
synergizes with the IL-4 signal but has no or little effect by itself
(13). A number of studies have identified the minimal regulatory region in the IgE germline promoter responsible for mediating IL-4-induced activation of transcription (14-17). The pivotal transcription factor
appears to be STAT6 (18, 19), which interacts with DNA upon cytokine
induction. Latent STAT6 is activated in the cytoplasm by tyrosine
phosphorylation. In its activated state, it can dimerize, translocate
to the nucleus, and bind to a specific DNA sequence in the regulatory
region of the IgE germline gene (20). There it functionally interacts
with other constitutively bound factors such as C/EBP (21), PU.1 (22),
and two different sets of NF-
B/Rel family members (23-25). NF-
B
proteins are involved in the synergistic function of the costimulatory
CD40 signal (26). Based on these data, inhibition of IgE isotype
switching and therefore IgE synthesis represents an attractive
therapeutic target.
Synthetic oligonucleotides are attracting increasing interest as tools
for specific manipulation of gene expression and have potential
therapeutic applications. The "antigene" (27-29) strategy is
based on inhibition of transcription of the selected gene by oligonucleotide-directed intermolecular DNA triple-helix formation. A
number of studies have demonstrated DNA triplex structures with critical polypurine:polypyrimidine cis-acting DNA elements found in the
regulatory region of genes and the prevention of DNA binding of
regulatory proteins (30-35). Recently, the synthesis and
physicochemical properties of chemically modified oligonucleotides
containing 2'-aminoethoxy-substituted riboses was reported (36). Such
triplex-forming oligonucleotides (TFO) assemble into very stable
DNA-triplexes and display a > 1000-fold higher association rate
constant compared with their unmodified counterparts (37).
The STAT6 binding site and the composite PU.1/NF-
B1 element in the
IgE germline promoter are composed of an almost perfect DNA polypurine
stretch. To explore the possibility, if the "antigene" approach can
be applied to this regulon, 2'-aminoethoxy-modified oligonucleotides
were tested for their ability to form DNA triplex structures with this
region. The TFOs formed DNA triple-helix structures and inhibited the
interaction of STAT6, NF-
B, and PU.1 in a dose-dependent
fashion with high specificity and selectivity. As a functional
consequence, the activity of the IgE germline promoter to drive
transcription of a reporter gene was blocked.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Reagents--
The human B-cell line DG75 was
carried in Iscove's modified Dulbecco's medium supplemented with 10%
heat-inactivated fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 µg/ml streptomycin. Purified human
recombinant IL4 was obtained from Novartis AG (Basel, Switzerland) with
a specific activity of 0.5 units/ng. The fusion protein composed of
soluble mouse CD40 ligand and soluble mouse CD8 (sCD40L-sCD8) (38), a
kind gift of Dr. Peter Lane, was purified by immuno-affinity chromatography. The three TFOs were synthesized as described (36) and
dissolved in 8% ammonium hydroxide. The MeC-3654 oligonucleotide was
synthesized using 5'-methylcytidine (MeC) as building block like in the
other three TFOs. It is identical in sequence to the 3654 TFO but does
not contain the 2'-aminoethoxy modification. All oligonucleotides were
characterized by analytical anion-exchange high performance liquid
chromatography and matrix-assisted laser desorption-time of flight.
Transient Transfection--
DG75 cells were cultured in fresh
medium for 24 h at 37 °C, then harvested and washed twice in
cold RPMI 1640. Before electroporation, 20 µg of the supercoiled
reporter plasmid LUCwt (22) was mixed with 1 µg of STAT6 expression
vector (22), 1 µg of the Renilla luciferase expression
vector pRL-TK (Promega, Madison, WI), and increasing molar excess of
TFOs in a final volume of 50 µl in phosphate-buffered saline. After
45 min at room temperature, 1 × 107 cells in 250 µl
of cold RPMI 1640 were added and electroporated at 1500 microfarads and
240 V using a Bio-Rad Gene PulserTM. Immediately after transfection,
700 µl of warm culture medium was added and the cells were diluted
with 3 ml of complete culture medium. Aliquots were cultured for
24 h in the presence or absence of induction mixture consisting of
25 units/ml IL-4, 1 µg/ml sCD40L-sCD8 fusion protein (38), and 1 µg/ml anti-CD8 monoclonal antibody. Firefly and Renilla
luciferase activities were quantitated using the Dual Luciferase kit
(Promega) according to the instructions of the manufacturer. The
Renilla luciferase values were used for normalization of
differences in transfection efficiency. The normalized values were used
to calculate the ratio of firefly luciferase activity between induced
versus uninduced cells (induction factor).
