(Received for publication, August 23, 1995; and in revised form, October 11, 1995)
From the
Type I and III fibrillar collagens are the major structural
proteins of the extracellular matrix found in various organs including
the myocardium. Abnormal and progressive accumulation of fibrillar type
I collagen in the interstitial spaces compromises organ function and
therefore, the study of transcriptional regulation of this gene and
specific targeting of its expression is of major interest. Transient
transfection of adult cardiac fibroblasts indicate that the
polypurine-polypyrimidine sequence of 1(I) collagen promoter
between nucleotides -200 and -140 represents an overall
positive regulatory element. DNase I footprinting and electrophoretic
mobility shift assays suggest that multiple factors bind to different
elements of this promoter region. We further demonstrate that the
unique polypyrimidine sequence between -172 and -138 of the
promoter represents a suitable target for a single-stranded polypurine
oligonucleotide (TFO) to form a triple helix DNA structure. Modified
electrophoretic mobility shift assays show that this TFO specifically
inhibits the protein-DNA interaction within the target region. In
vitro transcription assays and transient transfection experiments
demonstrate that the transcriptional activity of the promoter is
inhibited by this oligonucleotide. We propose that TFOs represent a
therapeutic potential to specifically influence the expression of
1(I) collagen gene in various disease states where abnormal type I
collagen accumulation is known to occur.
In response to tissue injury or invasion, a healing response is invoked that ultimately leads to an accumulation of fibrillar type I collagen. This is true for many systemic organs and the heart. Such a healing response, when unabated and invoked in the absence of injury, leads to a progressive interstitial fibrosis that proves pathologic. Parenchymal cell function is compromised by a disproportionate concentration of type I collagen, a characteristic feature of interstitial fibrosis in different organs(1, 2, 3, 4, 5, 6, 7, 8, 9) . Various stages of organ dysfunction are marked by the activation and repression of type I collagen gene, thereby allowing for the design of specific agents to promote the necessary or adaptive phenotype or to repress the onset of pathologic interstitial fibrosis.
A wide array
of hormones, cytokines, and growth factors have been implicated in the
mediation of fibrous tissue
formation(10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) .
Many of these factors mediate their action through transcriptional
mechanisms. Therefore, the study of transcriptional regulatory elements
within the 1(I) and
2(I) collagen gene promoters and their trans-acting protein factors is of major interest. Effector
cells that bring about fibrosis include interstitial fibroblasts and
phenotypically transformed fibroblast-like cells termed myofibroblasts (21) .
Several cis-acting elements in the 1(I)
and
2(I) collagen genes located on both sides of the transcription
start site as well as their trans-acting factors have been
identified (for recent reviews, see (22, 23, 24) ). Very little is known about
the factor(s) binding to the -200 to -140 region of the
1(I) collagen promoter that includes the 35-bp (
)stretch of polypyrimidine sequence and an adjacent
purine-rich sequence (Fig. 1). These sequences are highly
conserved among mammals (25) and correspond to the DNase
I-hypersensitive regions around the transcriptional start site. It is
generally believed that DNase I hypersensitivity represents
nucleosome-free regions which can interact with various regulatory
proteins(26) . cis-acting elements in the -190
to -170, and -160 to -133 region of the mouse
1(I) promoter and trans-acting factors binding to these
elements in NIH-3T3 fibroblast nuclear extracts have been studied in
some detail (27) . Competition experiments in EMSAs provided
evidence that a single factor binds to both of these elements.
Furthermore, in transient transfection experiments, while a 3-bp
substitution mutation in the more distal element (from -194 to
-168) had little effect on the promoter activity, a 3-bp mutation
in the more proximal element (from -160 to -133) resulted
in a 4-fold increase in reporter gene expression, indicating that this
factor negatively regulates transcription (designated IF-1 in (26) ). In contrast, Brenner et al.(28) have
shown that deletion of both regions of the mouse
1(I) promoter
resulted in decreased promoter activity implying positive activation of
transcription.
Figure 1:
Sequences of the -230 to
-120 portion of the rat 1(I) collagen promoter and the
oligonucleotides used in this study. Restriction enzyme recognition
sites are marked. Asterisk indicates the site of radiolabeling
in DNase I footprinting experiments. The double-stranded
oligonucleotides in boxes were used in EMSAs. The underlined sequences marked a through f correspond to the protected areas seen in DNase I footprinting
studies. Arrowheads indicate sites of substitution mutations
used in the study by Karsenty and de Crombrugghe(27) .
