(Received for publication, September 19, 1996, and in revised form, December 11, 1996)
From the Departments of Type VII collagen is the major component of
anchoring fibrils, structural elements that stabilize the attachment of
the basement membrane to the underlying dermis. In this study, we have
dissected the human type VII collagen gene (COL7A1)
promoter to characterize the cis-elements responsible for
the expression of the gene in cultured fibroblasts and keratinocytes.
Using transient cell transfections with various 5 The collagens comprise a family of proteins that play a crucial
role in the maintenance of the extracellular matrix integrity. The more
abundant collagens, such as types I and III, have a widespread distribution throughout the body, whereas some minor collagens have a
strictly limited tissue location. Among the latter, type VII collagen
is found exclusively in the basement membrane zone of stratified
squamous epithelia, such as in the skin, various mucous membranes, and
the cornea of the eye (1, 2). Specifically, type VII collagen is the
predominant, if not the exclusive, component of anchoring fibrils,
attachment structures that play a critical role in ensuring stability
to the association of the basement membrane zone to the underlying
papillary dermis (3, 4). Although it is generally believed that the
basement membrane is of epithelial origin, differentiated fibroblasts
adjacent to epithelial tissue in vivo produce basement
membrane components in general and type VII collagen in particular and
assist in basement membrane assembly and anchoring fibril formation on
the dermal side of the basement membrane (5). Therefore, it is
considered that the two main cell types producing type VII collagen are
keratinocytes and fibroblasts. Type VII collagen is a homotrimer,
[ Synthesis of functional anchoring fibrils is of critical importance in
providing integrity to the cutaneous basement membrane zone, and
abnormalities in these structures clinically manifest as dystrophic
forms of epidermolysis bullosa, a group of bullous diseases
characterized by cutaneous fragility and tendency to sub-basal lamina
densa blister formation (8). In fact, recent cloning of
COL7A1 genomic sequences in our laboratory (9, 10) allowed
us to demonstrate that mutations within this gene are associated with
different forms of dystrophic epidermolysis bullosa (11, 12). Analysis
of the 5 In this study, we have investigated the molecular mechanisms regulating
the activity of human COL7A1 in dermal fibroblasts and
epidermal keratinocytes, two principal cell types in the skin expressing the type VII collagen gene (2, 4).
Human dermal fibroblast cultures, established
by explanting tissue specimens obtained from neonatal foreskins, were
utilized in passages 3-6. The cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated fetal
calf serum, 2 mM glutamine, and antibiotics (100 units/ml
penicillin, 50 µg/ml streptomycin-G, and 0.25 µg/ml FungizoneTM).
Human epidermal keratinocyte cultures, initiated by explanting foreskin
specimens, were grown in serum-free, low calcium (0.15 mM)
keratinocyte growth medium (Clonetics Corp., San Diego, CA),
supplemented with epidermal growth factor, hydrocortisone, insulin, and
bovine pituitary extract. Keratinocyte cultures were utilized in
passages 1 and 2 to avoid differentiation inherent to prolonged
subculturing of these cells. Drosophila melanogaster
Schneider SL2 cells (14), kindly provided by Dr. James B. Jaynes
(Jefferson Medical College), were grown in Schneider medium (Life
Technologies, Inc.) supplemented with antibiotics (100 units/ml
penicillin, 50 µg/ml streptomycin-G, 0.25 µg/ml FungizoneTM) and
12% heat-inactivated fetal calf serum.
To study the transcriptional regulation
of human type VII collagen gene (COL7A1) expression,
transient transfection experiments were performed with various
COL7A1 promoter 5 pPacSp1, an expression vector for Sp1 driven by the actin promoter
(16), a kind gift of Dr. Robert Tjian (University of California,
Berkeley, CA), was used to express Sp1 in Drosophila SL2
cells deficient in Sp1. Empty pPac0 was used as a control.