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assay (EMSA)--
Nuclear extracts from DG75 cells stimulated
with 400 units/ml IL-4 for 2 h were prepared as described
previously (39). Double-stranded (ds) DNA was end-labeled using
[
-32P]dCTP (3000 Ci/mol) (Amersham Pharmacia Biotech)
and Klenow polymerase (Life Technologies, Inc.). The labeled product
was purified by size exclusion chromatography (G-25 Sephadex
MicrospinTM columns, Amersham Pharmacia Biotech) and polyacrylamide gel
electrophoresis. The binding reaction was done in a total volume of 20 µl containing 2 mM HEPES (pH 7.9), 50 mM
NaCl, 10 mM KCl, 10 mM Tris·HCl (pH 7.5), 1.5 mM EDTA, 20 µg of bovine serum albumin, 15% glycerol, 4 µM ZnCl2, 5 mM dithiothreitol,
0.5 mM phenylmethylsulfonyl fluoride, 25 fmol of
end-labeled dsDNA, 2 µg of poly(dI-dC) (Amersham Pharmacia Biotech),
and 5 µg of nuclear extracts. The reaction mixture was incubated for
30 min on ice and electrophoresed in a 4% native polyacrylamide gel at
4 °C in 1× TBE at 200 V for 1 h. Gels were dried and exposed
to x-ray films at
70 °C (Kodak X-Omat MR).
320 fmol of recombinant NF-
B p50 (Promega) was incubated with 2 fmol
of labeled ds-probe for 30 min at room temperature before 3.2 pmol of
TFOs were added for various time periods.
 |
RESULTS |
The DNA region of the IgE germline promoter encompassing the
STAT6 site and the composite PU.1/NF-
B element represents an almost
perfect polypurine DNA stretch. To investigate the biologic properties
of 2'-aminoethoxy modified TFOs on protein binding and gene induction,
complementary polypyrimidine TFOs were synthesized (Fig.
1). The 21-mer 3654 TFO covers the 3'
half of the STAT6 site, the entire PU.1 motif, and the 5' half of the
NF-
B1 element. The 15-mer 3651 oligonucleotide is shorter at the 3'
end and does not cover the NF-
B1 site. Both TFOs contain one
mismatch with the promoter sequence at position
82. The cytidine at
that position in the TFO was chosen for optimized binding (39). To
determine whether protein binding to this region can be blocked by
TFOs, EMSA experiments were carried out using a radiolabeled
ds-oligonucleotide probe spanning nucleotides
106 to
55 of the IgE
germline promoter (22). Increasing concentrations of the two specific
2'-aminoethoxy-modified polypyrimidine oligonucleotides (3654 and 3651)
or a 2'-aminoethoxy modified control oligonucleotide representing an
irrelevant sequence (3595) were pre-incubated with the radiolabeled
probe for 45 min at room temperature. Then, nuclear extract prepared
from IL-4-treated DG75 cells was added for 30 min on ice. In the
absence of TFOs, three distinct protein bands were observed,
corresponding to a large composite STAT6/NF-
B complex, a predominant
NF-
B, and a faster migrating PU.1 complex (Fig.
2) (22). Increasing concentrations of
both 3654 and 3651 triplex oligonucleotides led to a
dose-dependent reduction of all three complexes. A 270-fold
molar excess of oligonucleotide 3651 led to almost complete
disappearance of the STAT6/NF-
B and the PU.1 complex and a 50%
reduction of the NF-
B band. The same effect was observed with a
90-fold molar excess of the longer 3654 oligonucleotide, showing that
the increased length of the TFO positively affected its inhibitory
behavior. Increasing amounts of TFO led to the appearance of a new band
representing the DNA triple helix, which migrated just above the free
radiolabeled duplex and became more prominent with increasing TFO
concentrations (Fig. 2, lower panel). The
intensity of the triple helix band inversely correlated with the
intensity of the nucleoprotein complexes, demonstrating a direct
relationship between inhibition of protein factor binding and formation
of DNA triplex structures. The observed inhibition of protein binding
was sequence-specific since the control TFO 3595 had no effect.