Single-stranded oligonucleotides Oligo Col TFO, Oligo Control, and
double-stranded Oligo C-1 were used in the triple helix experiments (Fig. 4Fig. 5Fig. 6).
Figure 4:
Gel mobility shift analysis of
oligonucleotide-directed triplex formation on the 1(I) collagen
promoter target. Double-stranded target oligonucleotide Oligo C-1 was
end-labeled and incubated alone (lanes 1 and 6) or
with an excess of unlabeled single-stranded Oligo Col TFO (lanes
2-5), and Oligo Control (lanes 7-10). The
concentration of the oligonucleotide added to approximately 1 nM target DNA is indicated above each lane. D, duplex DNA; T, triplex DNA.
Figure 5:
Electrophoretic mobility shift analysis of
the effect of triplex-forming oligonucleotide Oligo Col TFO on the
binding of protein factor(s) present in RCF nuclear extracts to the
target region of the 1(I) collagen promoter. Radiolabeled
double-stranded target Oligo C-1 (2.5 nM) (lanes
3-12) was preincubated with increasing concentrations of
single-stranded Oligo Col TFO (lanes 5-8), and Oligo
Control (lanes 9-12). Samples were then incubated with
RCF nuclear extracts (lanes 4-12), and electrophoresed
in a polyacrylamide gel. Radiolabeled single-stranded Oligo Col TFO was
also incubated with RCF nuclear extracts to exclude binding of protein
factors to single-stranded DNA (lane 2). Protein-DNA complexes
are indicated by arrows.
Figure 6:
In vitro run-off transcription assay
showing the effect of promoter-targeted triplex-forming Oligo Col TFO
on the transcriptional activity of the 1(I) collagen promoter. The
-1000 to +100 sequence (lanes 1-6) and the
-150 to +100 sequence (lanes 7-10) of the
1(I) collagen gene, and, the -600 to +300 fragment of
the CMV IE gene (lanes 11-16) were used as templates in in vitro transcription assays. Oligonucleotides at the
indicated concentrations were incubated with these templates in
separate reactions, followed by HeLa nuclear extract-initiated
transcription. Radiolabeled transcription products of expected sizes (100 and 300 nucleotides, respectively) are
shown.
The 35-bp stretch of pyrimidines located from
-172 to -138 of the rat 1(I) collagen promoter could
potentially serve as a target for a novel antisense strategy, namely
the triplex strategy, which employs single-stranded DNA
oligonucleotides that bind to the major groove of a double-stranded DNA
to form a triple helix in a sequence-specific manner. These complexes
have been shown to inhibit sequence-specific DNA-binding proteins,
thereby affecting the transcriptional activity of various promoters in
both in vitro and in vivo experiments(29, 30, 31, 32) .
In the present study, we provide evidence for the presence of
multiple factors in rat cardiac fibroblasts that specifically interact
with the -198 to -138 sequence of the rat 1(I)
collagen promoter. We further show that a single-stranded polypurine
oligonucleotide can form a triple-helix structure with the long
polypyrimidine segment of the promoter, inhibits the protein DNA
interaction in this region and in an in vitro transcription
system specifically blocks the transcriptional activity of the long
1(I) promoter. In addition, this triple helix-forming
oligonucleotide significantly inhibits the expression of a reporter
gene, CAT, driven by the rat
1(I) promoter in rat cardiac
fibroblasts.
Figure 2:
DNase I footprint of a segment of the rat
1(I) collagen promoter. The BglII-HaeIII
fragment (-222 to -123) of plasmid pCol 1.1 labeled at 3`
end of the noncoding strand was incubated without or with nuclear
extracts of rat cardiac fibroblasts and HeLa cells, and was treated
with DNase I for 30 s (lane 1) or 1 min (lane 2) in
the absence of nuclear extracts; lanes 3 and 4, 30
µg of RCF nuclear extracts and DNase I digestion for 1 or 2 min,
respectively; lanes 5 and 6, 30 µg of HeLa
nuclear extracts and DNase I digestion for 1 or 2 min, respectively.
Different areas of protection are marked by brackets. Numbers
on the right correspond to base pairs upstream of the start of
transcription.