Transient cell transfections of human
dermal fibroblasts were performed with calcium phosphate/DNA
co-precipitation procedure (17). Briefly, cultured cells were
transfected with 10 µg of plasmid DNA and 2 µg of the
pRSV- Basal keratinocytes grown in keratinocyte growth medium were
transiently transfected with a liposome-based method (DOTAP, Boehringer
Mannheim), according to the manufacturer's protocol. Sixteen
hours after transfection, medium was replaced and cells were
incubated for 40 h. At the end of incubation, the cells were harvested by scraping and lysed by three cycles of freeze-thawing in
200 µl of reporter lysis buffer. Aliquots corresponding to identical
Drosophila SL2 cells were transfected with 2 µg of The wild type (WT) fragment
spanning the region between nucleotides
Nuclear extracts were isolated from human dermal fibroblasts or
epidermal keratinocytes using a small scale preparation (20). Samples
were aliquoted and stored at Nuclear extracts (5 µg) were incubated for 20 min on ice in binding
reaction buffer (10 mM HEPES-KOH, pH 7.5, at 4 °C, 4% glycerol, 40 mM KCl, 0.4 mM EDTA, and 0.4 mM dithiothreitol) in the presence of 1 µg of poly(dI-dC)
prior to the addition of gel-purified, 5 To localize
the cis-acting elements involved in the basal activity of
the COL7A1 promoter, human dermal fibroblast and epidermal keratinocyte cultures were transiently transfected with several 5
To elucidate the transcriptional mechanisms of
type VII collagen gene expression, and in particular, the role played
by the region of promoter comprised between residues
In parallel studies, fragment I was used as a probe in gel
mobility shift assays. An electrophoretic pattern similar to that
obtained with radiolabeled WT was observed, with the exception that the
slowest migrating complex appeared as a doublet instead of a single
band; these bands were identified as shifts 1 and 2 (Fig. 3,
lanes 6-9). The formation of all three distinct DNA-protein complexes 1, 2, and 3 was abolished by the addition of a 60-fold molar
excess of unlabeled oligonucleotide I (Fig. 3, lane 7) but not by a 60-fold molar excess of unlabeled oligonucleotides II or III (Fig. 3, lanes 8 and
9, respectively), further indicating that these three
complexes specifically bind to fragment I. No specific
DNA-protein complex was detected when either fragment II
(Fig. 3, lanes 10-13) or fragment III (data not
shown) was used as a probe. Similar results were obtained when nuclear
proteins extracted from either dermal fibroblasts (see Fig. 3) or
epidermal keratinocytes (data not shown) were used in gel mobility
shift assays with the various radiolabeled probes. It should be noted
that the electrophoretic pattern generated by nuclear extracts from
fibroblasts is similar to that of keratinocyte extracts, both
qualitatively and quantitatively (Fig. 4, lanes
3 and 6 versus lanes 2 and 5), suggesting
that the regulatory mechanisms controlled by this region of
COL7A1 promoter may be similar in both cell types.
The next set of experiments was designed to
further identify the cis-acting element(s) responsible for
the binding of fibroblast nuclear extracts to the 34-bp segment of the
COL7A1 promoter located between residues
The critical role of box A was further confirmed in competition
experiments with unlabeled oligonucleotides I To further understand the
importance of box A in the transcription of COL7A1, an
oligonucleotide, ImA, containing a double mutation in box A
(in bold), GGGTGGGG
To evaluate the contribution of the GT box identified between residues
GT boxes,
such as found in box A, have been previously described in other gene
promoters and shown to be potential binding sites for the transcription
factor Sp1 (21), although more classical Sp1-binding sites generally
consist of GC boxes (22, 23). Further, the electrophoretic pattern
observed in mobility shift assays with radiolabeled oligonucleotide
I as a probe closely resembles that of Sp1 binding. To
ascertain that the GT box between residues
These observations prompted us to explore the possibility that Sp1 or
an Sp1-related protein may interact with the GT box located between
nucleotides Recently, several novel factors binding to either GC or GT boxes have
been identified by cDNA cloning, and many of these proteins are
closely related to Sp1 (24-27). Indeed, three of these proteins have
been characterized in detail and designated Sp2, Sp3, and Sp4, putative
new members of the Sp1 multigene family. These proteins are predicted
to contain zinc finger and trans-activation domains similar
to those found in Sp1. Among these factors, Sp3 is strikingly homologous to Sp1 (26). However, a polyclonal antibody against Sp3 did
not supershift any of the DNA-protein complexes formed by incubating
nuclear extracts from fibroblasts with either WT or I
oligonucleotides, indicating that Sp3 does not participate in the
formation of these shifts (data not shown). Taken together, these
results suggest that Sp1 is the transcription factor involved in the
binding to the GT box located between nucleotides To
formally prove that Sp1 binds to the 8-bp GT-rich box of the
COL7A1 promoter (box A), gel mobility shift assays were
performed using human recombinant Sp1 protein (Fig. 8).