Similarly, the unmodified polypyrimidine single-stranded
oligonucleotide corresponding to position
106 to
55 of the IgE
germline promoter sequence had no effect. The simultaneous presence of
protein and TFO never led to the appearance of a slow migrating band
indicative of a tetrameric complex consisting of ds-probe, TFO, and
protein. This suggested that protein and TFO binding occurred in a
mutually exclusive fashion.

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Fig. 1.
The IgE germline proximal promoter and
oligonucleotides used in this study. A schematic view of the human
IgE germline promoter is shown. The locations of known cis-acting
elements and the corresponding DNA binding factors are depicted as
black boxes. The start site of transcription is
marked with a bent arrow. Below, the
nucleotide sequence between position 92 and 62 relative to the
major start site of transcription is given along with the sequences of
three TFOs used in this study.
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Fig. 2.
Gel mobility shift assay of protein binding
and triplex formation. An EMSA experiment with ds-probe 106/55 is
shown. The name of the TFO and the molar excess versus the
ds-probe are depicted above and below the
brackets, respectively. The nature of the individual bands
is shown at the left. The lower panel
represents a shorter exposure (12 h) of the lower third of the gel to
better discriminate between the duplex and the triplex DNA.
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|
Further confirmation for the sequence specificity of the TFOs
came from experiments in which a different radiolabeled probe was used.
Increasing amounts of the oligonucleotides were pre-incubated with
another IgE germline promoter probe spanning positions
57 to
3
(41). This DNA (57/3) contains the NF-
B2 element, which is very
similar to the PU.1/NF-
B1 sequence in the 106/55 probe (23). This
sequence produces a complex banding pattern consisting of at least six
different complexes, of which one has been identified to contain
NF-
B family members (Fig. 3) (23).
Even a very high excess of the TFOs did not lead to DNA triplex
formation or to changes in nucleoprotein complex formation compared
with the positive 106/55 control, demonstrating high sequence
specificity of the 3654 oligonucleotide. Identical results were
obtained using a composite STAT6/NF-
B site (42) from the human
eotaxin gene promoter (data not shown).

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Fig. 3.
Specificity of the TFOs for its ds-target
DNA. An EMSA experiment with ds-probes 57/3 and 106/55 as positive
control is shown. On top, the name and the relative
concentration of two TFOs are depicted. The position of the NF- B
containing complex is marked at the left.
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To characterize the triplex-forming reaction in more detail, the time
of addition of the TFO relative to the nuclear extract was varied. The
106/55 ds-probe was either pre-incubated with a 300-fold molar excess
of the 3654 TFO on ice for 45 or 15 min or added simultaneously with
nuclear extract or was pre-incubated with the proteins for the same
time periods before addition of the TFO. After addition of all
components, all samples were further incubated for another 30 min on
ice. The results demonstrated that triplex formation was strongest when
the probe was pre-incubated with the TFO for 45 min and weakest when
added 45 min after the nuclear extract (Fig.
4, lower panel).
Similar to the data shown in Fig. 2, increasing amounts of triplex DNA
correlated with reduced protein binding. It should be noted that, even
in the samples in which the TFO was added after the extract, all three
nucleoprotein complexes were significantly reduced, suggesting that the
TFO could interfere with already formed nucleoprotein complexes (Fig. 4, upper panel). Again, this effect was not
observed with the 3595 control TFO.

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Fig. 4.
Triplex formation as a function of time.
An EMSA experiment with ds-probe 106/55 is shown. On top,
the time of TFO addition relative to the nuclear extract is shown. The
lower panel represents a shorter exposure (3 h)
of the lower third of the gel.
