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INTRODUCTION |
Triplex-forming oligonucleotides
(TFOs)1 recognize specific
sequences in double-stranded DNA and bind in the major groove at homopurine stretches. Specificity arises from the formation of Hoogsteen or reverse-Hoogsteen hydrogen bonds between the bases of the
pyrimidine or purine TFO and the bases of the purine strand of the
duplex (1-5). Because of their sequence-specific DNA binding property,
TFOs have potential for manipulating gene structure and function in
living cells.
TFOs can inhibit transcription by interfering with regulatory protein
binding or by blocking mRNA elongation by polymerases (4, 6-14).
Alternatively, TFOs can be used for targeted gene modification. They
are capable of directing site-specific DNA damage by delivering a
mutagen to a specific site (1, 15-18). It has also been shown that TFO
binding alone, without a tethered mutagen, can stimulate repair and
recombination and induce site-specific genome changes in cells through
a repair-dependent process (18-30).
The ability of TFOs to induce mutagenesis is relatively efficient when
they are allowed to bind to their target in plasmid DNA in
vitro prior to the transfection of cells (19), but the effect of
the TFO is diminished when intracellular binding is required (18). The
in vivo efficacy of TFOs is potentially limited by multiple
factors in the cellular environment. For example, the presence of
single-strand nucleases can lead to rapid degradation of
oligonucleotides if the 3'-end is not protected (31). The neutral pH
inside cells is suboptimal for triplex formation by pyrimidine TFOs
because of the requirement for protonation of cytosine at the N3
position (32), and high potassium levels inhibit purine motif triplex
formation (33-35).
An additional limiting factor may be the competition with DNA-binding
proteins. Because of the complex structure into which chromosomal DNA
is packaged by histones and other proteins, there has been substantial
debate in the field as to whether chromosomal DNA would be accessible
to TFOs. In studies conducted in vitro, triplexes did not
form on DNA sequences already organized into nucleosomes (36, 37)
except at sites located toward the ends of nucleosomal DNA fragments
(38). Conversely, pre-formed triplexes blocked nucleosome formation on
DNA fragments in vitro (36, 37). In Xenopus
oocytes, Bailey and Weeks (39) found that a specific TFO could inhibit
reporter gene expression on a chromatinized plasmid only when multiple
target sites were introduced into the promoter region in a pattern
designed so that not all of the sites could be simultaneously bound by
a histone octamer. Together, these studies suggested that nucleosome
formation and TFO-mediated triplex formation may be competing processes.
Despite these studies, several lines of evidence support the ability of
TFOs to target chromosomal sites. TFOs have been shown to induce
site-specific chromosomal mutagenesis both in yeast (24) and mammalian
cells (22, 23) and even in mouse tissues following systemic
administration of TFOs in mice (25). Other studies have reported direct
demonstrations of triplex formation on chromosomal targets using a
number of methods for physical detection, including restriction
protection, ligation-mediated PCR, and primer extension (40-43).
However, some questions have been raised about possible artifacts in
certain detection assays (44). In particular, Becker and Maher (44)
showed that unbound oligonucleotides present in cells could persist
following cell lysis and thereby influence the apparent detection of
in vivo formed triplexes in a ligation-mediated PCR
assay (44).
We hypothesized that the apparent discrepancies between the in
vitro and in vivo studies of triplex formation on
chromatinized targets may reflect the dynamic nature of chromatin
structure in vivo. To test this hypothesis, we set out to
examine the effect of transcription on triplex formation in
vivo based on the concept that transcription is a key factor in
altering chromatin structure in living cells. Here, we report the
development of an assay to detect triplex formation at a chromosomal
site in mammalian cells. The assay depends on resistance to restriction
enzyme cleavage at the target site conferred by triple helix-directed
psoralen crosslinks. Using this assay, we tested the effect of
transcription on TFO binding at a chromosomal site where transcription
can be specifically regulated by hormone treatment to activate a
specialized transcription factor. We show here that transcriptional
activity at a chromosomal site can substantially influence the
accessibility of that site to binding by a TFO, especially at low TFO
concentrations. In addition, even in hormone-treated cells when RNA
polymerase activity is poisoned by
-amanitin treatment, TFO
targeting is reduced, further demonstrating a direct effect of
transcription, and not just transcription factor binding, on the
targeting of chromosomal sites by TFOs.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides--
Psoralen-conjugated oligonucleotides were
synthesized by either Midland Certified Reagent Co. (Midland, TX) or
Oligos, Etc. (Wilsonville, OR) and were gel or high pressure liquid
chromatography (HPLC) purified as described previously (22). These
oligonucleotides were synthesized with a propylamine group (Glen
Research) on the 3'-end to prevent degradation by nucleases (45).