Indeed, recombinant Sp1 effectively bound to probes WT (Fig. 8,
lane 2) and I (Fig. 8, lane 4) and to
the consensus Sp1-binding site oligonucleotide (Fig. 8, lane
8). However, this DNA-Sp1 interaction was not noted when
oligonucleotide ImA, in which the GT box has been altered,
was used as a probe (Fig. 8, lane 6). Together with the results of competition and supershift assays described above, these
data unequivocally demonstrate that Sp1 is the protein binding to the
GT-rich sequence located between nucleotides
These results clearly identify that all major DNA-protein complexes
observed with either probe WT (shifts 1 and 3) or probe I
(shifts 1 and 2) are due to Sp1 binding. Because the formation of
multiple complexes between Sp1 and a single DNA probe has been
previously reported (24, 28), we speculate that the fastest migrating
complex (shift 3) is a minor degradation product of the Sp1 protein, as
described previously (29). The two larger complexes observed with
radiolabeled oligonucleotide I (shifts 1 and 2) are likely
to represent two states of differential phosphorylation of Sp1 (30). At
this point, we cannot explain the presence of a single upper band when
oligonucleotide WT is used as a probe.
As a critical test for the requirement of Sp1 in
COL7A1 promoter activity, the
The interest in studying the regulation of COL7A1 gene
expression is motivated by the fact that this gene product is essential for the integrity of the attachment of the epidermis to the underlying dermis. Indeed, structural abnormalities, paucity, or even absence of
type VII collagen/anchoring fibrils manifest clinically as the
dystrophic forms of epidermolysis bullosa, a group of blistering skin
diseases characterized by cutaneous fragility and tendency to sub-basal
lamina densa blister formation (31). In the present study, we provide
compelling evidence for the role of the transcription factor Sp1 in
maintaining high expression of COL7A1 in both fibroblasts and keratinocytes, the two principal cell types expressing type VII
collagen gene in the skin (2, 4).
It has been previously assumed that the transcription initiation of
eukaryotic genes could be directed either by the TATA sequence as in
tissue-specific genes or by GC/GT boxes as is the case in several
TATA-less housekeeping genes. In the latter case, the TATA-binding
protein, TFIID, does not directly interact with the DNA but is
positioned indirectly to the correct initiation site via
protein-protein interactions. In this context, Sp1 has recently been
found to be able to recruit TATA-binding protein, thereby positioning
the initiation complex to the correct start site (32-34). The Sp1
binding site crucial for the maintenance of the high transcriptional
activity of human COL7A1 is located at position Although Sp1 was originally described as a ubiquitous transcription
factor regulating housekeeping genes, recent observations indicate that
its expression can be regulated during development (35) and that it may
be important for the cell type-specific regulation of gene expression,
as exemplified by studies on the keratinocyte-specific keratin K3 gene
(36), demonstrating a direct correlation between the levels of Sp1 and
the expression of K3. In the case of type VII collagen, Sp1 binding to
box A was identical in both fibroblasts and keratinocytes (see Fig. 4),
contrasting with the results of Regauer et al. (36) on K3 gene expression. Both cell types have been shown to express and synthesize type VII collagen (5, 36-38), and although basal expression
of COL7A1 in vitro may be higher in keratinocytes (39, 40),
there is clear indication that fibroblasts participate in producing
basement membrane components, including type VII collagen in
vivo and assisting in basement membrane assembly (5).
The Sp1 binding elements have also been suggested to play a role in the
regulation of the expression of other collagen genes. For example, Sp1
sites between nucleotides In summary, using a series of 5 We thank Dr. Robert Tjian (University of
California, Berkeley, CA) for the generous gift of the pPacSp1
expression vector and Dr. James B. Jaynes (Jefferson Medical College)
and Dr. John Varga (University of Illinois in Chicago, IL) for the gift
of Drosophila SL2 cells. Expert technical assistance of
Ying-Jee Song is gratefully acknowledged.