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To corroborate these results, the stability of the triplex structures
at two different temperatures was measured. The 106/55 probe was
simultaneously incubated with nuclear extract and a 300-fold molar
excess of the 3654 TFO. Then the mixtures were incubated for different
time periods on ice or 37 °C and subsequently analyzed. Incubation
on ice in the absence of TFO led to a gradual increase in NF-
B and
STAT6/NF-
B protein binding, while the PU.1 band remained constant.
The STAT6/NF-
B complex became detectable only after 60 min,
suggesting a slower on-rate than the one of the other two proteins
(Fig. 5A, upper
panel). In the presence of the TFO, a gradual increase in
triplex formation was apparent at the expense of free duplex DNA probe
within the first 60 min. Thereafter, no significant amounts of free
ds-probe could be detected (Fig. 5A, lower
panel). Simultaneously, at the longer time points, a gradual
loss of protein binding was observed. At 240 min, no PU.1 and
STAT6/NF-
B bands were measurable and the NF-
B complex was
strongly reduced. It should be noted that inhibition of protein binding
was only observable when all the free available ds-duplex was converted
to the triplex form (Fig. 5A). When the mixtures were
incubated at 37 °C in the absence of TFO, not much difference in
NF-
B and PU.1 protein binding was seen with time, suggesting that
the interaction with the probe occurred much faster, was nearly
complete even after 10 min and remained stable during the experiment
(Fig. 5B). The STAT6/NF-
B complex was not formed at the
higher temperature possibly due to instability under the experimental conditions used. In the presence of the 3654 oligonucleotide, most of
the available duplex DNA was converted to the triple helix structure
already after 10 min, probably due to an increased association rate at
37 °C. Thereafter, a slight and constant increase of the triplex
signal over the observation period could be measured (Fig. 5B, lower panel). Protein binding was
severely reduced at 10 min and resembled the one observed at 240 min
upon incubation on ice. At later time points, an additional decrease of
the remaining NF-
B signal occurred. This suggested that, in the
absence of excess free dsDNA, the TFO actively displaced the remaining
NF-
B molecules from the DNA probe and thus may explain the increase of triplex DNA at the later time points. In addition, the triplex structures appeared to be stable, since no duplex DNA or PU.1 protein
binding could be detected over time. To further substantiate the
possibility that the TFOs can displace dsDNA-bound protein, recombinant
NF-
B p50 was incubated with the radiolabeled 106/55 probe such that
all available DNA was occupied by protein. Then, 3654 TFO was added at
a 10-fold higher molar concentration than the protein and incubated for
various time periods at room temperature (Fig.
6). In the absence of TFO, p50 produced
two nucleoprotein complexes. The lower band likely represents a p50
homodimer, based on the finding that it migrated at the same position
as the NF-
B complex in crude nuclear extracts, which contains p50
(Ref. 23, and data not shown). The upper complex may consist of a
higher order p50 multimer. The presence of 3654 TFO led to increasing formation of triplex DNA in a time-dependent fashion. At
the same time, a loss of intensity of protein binding was observed,
which was most prominent at the longest time point. This effect was specific since the irrelevant 3595 TFO did not lead to triplex formation. The experiment confirms the importance of the 2-aminoethoxy substitution to mediate triplex formation since an oligonucleotide with
the same sequence but lacking this side chain (MeC-3654) was not able
to displace p50. Identical results, albeit weaker, were obtained when
equimolar amounts of TFO and p50 was used (data not shown). These
experiments demonstrated that the specific TFO could efficiently
displace DNA-bound NF-
B p50 by forming DNA triplex structures, which
are then inaccessible for the protein.

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Fig. 5.
Triplex formation as a function of
temperature. EMSA experiment with ds-probe 106/55, mixed with TFO
and nuclear extract at the same time. On top, the incubation
time is shown on ice (A) or at 37 °C (B). The
lower panels represent short exposures of the
lower third of the gels (2 h) to visualize duplex and triplex
DNA.
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Fig. 6.
NF- B displacement
from dsDNA by 2'-aminoethoxy-modified TFOs. EMSA experiment with
ds-probe 106/55 and recombinant NF- B p50. The probe was
pre-incubated with saturating amounts of p50 before a 10-fold excess of
TFO was added for various times. MeC-3654, identical to 3654 in sequence and containing MeC, but lacking all the 2'-aminoethoxy
modification.