Oligonucleotides for cloning were synthesized at the Keck Facility
(Yale University). Their sequences are the following: MM1 (5'-CTA GGA
TCC TTC CCC CCC CTC CTC CCC CTC CCC CTC-3') and MM2 (5'-AGC TGA GGG GGA
GGG GGA GGA GGG GGG GGA AGG ATC-3').
Plasmid Constructs--
Plasmid pMM2 was derived from pIND/LacZ
(Invitrogen) and was constructed to contain a G:C bp-rich site
amenable to triplex formation in the purine motif. The polypurine
duplex target site was created by annealing oligonucleotides MM1 and
MM2. These oligonucleotides were engineered to contain a
BamHI restriction site overlapping the polypurine sequence
at one end. pMM2 was generated by cloning the synthetic duplex into the
HindIII site within the inducible expression cassette in
pIND/LacZ.
Cell Lines--
Cells in which transcription at the TFO binding
site could be manipulated were generated by transfecting ECR-293 cells
(Invitrogen), which stably express the hormone-responsive ecdysone
receptor heterodimer, with ScaI-linearized pMM2 plasmid DNA
using GenePORTER reagent (Gene Therapy Systems, San Diego, CA). Stable
clones with chromosomally integrated pMM2 DNA were selected by colony
formation upon 2 weeks of growth in selective medium consisting of high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mM
L-glutamine, 1% penicillin-streptomycin, 400 µg/ml
zeocin, and 400 µg/ml G418. One resulting cell line, EC293-6, was
chosen for further study. In this cell line, stable chromosomal
integration of the pMM2 DNA was confirmed by genomic Southern
analysis. The target sequence remained stably integrated in the
genome of EC293-6 cells in culture for several months as determined by
genomic Southern analysis (data not shown).
Induction of Transcription and Expression
Analysis--
Transcription at the target locus was specifically
induced by treatment of the cells with the ecdysone analog, ponasterone A. Ponasterone A was dissolved in 95% ethanol at a concentration of 1 mM and added to the growth media of EC293-6 cells at a
final concentration of 5 µM. Cells were incubated at
37 °C for 20 h, extracts were prepared, and induced
-galactosidase activity was detected using the Galacto-Star
chemiluminescent assay (TROPIX, Inc., Bedford, MA). A luminometer was
used to measure light signal output. For direct visualization following
induction with 5 µM ponasterone for 20 h at
37 °C, cells were fixed with 2% formaldehyde and 0.2%
glutaraldehyde in PBS at 4 °C for 5 min, washed three times with
PBS, and stained with 20 mM potassium ferricyanide, 20 mM potassium ferrocyanide, 2 mM
MgCl2, and 0.5 mg/ml X-gal in PBS. After incubation for
2 h at 37 °C, the cells were visualized by light microscopy.
Inhibition of Transcription--
To inhibit transcription,
-amanitin was dissolved in water at a concentration of 1 mg/ml and
added to the growth media of EC293-6 cells at a final concentration of
10 µg/ml. The cells were incubated at 37 °C for 24 h. If
cells were exposed to
-amanitin and ponasterone, ponasterone
was added 12 h after
-amanitin addition.
Oligonucleotide Transfection--
Cells were transfected by
permeabilization with digitonin as described by Giovannangeli et
al. (41). Oligonucleotides, either pso-AG30 or pso-SCR30, were
added to the digitonin-permeabilized cells at final concentrations of
0-20 µM. Following a 1.5 h incubation of the cells
at 37 °C in suspension, the cells were irradiated with long
wavelength UV light (UVA; using a broad band UVA light source (320-400
nm, centered at 365 nm; Southern New England Ultraviolet, Branford, CT)
as described previously in Ref. 46) for psoralen photoactivation to
crosslink bound psoralen-TFOs to the chromosomal DNA. The irradiation
took place over 6 min, corresponding to a total dose of 1.8 J/cm2. UVA doses were determined by radiometry
(International Light, Newburyport, MA), as described previously
(46).