Dermatology and Cutaneous
Biology and
Biochemistry and Molecular Pharmacology,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
end deletion
COL7A1 promoter/chloramphenicol acetyltransferase reporter
gene plasmid constructs, we determined that the region between
nucleotides
524 and
456, relative to the transcription start site,
is critical for high promoter activity in both cell types studied. Gel
mobility shift assays using several DNA fragments spanning this region
identified a GT-rich sequence between residues
512 and
505,
necessary for the binding of nuclear proteins to this region of the
promoter. Point mutations abolished the binding of nuclear proteins in
gel shift assays and drastically diminished the activity of the
promoter in transient cell transfections. Supershift assays with
antibodies against various transcription factors including Sp1, Sp3,
c-Jun/AP-1, and AP-2, and competition experiments with oligonucleotides
containing consensus sequences for Sp1 and AP-1 binding identified Sp1
as the transcription factor binding to this region of the
COL7A1 promoter. Indeed, recombinant human Sp1 was shown to
bind the COL7A1 promoter GT-rich element but not its
mutated form in gel mobility shift assays. In addition, co-transfection
of pPacSp1, an expression vector for Sp1, together with the
COL7A1 promoter/chloramphenicol acetyltransferase construct into Sp1-deficient Drosophila Schneider SL2 cells
unequivocally demonstrated that Sp1 is essential for high expression of
the COL7A1 gene. These data represent the first in-depth
analysis of the human COL7A1 promoter transcriptional
control.
1(VII)]3, and each
-chain consists of a central
collagenous domain of ~145 kDa flanked by noncollagenous segments (6,
7). The tissue form of type VII collagen has been suggested to be an
anti-parallel dimer, associated through overlapping carboxyl-terminal
regions between the individual molecules. These anti-parallel dimers
then aggregate laterally to form the anchoring fibrils (4).
end sequences of both human and murine COL7A1
genes has revealed a promoter without either a canonical TATA or a CAAT
box, upstream of the transcription initiation site (13), a feature
usually associated with so-called housekeeping genes.
Cell Cultures
deletion fragments cloned into
promoterless pBS0CAT vector (15). A mutated promoter construct,
524m,
was generated by polymerase chain reaction amplification using a 5
end
primer containing a double point mutation (in bold) in the GT box
located between nucleotides
512 and
505, GGGTGGGG
GTTTGGGG, and a XhoI restriction site. At the 3
end, a primer upstream of position +92 containing a HindIII
restriction site was utilized. The resulting amplification product was
cloned as a XhoI/HindIII fragment into pBS0CAT.
The fidelity of both polymerase chain reaction and subcloning procedure
was ensured by automated sequencing (ABI).
-galactosidase plasmid DNA to monitor the transfection
efficiencies (18). After glycerol shock, the cells were placed in
Dulbecco's modified Eagle's medium containing 1% fetal calf serum.
After 40 h of incubation, the cells were rinsed once with
phosphate-buffered saline, harvested by scraping, and lysed in 200 µl
of Reporter Lysis Buffer (Promega, Madison, WI).
-galactosidase activity were used for each
CAT1 assay, using
[14C]chloramphenicol as substrate (19).
524
COL7A1 promoter/CAT reporter gene plasmid construct together
with 6 µg of either pPac0 or pPacSp1 by the calcium phosphate/DNA
co-precipitation method. The DNA precipitates were left on the cells
for 48 h. The cells were then collected by scraping, rinsed twice
in phosphate-buffered saline and lysed by three cycles of
freeze-thawing in 200 µl of reporter lysis buffer. CAT assay was
performed as described above with identical amount of protein in each
sample. The acetylated and nonacetylated forms of
[14C]chloramphenicol were separated by thin layer
chromatography and visualized by autoradiography. The promoter
activity, reflected by CAT activity, was determined for each sample by
expressing the radioactivity in the acetylated forms of
[14C]chloramphenicol as a percentage of total
radioactivity.
524 and
457 was generated
by polymerase chain reaction amplification using the plasmid
722
COL7A1 as template. The polymerase chain reaction product
was then run on a 2% agarose gel, electroeluted, and precipitated
overnight at
80 °C. The oligonucleotides I, I
A, I
B, ImA, II and
III, spanning short segments of the WT fragment (Fig.
1), as well as Sp1 and AP-1, were synthesized on an
Applied Biosystem automated DNA synthesizer. The nucleotide sequences
and a schematic representation of the oligonucleotides used in this
study are shown in Fig. 1, panels A and B,
respectively.
Fig. 1.
Oligonucleotides used in this study.