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To evaluate the observed inhibitory effect of the TFOs on
IL-4/CD40-induced expression of the human IgE germline gene, transient transfection experiments were carried out. A reporter gene plasmid (LUCwt) in which the human IgE germline promoter drives expression of
the luciferase gene was used (22). IL-4-induced reporter gene
expression in this construct is mediated by a functional cooperation of
STAT6 with PU.1 and NF-
B. Cross-linking of CD40 leads to further
stimulation in a synergistic fashion. This effect is also mediated by
the PU.1/NF-
B1 element
(26).2 The LUCwt DNA was
mixed with increasing concentrations of TFOs and then transfected into
DG75 cells by electroporation. STAT6 expression vector was also
included to maximize IL-4 inducibility (22, 43). The transfectants were
induced with a mixture containing IL-4, a fusion protein consisting of
soluble murine CD40 ligand and soluble murine CD8 (38), and anti-CD8
antibodies to cross-link the CD40 receptor for 24 h. IgE germline
promoter activity was inhibited in a dose-dependent manner
by the 3654 TFO compared with the solvent control. The shorter 3651 oligonucleotide also inhibited reporter gene expression but with less
efficiency (Fig. 7). This is in line with
the results obtained in the EMSA assays, in which the 3651 TFO was also
less effective compared with the 3654 oligonucleotide. In contrast, the
3595 control TFO had no effect. These data demonstrated that the loss
of transcription factor binding due to triplex formation correlated
strongly with the inhibition of gene induction. It also underscores the
importance of this regulatory region of the promoter as mediator of
IL-4/CD40 triggered activation of IgE germline gene expression.

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Fig. 7.
Inhibition of IL-4-induced IgE germline gene
transcription by TFOs. Activity of a human IgE germline promoter
luciferase construct (LUCwt) in transiently transfected DG75 cells. The
induction factor shown is defined as the ratio of firefly luciferase
activity in stimulated versus uninduced transfectants. One
representative experiment out of four is shown. SC, solvent
control containing a 400-fold molar excess of an unmodified irrelevant
29-mer oligonucleotide.
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 |
DISCUSSION |
An almost perfect polypurine stretch in the promoter region of the
IgE germline gene is part of the critical regulatory region involved in
activation by IL-4 and CD40 triggering. The transcription factors
binding to this sequence after activation have been identified to be
STAT6, PU.1, and members of the NF-
B family such as p50 (22-24,
26). 2'-Aminoethoxy-modified oligonucleotides complementary to this
polypurine target formed a triplex structure in a
dose-dependent fashion. Triplex DNA formation occurred with
high sequence specificity since two other similar targets were not
recognized. The binding affinity of the specific TFOs was more than
800-fold higher than that of an unrelated TFO (3595). These numbers are
in good agreement with previous results obtained in surface plasmon
resonance experiments (36). In addition, the binding reaction in the
EMSA assays were carried out at pH > 7, which does not favor the
formation of protonated cytidines, even considering that
5'-methylcytidine was used in the synthesis of the TFOs. These results
support previous findings showing that the interaction of the
2'-aminoethoxy side chains of the TFO with phosphate groups of the
duplex DNA is a very important contribution to the Hoogsteen hydrogen
bonds for the total affinity of the observed DNA triplexes (36, 37).
Although the shorter 3651 TFO does not hybridize directly with the
NF-
B site, protein binding was inhibited to a similar degree as PU.1
or STAT6. This suggests either that the triplex structure affects the
conformation of the neighboring duplex to an extent that precludes
binding of NF-
B proteins or that steric hindrance does not allow
access of the proteins. Alternatively, the presence of STAT6 or PU.1 may be a pre-requirement for NF-
B nucleoprotein complex formation. This alternative seems unlikely, however, because ds-oligonucleotides containing point mutations that specifically abolished protein binding
of STAT6 or PU.1 were still able to bind NF-
B factors (22).
A time kinetic at 37 °C demonstrated that the simultaneous
incubation of protein and TFO with dsDNA led to an increase in triplex
DNA at the expense of protein binding in the absence of free ds-probe.