Cell Lysis and Preparation of Genomic DNA--
Immediately
following UVA irradiation, cells were washed with PBS and resuspended
at a concentration of 5 × 106 cells/ml in lysis
buffer (50 mM Tris-Cl, pH 7.5, 20 mM EDTA, 100 mM NaCl, 0.1% SDS), heated to 60 °C for 15 min, and
treated with proteinase K at a concentration of 100 µg/ml overnight
at 37 °C. Lysates were extracted once with phenol (equilibrated with Tris) and twice with chloroform/isoamyl alcohol (24:1). The DNA was
ethanol precipitated and resuspended in 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0, and 100 mM KCl. Once in
solution, genomic DNA samples were incubated at 60 °C for 2 h
to disrupt non-covalent triplexes. Unbound and un-crosslinked
oligonucleotides were removed by filtration through a Centricon-100
(Millipore, Bedford, MA).
Restriction Protection and Southern Analysis--
Following
lysis and specialized sample preparation as above, genomic DNA from
EC293-6 cells was digested with BglII, EcoRI, and
BamHI and analyzed by Southern blotting. The 3.1-kb
HindIII/EcoRI fragment of pIND/LacZ (Invitrogen)
was used as a probe. Band intensities were quantified by a
PhosphorImager (Amersham Biosciences).
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RESULTS |
Experimental Design--
To study the effect of transcription on
triplex formation, we created a cell line in which we could manipulate
transcription at the TFO target site in genomic DNA. Human 293 cells,
previously engineered to express the ecdysone receptor heterodimer,
were stably transfected with a linearized vector (pMM2), containing a
target site for TFO binding downstream of an ecdysone-inducible promoter and upstream of a lacZ reporter gene (Fig.
1A), and stable clones with
the vector sequence integrated within a chromosomal locus were selected
by long term growth in G418. One cell line, EC293-6, was selected for
further study, and chromosomal integration of the vector DNA was
confirmed by genomic Southern analysis (data not shown). In the EC293-6
cells, it was also confirmed that transcription across the TFO target
site could be specifically induced by treatment of the cells with
ponasterone, an ecdysone analog. Ponasterone binds to the ecdysone
receptor heterodimer in the cells, and this causes the receptor to bind
to the ecdysone response element situated upstream of the
Phsp minimal promoter, the TFO target site, and the
-galactosidase coding region (in that order), thereby activating transcription downstream. We measured
-galactosidase activity in
these cells before and after induction with ponasterone. There was very
little
-galactosidase expression in uninduced cells, but
-galactosidase expression was induced ~250-fold above background in cells exposed to 5 µM ponasterone for 20 h (Fig.
1B). Direct staining revealed that expression was induced in
almost all of the cells (Fig. 1C). Hence, transcription
across the TFO target site could be specifically induced by ponasterone
treatment of the EC293-6 cells.

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Fig. 1.
Experimental design to study the influence of
transcription on chromosome targeting by TFOs. A,
ecdysone-inducible transcription at the third strand target site. A
human 293 cell sub-clone (EC293-6) expressing the ecdysone receptor
heterodimer was engineered to contain an expression cassette
incorporating a third strand binding site downstream of an
ecdysone-inducible promoter and upstream of a lacZ
reporter gene within a chromosomal locus, as indicated.