Figure 3:
Binding of RCF nuclear factors to Oligo
C-1 and Oligo C-2 regions of the rat 1(I) collagen promoter. DNA
binding was analyzed by EMSA. Labeled double-stranded Oligo C-1 (lanes 1-7) and Oligo C-2 (lanes 8-14)
were incubated with nuclear extracts of RCF (except for lanes 1 and 8 where bovine serum albumin was used) and
fractionated on 6% polyacrylamide gel. Competition experiments were
performed with increased amounts of unlabeled Oligo C-1 (lanes
3, 4, 12, and 13), Oligo C-2 (lanes
5, 6, 10, and 11), and Oligo C-3 (lanes 7 and 14). Arrows indicate the slow
migrating protein-DNA complexes.
Substantial homology exists in the sequences of the different binding sites as we discussed above. The experiments of Karsenty and de Crombrugghe (27) suggested the presence of negative regulatory factors in NIH 3T3 cell nuclear extracts that bind to both oligonucleotides of the mouse promoter corresponding to Oligo C-1 and C-2. Therefore, we performed cross-competition experiments using unlabeled Oligo C-1 to compete the binding of labeled Oligo C-2 and vice versa (Fig. 3, lanes 5 and 6 and lanes 12 and 13). The results show that Oligo C-1 was able to completely compete with the binding of all three complexes to Oligo C-2 probe (Fig. 3, lanes 12 and 13) indicating that Oligo C-1 contains all the elements required for binding of nuclear factors to Oligo C-2. Once again, a differential competition pattern among the three complexes can be seen (the middle complex is competed at lower molar excess of Oligo C-1 than the top and lower ones). By contrast, when unlabeled Oligo C-2 was used to compete with the binding of nuclear factors to Oligo C-1 probe, complete competition was not seen (Fig. 3, lanes 5 and 6). Only the slower moving complex was competed, and the lower complex showed no inhibition. In fact, it would appear that in the presence of unlabeled Oligo C-2, the binding of Oligo C-1 to nuclear factor(s) becomes more compact and slightly shifted. The results suggest that Oligo C-2 does not contain all elements necessary for the formation of complexes like Oligo C-1. It is also possible that Oligos C-1 and C-2 may contain some closely related elements that have different binding affinities to the same protein factor. The fact that the slower moving complex (Fig. 3, top arrow) is formed with both C1 and C2, it is very likely that this factor may be similar to 1F-1(27) . The other complex may be more specific to rat cardiac fibroblasts. Further analyses with mutant oligonucleotides and both mouse and rat nuclear extracts are required to identify the nature of these factors.
Figure 7:
Inhibition of 1(I) collagen promoter
directed transcription by Oligo Col TFO in adult rat cardiac
fibroblasts. a, pColCAT220 (lanes 1-4) or
pColCAT140 (lanes 5-8) reporter constructs were
transfected into RCF cells. Two h later cells were retransfected with
Oligo Col TFO or Oligo Control as indicated. Cell lysates were assayed
for CAT activity 24 h later. These data are representative of three
independent experiments. cpm/µg represents the acetylated
counts/µg of protein. The percentage acetylation was calculated as
(radioactivity in the acetylated areas)/(total extracted from thin
layer plates)
100. b, histogram showing the results of
transient transfection. After adjusting for
-galactosidase
activity to normalize transfection efficiency, the CAT assay counts
from treated plates were divided by full activity counts to generate
percent activity.
Two previous studies on the regulatory elements of the
1(I) collagen gene have shed considerable light on the cis-acting elements, trans-acting factors and their
functional properties in both in vitro and in vivo experiments. Data from Karsenty and de Crombrugghe (27) have shown two distinct binding sites (from -190 to
-170, and from -160 to -133) within the mouse
1(I) collagen promoter. Competition experiments coupled with
substitution mutation analyses indicated that the same factor contained
in NIH-3T3 nuclear extracts bound to both of these sites. DNA
transfection experiments using 3-bp substitution mutants in these
polypyrimidine and purine-rich sites suggested that this factor acted
as a transcriptional inhibitor (designated IF1)(27) . Our data
indicate the presence of multiple binding sites within the sequence
-190 to -130. EMSAs identified two distinct complexes bound
to Oligo C-1 and three slow migrating bands when Oligo C-2 was used as
probe. It appears that at least one factor binding to C1 is unique as
it is not competed out by C-2. While some of these proteins are similar
to the ones described by Karsenty and de Crombrugghe(27) ,
distinct differences are also evident. These multiple bands could be
produced by the interaction of different size proteins with or by the
formation of homo- or heterodimers. The possible presence of multiple
factors and binding elements within these two regions may offer an
alternative solution to the seemingly conflicting data shown in the two
studies cited, regarding the opposite functional activity of these
promoter elements. Karsenty and de Crombrugghe (27) introduced
substitution mutations into both proximal and distal elements, while
leaving other potentially positive binding elements intact. On the
other hand, both we and Brenner et al.(28) used
deletion mutations of longer segments of the promoter, thereby
eliminating the binding of both negative and putative positive trans-acting factors. It should be pointed out that deletions,
as opposed to point mutations, disrupt the normal organization of the
promoter and enhancer and therefore may affect the interactions of
different factors. Nevertheless, our studies clearly indicate the
presence of some positive regulatory factors that interact with the
triplex-forming sequence. The data of Karsenty and de Crombrugghe (27) indicated the binding of a single factor binding to both
sites (-190 to -170 and -160 to -130). Since
TFO used here is specific to -170 to -141, it is possible
that the binding of the negative factor to both sites is essential to
exert its maximal effect. Some of the differences between these studies
may be due to inherent differences in the promoter sequences and also
in the host cells used. For instance, while the proximal polypyrimidine
sequence is identical between rat and mouse
1(I) collagen
promoter, the distal polypurine sequence shows only 80% homology.