A, sequences of the various double-stranded oligonucleotides
used in gel mobility shift assays, spanning the region from 524 to
457 of the human COL7A1 promoter. Only the upper strand is
shown. Oligomers I
A, I
B, and
ImA, respectively, correspond to fragment I
devoid of box A or box B or mutated in box A (see "Results"). Mutated bases are indicated by asterisks. I
A
and I
B were synthesized based on a computer search for
potential transcription factor binding sites within the sequence
spanned by oligonucleotide WT. ImA was synthesized after
DNA-protein interaction assays showed the importance of box A. Consensus motifs for Sp1 and AP-1 transcription factors are
underlined. NA, not applicable. B,
schematic representation of the oligonucleotides. Note that fragments
I and II collectively cover the entire WT
sequence but do not overlap and that oligonucleotide III
does not contain box A. NA, not applicable.
[View Larger Version of this Image (29K GIF file)]
80 °C until use. The protein concentration in the extracts was determined using a commercial assay
kit (Bio-Rad).
end-labeled oligomers
(0.05-0.1 pmol, 2-6 × 104 cpm) for another 20-min
incubation at 4 °C. For competition experiments, a 1-60-fold molar
excess of unlabeled DNA was added to the binding reaction. For
supershift experiments, nuclear extracts were incubated overnight at
4 °C with 2 µg of polyclonal antibodies specific for c-Jun, AP-2,
Sp1, or Sp3 (TransCruzTM Gel Supershift Reagents, Santa Cruz
Biotechnologies, Santa Cruz, CA) prior to the binding reactions. In
experiments with human recombinant Sp1 (Promega Corp., Madison, WI),
the amount of poly(dI-dC) per reaction was decreased to 500 ng, and
bovine serum albumin (10 µg) was added to the samples. Samples were
then separated by electrophoresis on a 4% polyacrylamide gel in
0.5 × Tris borate-EDTA buffer at 200 V for 2 h at 4 °C,
fixed for 1 h in 30% methanol-10% acetic acid, vacuum-dried, and
autoradiographed.
Deletion Analysis of the Human COL7A1 Promoter
end
deletion/CAT reporter gene constructs spanning the COL7A1 promoter region from positions
722 to +92 relative to the
transcription start site, +1. A high level of activity was detected
with constructs
722 and
524 in both cell types (Fig.
2, panels A and C). Subsequent 5
deletion of 68 bp to position
456 led to a dramatic reduction (70-90%) of the promoter activity, whereas additional deletions to
positions
396 and
230 had no further effect (Fig. 2, panels B and D). These data suggest that the DNA sequences
located between residues
524 and
456 are essential in providing
high basal activity to the human COL7A1 promoter constructs
in both dermal fibroblasts and epidermal keratinocytes.
Fig. 2.
Deletion analysis of the human
COL7A1 promoter. Confluent fibroblast (panels
A and B) and keratinocyte (panels C and D) cultures were transfected with various 5 deletion
constructs of the COL7A1 promoter linked to the CAT gene as
described under "Material and Methods." A and
C, autoradiograms of representative experiments in each cell
type. AC, acetylated form of
[14C]chloramphenicol; C, unacetylated form of
[14C]chloramphenicol. The position of the 5
end of each construct is indicated below each duplicate.
B and D, graphic representations of the
means ± S.D. of three separate experiments, each performed with
duplicate samples in each cell type. The values are expressed as
relative CAT activity with the activity of the longest fragment,
722COL7A1, arbitrarily set as 1. The position of the 5
end of each construct is indicated below each histogram bar.
[View Larger Version of this Image (54K GIF file)]
524/
491 Region of Human
COL7A1 Promoter
524 and
456,
we analyzed the binding of nuclear factors to this region. For this purpose, we designed a series of oligonucleotides spanning this whole
region. Their sequences and relative positions are depicted in Fig. 1.
Gel mobility shift assays were performed either with the
oligonucleotide WT, containing the entire sequence of the
524/
457
region of the COL7A1 promoter, or with three shorter oligonucleotides, I, II, and III,
spanning the regions
524/
491,
490/
457, and
500/
475,
respectively (see Fig. 1). Incubation of the radiolabeled WT
oligonucleotide with fibroblast nuclear extracts resulted in the
formation of two major DNA/protein complexes, identified as shifts 1 and 3 (Fig. 3, lane 2). The formation of
these two complexes could be abolished by the addition of a 60-fold
molar excess of unlabeled oligonucleotide I (Fig. 3,
lane 3), but there was no competition by the addition of the
same molar excess of the unlabeled oligonucleotides II or
III (Fig. 3, lanes 4 and 5,
respectively). Together, these data indicate that binding of nuclear
proteins to the
524/
456 region of human COL7A1 promoter
occurs within the region spanned by oligonucleotide I, from
position
524 to
491.