E/GRE, ecdysone/glucocorticoid-responsive
element. B, ponasterone-induced expression of the
lacZ reporter gene. EC293-6 cells were grown in the absence
or presence of 5 µM ponasterone, an ecdysone analog, for
20 h, and -galactosidase activity was measured in cell lysates
using a chemiluminescent substrate. The bars indicate light
signal output. RLU, relative light unit. C,
direct staining of ponasterone-induced EC293-6 cells for
-galactosidase activity by histochemical methods. D,
sequence of the triplex target site within the lacZ reporter
construct and corresponding oligonucleotides. Pso-AG30 binds to the G:C
bp-rich target site in an anti-parallel orientation relative to the
purine strand of the duplex. Pso-SCR30 is a control oligonucleotide
with the same base composition as pso-AG30 but with an altered sequence
that creates 12 mismatches. All TFOs were conjugated to psoralen at the
5'-end and synthesized with a 3'-propylamine group. The third strand
binding site overlaps a BamHI restriction site in the
duplex. Restriction sites and the lengths of the fragments that would
be obtained after BglII, BamHI, and
EcoRI digestion of the DNA are indicated.
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In previous work, we had found that the 30-nucleotide, G-rich TFO,
AG30, could mediate site-directed mutagenesis and recombination in the
supFG1 reporter gene in chromosomal DNA in mammalian cells and mice (22, 25, 30). We therefore chose the 30-bp supFG1 polypurine site as a suitable target sequence to test the influence of
transcription on TFO binding. As explained above, the inducible expression cassette in the EC293-6 cells was designed to contain this
30-bp target site at the position indicated in Fig. 1A so that transcription induced by ponasterone would occur through the 30-bp
site. In addition, the site was designed to overlap a BamHI
restriction site at one end. In this arrangement, triple helix
formation by the 5'-psoralen-conjugated TFO, pso-AG30, would be
expected to position the psoralen for photoreaction at the 5' ApT
3'-sequence within the BamHI recognition site (Fig.
1D). Because triplex-directed psoralen adducts have been
shown to block restriction enzyme cleavage if the adduct is formed
within the recognition site (41, 47), we used this substrate as the
basis for an assay to directly measure intracellular triplex formation. We expected that triplex-directed psoralen adducts could be measured by
the extent of BamHI resistance at the target site in a
quantitative genomic Southern blot analysis.
However, we were concerned that unbound or un-crosslinked TFOs present
in cell lysates might bind to the genomic DNA in vitro after
lysis and so influence the apparent detection of in vivo formed triplexes in the restriction protection assay. We therefore developed a genomic DNA purification procedure to eliminate unbound oligonucleotides and disrupt non-covalent triplexes, thereby allowing persistence only of covalent psoralen third strand adducts in the
genomic DNA. To achieve this, the genomic DNA samples were incubated in
a buffer containing 10 mM Tris, 1 mM EDTA, and
100 mM KCl at 60 °C for 2 h, and unbound and
un-crosslinked oligonucleotides were removed via size filtration prior
to restriction digestion. Under these conditions, triplex formation is
inhibited because EDTA chelates Mg2+, which is important
for stabilizing triple helices via charge neutralization, and the high
concentration of K+ favors the self-aggregation of the
G-rich TFO. In addition, non-covalent triplexes formed by AG30 melt at
60 °C while duplexes remain intact, and the size filtration
separates the free oligonucleotides from the genomic DNA. This approach
was tested and validated via a series of in vitro
experiments on defined DNA samples (data not shown).
Transcription Dependence of TFO Binding at a Chromosomal
Site--
To study the influence of transcription on TFO binding at
the chromosomal site in EC293-6 cells, the cells were either treated or
not with ponasterone to induce transcription at the TFO target site.
They were subsequently transfected with selected oligonucleotides at a
concentration of 20 µM via permeabilization with
digitonin to achieve high levels of intracellular delivery. The
oligonucleotides included either pso-AG30 or a control oligonucleotide,
pso-SCR30, which has the same base composition as pso-AG30 but
possesses a scrambled sequence creating 12 mismatches so that it is not capable of binding to the target site as a third strand (Fig. 1D and Ref. 22). One and a half hours after transfection,
the cells were irradiated with long wavelength UV light. A total UVA dose of 1.8 J/cm2 was given over a period of 6 min to fix
the bound TFO to its binding site via photoactivation of the psoralen moiety.
Immediately following UVA irradiation, the cells were lysed for
preparation of genomic DNA using the protocol described above. The
genomic DNA was digested with BglII, BamHI, and
EcoRI, and the samples were analyzed by Southern blotting
using a probe spanning the lacZ gene (Fig.