This effect was even evident when the TFO was added after the nuclear
extract. This strongly suggested competition of the TFOs with the
transcription factors for duplex DNA binding. The Kd
value for a 15-mer 2'-aminoethoxy-modified oligonucleotide has been
determined to be 0.9-4.8 × 10
9
M (36). This interaction is considerably weaker than the
one measured for NF-
B proteins
(10
12-10
13
M) (44) and thus could argue against a competition
mechanism. However, it is difficult to compare these numbers since
different methods, conditions, oligonucleotide lengths, and ds-targets
were used to determine these values. Therefore, it is possible that, under the conditions used in this study, the 21-mer 3654 TFO has a
binding affinity similar or higher than NF-
B, making a dsDNA interaction with the TFO over that with protein more likely. The observed shift from nucleoprotein complexes to DNA-triplex structures with time may also be driven by the large excess of TFO molecules compared with the amount of protein and perhaps by a possible difference in half-lives of the complexes. It has been shown that the
half-life of NF-
B proteins on its target was 45 min (45). In the
absence of free ds-target DNA, shorter-lived dissociating protein-DNA
complexes may be converted to DNA-triplexes with a longer half-life. In
fact, it has been measured that a 2'-aminoethoxy-modified TFO
dissociated at one fortieth the rate from its target duplex than an
unmodified oligonucleotide with the same sequence (36). In addition,
triplex DNA formed with a 2'-aminoethoxy-modified TFO was stable at
55 °C (37), whereas NF-
B binding was not possible anymore at this
temperature (44). It is also possible that the 3654 TFO first binds to
the adjacent STAT6 binding site and then "zips" through the NF-
B
site, competing for both bases and phosphates binding, resulting in
efficient NF-
B displacement. Such a mechanism is not possible with
unmodified oligonucleotides, as they can only interact weakly with the
bases of the dsDNA target. Although our experiments cannot discriminate
between these various possibilities, it is worth noting that the TFOs
were capable of displacing DNA-bound p50 protein. To our knowledge this
is the first demonstration of an NF-
B binding inhibitor that exerts its biological activity on a pre-formed NF-
B nucleoprotein complex as opposed to an experimental set-up in which the inhibitor was pre-incubated with the dsDNA (35, 46, 47).
The simultaneous transfection of a reporter gene plasmid and TFO led to
inhibition of reporter gene activity after stimulation in a
dose-dependent fashion. This result demonstrated that loss of DNA-protein interaction resulted in inhibition of IgE germline promoter activation and thus underscores the importance of the targeted
promoter region in activating gene expression. Although our data do not
prove the existence of triplex structures within the transfected cells,
it is worth noting that the amounts of triplex oligonucleotide
necessary to inhibit gene expression by 50% was similar to that
required to block protein binding to DNA to the same extent. This
suggests that the mechanism(s) responsible for the inhibitory
effect was due to blockade of critical nucleoprotein complex formation
caused by the presence of a local DNA-triplex structure in the IgE
germline promoter.
Overall, this first demonstration of the biological effects of
2'-aminoethoxy-modified oligonucleotides represents a step forward to
overcome the lack of binding affinity and the slow association rate for
the DNA target classically associated with conventional
oligonucleotides. With this type of chemically modified oligonucleotides, displacement of already DNA-bound transcription factors may become feasible in a therapeutic setting. This may be
important not only for inducible genes such as the IgE germline gene
but also for constitutively active genes whose activity is associated
with the disease phenotype. Further efforts are aimed to explore the
properties of these oligonucleotide derivatives with regard to nuclease
resistance and cellular uptake.
 |
ACKNOWLEDGEMENTS |
We thank Walter Grimling for skillful artwork
and Marcel Blommers for support and critically reading the manuscript.
 |
FOOTNOTES |
*
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. Tel.:
43-186634-730; Fax: 43-186634-582; E-mail:
max.woisetschlaeger@pharma.novartis.com.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010260200
2
A. M. Stütz, and M. Woisetschläger, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
IL, interleukin;
TFO, triplex-forming oligonucleotide;
STAT, signal transducer and
activator of transcription;
ds, double-stranded;
EMSA, electrophoretic
mobility shift assay;
MeC, 5'-methylcytidine.
 |
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