Oligonucleotides provide novel reagents for inhibition of gene
expression because of their high affinity binding to specific
nucleotide sequences. Two strategies for oligonucleotide reagents have
been used. The best known involves antisense oligonucleotides, which
bind mRNA to inhibit its processing or translation. The second is the
triplex strategy, which employs single-standard DNA oligonucleotides
that bind to the major groove of a double-standard target DNA (for
review, see (40) and (46) ). The advantages of the
triplex approach include fewer and less degenerative targets, thus
offering the potential for low dose long-acting therapeutics. The major
limitation of the application of oligonucleotide-directed triplex
formation to naturally occurring sequences is the requirement for
predominantly purine:pyrimidine tracts. The long polypyrimidine
sequence of the 1(I) collagen promoter represents a unique
structure that provides an attractive target for the design of
sequence-specific DNA binding agents, which may influence transcription
of this biologically important gene. Although most studies have
employed pyrimidine-rich TFOs, we chose to use a 30-mer polypurine
oligonucleotide corresponding to the noncoding strand of the promoter
between -170 and -140 because of its binding stability at
physiological pH. It has been suggested that triplex formation is based
on the assembly of G-GC, T-AT, and A-AT
triplets(39, 41, 46) . The orientation of the
purine type TFOs in the major groove of the double helical DNA has
initially been a matter of controversy. In the first description of
triplex formation in the promoter of the human c-myc gene, it
was implied that the TFO was bound parallel to the purine
strand(31) . Later evidence suggested that the TFO in that
study could potentially bind either parallel or antiparallel and make
similar base contacts with the duplex(42) . This is because the
c-myc target is pseudopalindromic. We used a TFO that is in
parallel orientation with the purine strand and similarly has
pseudopalindromic sequences. The binding orientation therefore is
likely to be antiparallel. This may explain why slightly higher K
values were observed in gel mobility shift
assays. The repression of the in vitro transcription of the
collagen promoter by the specific TFO was complete even at lower
concentration than was predicted by TFO titration experiments. This
difference could be the result of stabilization of the template and the
triplex by components of the nuclear extract (e.g. proteins
and polyamines).
To evaluate the effect of triple helix formation on
the transcriptional activity of the 1(I) promoter, we employed an in vitro transcription system using HeLa nuclear extracts. The
reason for this is that extracts from rat cardiac fibroblasts could not
sustain transcription due to an apparent RNase activity, which we were
unable to avoid even with the use of RNase inhibitors or different ways
of preparing nuclear extracts. The usefulness of the HeLa system in the
study of collagen gene expression is supported by the work of Furth et al.(43) , who showed that type I collagen mRNAs are
accurately initiated by HeLa cell RNA polymerase II. In HeLa cell
nuclei, significant amount of collagen mRNA is synthesized. However,
steady-state levels of mRNA are not detected, suggesting
post-transcriptional regulation of collagen synthesis in HeLa cells.
Furthermore, in the study of Brenner et al.(28) ,
deoxyribonuclease I footprints of the more proximal promoter from
-103 to -82 showed the same pattern of protection for both
HeLa and NIH 3T3 nuclear extracts. We also performed DNase I
footprinting assays on the promoter fragment from -220 to
-120 using HeLa nuclear extract. Fig. 2shows that the
protection pattern of HeLa (lanes 5 and 6) and RCF (lanes 3 and 4) nuclear extracts is identical,
lending further support to the usefulness of the HeLa transcription
system.