2). The appearance of a 3.6-kb fragment
indicates inhibition of BamHI cleavage due to TFO-directed
psoralen crosslinks at the overlapping site. In the absence of
crosslinks at that site, 3.1- and 0.5-kb fragments are produced (Fig.
1D). A 3.6-kb protected fragment is seen in samples from
cells incubated with pso-AG30 and UVA irradiated (Fig. 2A,
lanes 3 and 8), suggesting that intracellular
triplex formation can be detected at the target chromosomal site. Only
the specific TFO, pso-AG30, and not the control oligonucleotide,
pso-SCR30, produced site-specific adducts to inhibit BamHI
cleavage at the target site (compare Fig. 2A, lanes
3 and 8 with lanes 5 and 10). The
inhibition of BamHI cleavage must be due to target site
crosslinking in cells rather than binding of pso-AG30 in
vitro after lysis of the cells, because the 3.6-kb fragment is
absent in samples from cells transfected with pso-AG30 but not UVA
irradiated (Fig. 2A, lanes 2 and
7).

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Fig. 2.
Transcription increases the extent of triplex
formation at a target site in human cells, as detected using a
restriction protection assay. A, Southern blot analysis
of genomic DNA from treated cells. EC293-6 cells grown in either the
absence (lanes 1-5) or presence (lanes 6-10) of
ponasterone were transfected with pso-AG30 (lanes 2,
3, 7, and 8) or pso-SCR30 at a
concentration of 20 µM (lanes 4, 5,
9, and 10). In some cases as indicated, the cells
were irradiated 1.5 h later with long wavelength UV light. A total
UVA dose of 1.8 J/cm2 was given over a period of 6 min to
crosslink the bound TFO to its target site (lanes 3,
5, 8, and 10). Purified genomic DNA
was digested with BglII, BamHI, and
EcoRI and analyzed by Southern blotting. The probe detects a
3.1-kb fragment in unprotected, digested DNA. If the DNA is protected
from BamHI cleavage by TFO-directed psoralen adducts at the
third strand binding site, a longer fragment of about 3.6-kb results
(lanes 3 and 8). B, quantification of
restriction protection. The intensities of bands corresponding to the
protected and unprotected fragments were quantified for each sample
using a PhosphorImager. The bars indicate the percentage of
target site crosslinking as measured by the ratio of radioactive signal
detected in the protected band to the total signal for both bands. The
experiment was repeated three times, and the error
bars give standard error calculations for the percentage
crosslinking values.
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This experiment was repeated three times, and a representative gel is
shown. The intensities of bands corresponding to the protected and
unprotected fragments were quantified for each sample using a
PhosphorImager. The ratio of radioactive signal detected in the
protected band to total signal for both the 3.6- and 3.1-kb bands
provides an estimate of the extent of target site crosslinking, and
standard errors were calculated for the percentage crosslinking values.
Substantial binding (about 10%) was detected in the absence of induced
transcription, so even minimally or untranscribed regions can be
accessible to triplex formation, at least at high concentrations of
transfected TFOs (20 µM). Importantly, however, this
analysis revealed a 3-fold increase in target site crosslinking by
pso-AG30 when transcription was specifically induced at the target site (Fig. 2B). These results demonstrate that transcriptional
activity at a chromosomal site substantially influences the
accessibility of that site to oligonucleotide-mediated triplex formation.
Dose Dependence of Chromosomal Gene Targeting by TFOs--
The
experiment was repeated using varying concentrations of pso-AG30 and
quantified as above to determine the dose dependence of TFO binding at
the target site with or without induction of transcription. As the
concentration of pso-AG30 used to transfect EC293-6 cells was increased
from 0.2 to 20 µM, we detected an increase in the
abundance of the 3.6-kb protected fragment relative to the 3.1-kb
unprotected fragment in both ponasterone-induced and -uninduced samples
(Fig. 3). These data provide further
evidence that inhibition of BamHI cleavage is in fact TFO
dependent. These results are also quantitatively consistent with the
above experiments, as we saw a ~3-fold increase in target site
crosslinking in DNA samples from cells in which transcription was
induced at the target site relative to DNA from uninduced cells when we
used a 20 µM dose of pso-AG30 (Fig. 3). However, when
cells were transfected with pso-AG30 at a concentration of 10 µM, the level of targeting was almost 10-fold higher in
the ponasterone-induced cells than in the uninduced cells. At a 2 µM concentration of pso-AG30, we still detected
substantial TFO-mediated crosslinking in DNA from cells in which
transcription was induced at the target site (about 10%), but target
site crosslinking was less than 1% in DNA from uninduced cells (Fig.