Fig. 3.
Binding of fibroblast nuclear proteins to WT,
I and II regions of human COL7A1 promoter. Gel
mobility shift assays were performed with labeled oligonucleotides
spanning the region from 524 to
457 of the COL7A1
promoter. WT, lanes 1-5; probe I (
524 to
491), lanes 6-9; and probe II (
490 to
457), lanes 10-13. Details on the sequences and
relationships of the different probes are provided in the legend to
Fig. 1. Competition assays were performed with a 60-fold molar excess of unlabeled oligonucleotides, as indicated at the top of the figure.
DNA-protein complexes were separated from unbound oligonucleotides by
nondenaturing 4% acrylamide gel electrophoresis. Shifts 1, 2, and 3 indicate major DNA-protein complexes observed in this experiment. Free
probes refer to the unbound radiolabeled oligonucleotides. Note the
presence of several fast migrating bands with a pattern identical with
all three probes used. Because probes I and II,
which span WT, do not share any overlapping sequence, these bands are
likely to represent nonspecific binding.
[View Larger Version of this Image (89K GIF file)]
Fig. 4.
Comparison of fibroblast and keratinocyte
nuclear protein binding to fragments WT and I of the human
COL7A1 promoter. Gel mobility shift assays were
performed as described in the legend to Fig. 3 using 5 µg/lane of
nuclear extracts of either fibroblasts (F) or keratinocytes
(K) with either probe WT (lanes 1-3) or I (lanes 4-6).
[View Larger Version of this Image (79K GIF file)]
524/
491 Region
of COL7A1 Promoter
524 and
491
(oligonucleotide I). Computer analysis for sequence
homologies to known transcription factor binding sites was performed
(DNASIS software, Hitachi Software Engineering Co., Yokohama, Japan),
revealing two distinct areas of interest: (a) a 8-bp GT-rich
sequence (GGGTGGGG) located between nucleotides
512 and
505,
referred to as box A and (b) a potential
-interferon
response element, CAGGAGGC, located between nucleotides
502 and
495, referred to as box B. To determine whether these two boxes were
involved in the binding of nuclear proteins to fragment I,
two oligonucleotides were designed spanning the whole region covered by
oligomer I but devoid of either box A (I
A) or
box B (I
B), respectively (see Fig. 1). Gel mobility shift
assays performed in parallel with probes I,
I
A, or I
B indicated that deletion of the
8-bp region encompassing box A (probe I
A) abolished the
formation of DNA-protein complexes detected with probe I (Fig. 5A, lane 4 versus lane 2),
whereas deletion of box B (probe I
B) did not alter the
binding pattern obtained with probe I (Fig. 5A,
lane 6 versus lane 2). These data demonstrate that the
formation of all three DNA-protein complexes, shifts 1, 2, and 3, require box A between nucleotides
512 and
505.
Fig. 5.
Box A is critical for binding to fragment I. A, [32P]ATP-labeled oligonucleotides
I (lanes 1 and 2), IA (lanes 3 and 4), and I
B
(lanes 5 and 6) were used in gel mobility shift
assays in the absence or the presence of 5 µg/lane of fibroblast
nuclear extract as indicated to study their respective abilities to
bind transcription factors. Note the total absence of binding to probe
I
A, lacking box A, and the binding to I
B in
a manner identical to that to probe I. B, binding
of nuclear proteins to probe I was competed against a 1-, 20-, or 60-fold molar excess of unlabeled fragment I
B
(lanes 3-5) or I
A (lanes 6-8).
Note that only I
B, which contains box A, successfully
competes nuclear protein binding to probe I.
[View Larger Version of this Image (46K GIF file)]
A and I
B (Fig. 5B). Specifically, the binding
activity of nuclear extracts to probe I (Fig. 5B,
lane 2) was abolished by the addition of a 60-fold molar
excess of cold oligonucleotide I
B (Fig. 5B,
lane 5), but it was not altered by the addition of the same
molar excess of oligonucleotide I
A (Fig. 5B, lane 8).