The results of the transient transfection experiments
parallel the findings of the in vitro transcription assays.
Nevertheless, there are important differences that deserve further
discussion. First, the concentration of oligonucleotides relative to
plasmid template was markedly different between these two assays. One
possible explanation is that a much smaller proportion of plasmids are
actually expressed in cells after transfection is established and that
oligonucleotides are taken up by cells with much higher efficiency. The
intracellular environment may also be more stabilizing for the
formation of triple helix structure. Second, the maximum inhibition was
about 50%, as opposed to almost complete inhibition of in vitro transcription by Oligo Col TFO. At higher than 1 µM oligonucleotide concentration, there was a decrease in the
extractable protein concentration seen, and the expression of pSV2Gal
was also affected (data not shown). These nonspecific effects likely
represent toxicity and demonstrate the relatively narrow
``therapeutic range'' of TFOs. Chemical modification of
oligonucleotides may improve their potency and will likely widen the
margin of safety. Recently published data from Laptev et al.(44) showed the expression of human 1(I) collagen
gene can be effectively inhibited by modified antisense
oligonucleotides at 0.2 µM concentration targeted at
specific regions of the
1(I) mRNA. Due to the differences in
experimental conditions (cell line used; stable versus transient transfection; reporter construct; chemical modification
of oligonucleotides), it is difficult to extrapolate data for
comparison of relative efficiencies. Therefore, it will be important to
directly compare the efficacy of TFOs to antisense oligonucleotides
under the same test conditions.
The mechanism by which Oligo Col
TFO-directed triple helix formation inhibits the transcriptional
activity of 1(I) collagen promoter is not entirely clear from the
data presented here. One likely possibility is the
concentration-dependent interference of Oligo Col TFO with the
formation of complexes between cis-acting elements within the
target region and their cognate trans-acting factor(s). The
ability of triplex-forming oligonucleotides to compete with
site-specific DNA-binding proteins for binding to target sites, as the
mechanism accounting for transcriptional repression, has been
demonstrated in a number of in vitro and in vivo experiments. The close correlation observed in our study between
the ability of Oligo Col TFO to inhibit protein-DNA interaction (Fig. 5) and to repress promoter activity ( Fig. 6and Fig. 7) would support, but not conclusively prove, this
mechanism. However, considering that the polypyrimidine target site for
Oligo Col TFO has previously been shown to contain elements for binding
of a negative trans-acting factor (IF-1)(27) , one
would expect that inhibition of this factor to bind to its cis-element would result in transcriptional activation. To
reconcile these seemingly contradictory findings, the presence of
factor(s) with potential positive regulatory activity and an overall
positive transcriptional net effect within this target region could
once again be considered. Our DNase I footprinting and EMSA data
supports this notion, and so does the previously cited result of
Brenner et al.(28) , confirmed by our transient
transfection experiment, showing a 50% reduction of promoter activity
upon complete deletion of sequences corresponding to our TFO target
site. An alternative explanation for the inhibitory effect of Oligo Col
TFO on
1(I) collagen promoter activity could be adopted from the
studies by Maher et al.(45) , which showed that
site-specific DNA triple helices can repress transcription even when
the complexes do not overlap transcription factor binding sites. Their
results suggested other possible repression mechanisms including
effects on DNA flexibility, recruitment of inhibitory factors, or
alteration of chromatin structure. The results of our in vitro transcription assays, showing complete elimination of promoter
activity, as opposed to only partial inhibition that would be expected
if protein factor binding inhibition was primarily operational, support
these latter mechanisms. The only partial inhibition (around 50%) of
reporter gene expression by Oligo Col TFO in transient transfection
experiments demonstrates the differences and difficulties one
encounters when using intact cells. Apparent toxicity prevented us from
employing higher concentrations of oligonucleotides.
The
identification and characterization of genes that play important roles
in cellular processes leading to interstitial fibrosis have provided
excellent targets for transcriptional modulation. Because of the
ability of TFO's to selectively inhibit transcription of their
target genes in intact cells(29, 30, 32) ,
these oligonucleotides appear to have considerable potential as
therapeutic agents. We have identified the unique polypyrimidine of the
1(I) collagen promoter as a suitable target for a single-stranded
polypurine oligonucleotide to form a triple helix structure that could
effectively inhibit transcription in vitro and in cultured
cells. Further experiments to explore the potential therapeutic
applications of chemically modified TFOs in tissue culture and in
animal model systems are in progress.