3). These results indicate that at lower concentrations of
oligonucleotide, transcription-modulated accessibility of a chromosomal
site to TFO binding is of increased importance.

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Fig. 3.
Dose dependence of chromosomal gene targeting
by TFOs. EC293-6 cells grown in either the absence or presence of
ponasterone were transfected with concentrations of pso-AG30 ranging
from 0.2 to 20 µM. The cells were irradiated 1.5 h
later with long wavelength UV light. A total UVA dose of 1.8 J/cm2 was given over a period of 6 min to crosslink the
bound TFO to its target site. Purified genomic DNA was digested with
BglII, BamHI, and EcoRI and analyzed
by Southern blotting. The intensities of bands corresponding to the
protected and unprotected fragments were quantified for each sample
using a PhosphorImager. The percent target site crosslinking values
were determined by the ratio of radioactive signal detected in the
protected band to the total signal for both bands. The experiment was
repeated three times, and the error bars give standard error
calculations for the percentage crosslinking values.
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Requirement of Transcription through the Target Region for Enhanced
TFO Binding--
To further study the influence of transcription on
TFO binding, we conducted experiments to determine the relative
contributions to chromosomal accessibility of transcription through the
target site versus transcription factor binding nearby the
site alone. To do this, we used
-amanitin, an RNA polymerase
inhibitor, to block transcription through the target region even when
the ecdysone receptor was activated by ponasterone and capable of
binding to the response element. We first evaluated the effect of
-amanitin on expression at the target locus. EC293-6 cells were
treated with ponasterone,
-amanitin, or both, and
-galactosidase
activity was measured in cell extracts.
-galactosidase activity in
the untreated EC293-6 cells (Fig.
4A) was similar to the
background level (4.9 × 104 relative light
units by this scale) in the parental cell line ECR-293, which
does not contain the
-galactosidase expression cassette. As above,
ponasterone treatment stimulated high levels of lacZ
expression in the EC293-6 cells. However, when
-amanitin was present
in either ponasterone-treated or -untreated cells, lacZ
expression was prevented and in the range of background (Fig. 4A).

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Fig. 4.
Inhibition of RNA polymerase activity
substantially diminishes chromosomal TFO binding. A,
lacZ expression is inhibited in the presence of the RNA
polymerase inhibitor, -amanitin. EC293-6 cells were treated with
either -amanitin, ponasterone, both, or neither, and
-galactosidase activity was measured in cell lysates using a
chemiluminescent substrate. The bars indicate light signal
output. B, influence of -amanitin on TFO target site
binding. EC293-6 cells grown in the absence or presence of ponasterone
(P) and with or without -amanitin (A), as
indicated, were transfected with pso-AG30 at a concentration of 20 µM followed 1.5 h later by UVA irradiation to
crosslink the bound TFO to its target site. Purified genomic DNA was
digested with BglII, BamHI, and EcoRI
and analyzed by Southern blotting. The bars represent
percent target site crosslinking as measured by the ratio of
radioactive signal detected in the protected band to the total signal
for both bands.
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In parallel samples exposed to the same combinations of ponasterone and
-amanitin, EC293-6 cells were transfected with pso-AG30 at a
concentration of 20 µM and UVA irradiated 1.5 h
later. The genomic DNA was purified, and target site crosslinking was
analyzed by Southern blotting and quantified as above (Fig.
4B). We found that
-amanitin treatment of the cells led
to substantially reduced TFO binding even when ponasterone was added,
indicating that transcription through the target region (and not just
hormone-induced transcription factor binding) is important for the
enhanced chromosomal site targeting by TFOs (Fig. 4B).
However, we still detected some (~10%) target site crosslinking in
samples from cells treated with both
-amanitin and ponasterone. In
comparison, less crosslinking was seen in cells treated with
-amanitin in the absence of ponasterone (Fig. 4B).
Therefore, it is possible that transcription factor binding, by itself,
may alter local chromatin structure to some degree to facilitate TFO
binding at nearby sites. In the comparison of samples from cells not
treated with ponasterone,
-amanitin by itself was found to reduce
even the baseline level of TFO binding (Fig. 4B). This
reduction in TFO binding may reflect effects on chromatin accessibility
that are exerted at a distance because of transcription suppression at
flanking loci.
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DISCUSSION |
We have developed a restriction protection assay to physically
detect triple helix-directed psoralen crosslinks at a chromosomal site
in mammalian cells. We found that such an assay can be prone to
artifacts due to the persistence of un-crosslinked oligonucleotide in
cell lysates (data not shown), but the artifacts were eliminated by
modifying the DNA purification procedure. Using the modified protocol,
we provided direct evidence that the TFO, pso-AG30, could bind to a
chromosomal target site in human cells even in the absence of
transcription, with the degree of binding dependent on TFO dose. When
transcription was specifically induced at the target site, however, we
measured substantial increases in target site binding over a range of
TFO concentrations. However, the differential effect of transcription
on binding was greatest at the lower concentrations of the TFO.
By using
-amanitin to inhibit RNA polymerase II in the cells, we
further demonstrated that transcription through the target region, in
particular, plays a major role in the enhanced chromosomal targeting by
TFOs. However, transcription factor binding appeared to moderately
influence accessibility, perhaps via localized effects on chromatin
structure. In addition, a modest effect on accessibility due to overall
cellular transcriptional activity could be inferred from the effect of
-amanitin on the cells not treated with ponasterone. This effect may
reflect the ability of transcription at nearby sites to alter chromatin
structure or DNA topology at the target site, although altered
expression of a putative chromatin accessibility factor could be
another explanation.
DNA within the eukaryotic nucleus is organized and packaged into
chromatin by association with histone proteins. Nucleosomes are the
fundamental units of chromatin and consist of 146 base pairs of DNA
wound around an octamer of histone proteins, which includes two
molecules each of histones H2A, H2B, H3, and H4. Because there are 10 bases per turn of the DNA helix, at least some portion of the 30-base
pair AG30 target sequence would be on the inner face of the helix,
which is in contact with the histone octamer, possibly rendering it
inaccessible to TFO binding. Surprisingly, we detected 10% target site
crosslinking by pso-AG30 at the chromosomal locus even in the absence
of transcription, so chromatin structure in the basal state is not an
absolute barrier to triplex formation in vivo if a
sufficient concentration of oligonucleotide is present in the cell.
However, chromatin structure is dynamic in vivo due to
cellular processes such as transcription, replication, repair, and recombination. We found that the accessibility of a chromosomal site to
TFOs can increase at least 3-fold when transcription is specifically
induced at that site. The increase in binding at the transcribed site
was even greater when the TFO concentration was low. Because
intracellular delivery is a major limiting factor in the use of
oligonucleotides for genetic manipulation (48), we elected to use a
specialized method of transfection (digitonin permeabilization) to
maximize cellular uptake of oligonucleotides for our experiments.
Because other methods of transfection typically yield lower
intracellular oligonucleotide concentrations, transcription may be an
especially important determinant of TFO targeting efficiency when such
alternative methods are used.
Some previous studies were interpreted to suggest that there is no
difference in chromosomal binding of TFOs based on transcription (41,
43). However, these studies tested the effects of agents thought to
have global effects on transcription and did not directly measure
transcriptional activity at the specific target loci. Because the
regions examined in the previous studies were downstream of active
promoters, it is possible that basal transcriptional activity was
already relatively high at these other target sites.
In conclusion, our results demonstrate that at sufficient TFO
concentrations, triplex formation is possible in mammalian cells even
at non-transcribed chromosomal loci. Therefore, even unexpressed or
minimally expressed genes or intergenic regions may be targets for gene
modification by TFOs. However, transcription at a chromosomal site does
substantially increase its accessibility to TFOs, especially at lower
TFO doses, and so TFO-mediated gene targeting may be most effective
when transcriptional activity at the target region is high.