GTTTGGGG, was generated for
use in gel mobility shift assays. A 60-fold molar excess of unlabeled
oligomer ImA used as a competitor in a binding reaction with
radiolabeled oligomer I failed to compete with the binding
of nuclear proteins to probe I (Fig.
6A, lane 4), whereas an identical
amount of unlabeled oligonucleotide I abolished the binding
of all three complexes 1, 2, and 3 (Fig. 6A, lane 3 versus lane 2). Furthermore, no DNA-protein complexes were
detected when the mutated oligonucleotide ImA was used as a
probe in a parallel DNA-protein binding assay (Fig. 6A,
lane 6 versus lane 2). Collectively, these experiments
demonstrate that box A is responsible for the formation of three
separate DNA-protein complexes in the
524/
491 region of the human
COL7A1 promoter and that the two adjacent guanosine
nucleotides mutated within box A are critical for DNA-protein
interaction. Also, these data indicate that box B, identified by
computer sequence analysis, is not critical for basal expression of
COL7A1.
Fig. 6.
Mutation of box A abolishes transcription
factor binding and reduces COL7A1 promoter activity.
A, gel mobility shift assays were performed by incubating
probes I (lanes 1-4) or ImA
(lanes 5-6), the latter containing two point mutations (bold) in box A (GGGTGGGGGTTTGGGG),
with 5 µg/lane of fibroblast nuclear extracts as indicated.
Competition experiments were performed with a 60-fold molar excess of
unlabeled oligonucleotide I (lane 3) or
ImA (lane 4). Specific DNA-protein complexes are
indicated by arrows. Note that none of the complexes obtained with probe I are observed with probe
ImA. B, a double mutation corresponding to that
made in oligomer ImA (GGGTGGGG
GTTTGGGG) was introduced into the
524 COL7A1/CAT reporter gene construct to generate the
524m COL7A1 construct. The relative activity of
524,
524m, and
456 COL7A1 constructs was compared in parallel
transfections of fibroblasts (panel B) or keratinocytes
(panel C) as described under "Material and
Methods."
[View Larger Version of this Image (36K GIF file)]
512 and
505 to the basal activity of the human COL7A1 promoter, a nucleotide substitution identical to that introduced into
oligomer ImA (see above and Fig. 1) was created in the
524
COL7A1 promoter/CAT construct, generating the construct
524m (see "Materials and Methods"). The basal activity of the two
constructs was compared in transient cell transfections. Mutation of
the GT box led to a dramatic drop, ~70%, of the basal promoter activity as compared with that of the wild type
524 CAT construct, both in fibroblasts (Fig. 6B) and in keratinocytes (Fig.
6C). In fact, the CAT activity of the
524m construct was
comparable with that of the
456 construct, suggesting that the 8-bp
GT-rich sequence, located between nucleotides
512 and
505 and
designated as box A, is crucial in providing high basal activity of
COL7A1 promoter in both cell types.
512 and
505 of the
COL7A1 promoter is an Sp1-binding site, an oligonucleotide
containing a high affinity Sp1 recognition site (GC box) was used as a
competitor in gel mobility shift assays with probe I (Fig.
7A). The binding of nuclear factors to probe
I (Fig. 7A, lane 2) was competed away
by unlabeled homologous DNA (COL7A1 GT box), in a
dose-dependent manner (Fig. 7A, lanes
3-5), as well as the consensus Sp1 (GC box) oligonucleotide (Fig.
7A, lanes 6-8). In contrast, the binding was not
affected by a 60-fold molar excess of a consensus AP-1 oligonucleotide (Fig. 7A, lane 9), attesting to the specificity
of the competition by the Sp1 oligonucleotide.
Fig. 7.
Sp1 is the transcription factor binding to
box A. A, gel mobility shift assays were performed by
incubating fibroblasts nuclear extracts (5 µg/lane) with probe
I (lane 2). Competitions were carried out with
increasing concentrations (1-, 20-, and 60-fold molar excess) of
unlabeled fragment I (lanes 3-5) with consensus
Sp1 (lanes 6-8) or with a 60-fold molar excess of unlabeled
oligonucleotide containing a consensus AP-1 binding site (lane
9). B, gel mobility supershift assays were performed by
incubating fibroblast nuclear extracts (5 µg/lane) with polyclonal antibodies for Sp1, AP-2, or c-Jun prior to the binding reaction with
radiolabeled probes WT or I. Note that the Sp1 antibody displaces all three complexes bound to probes WT and I. Similar observations were obtained in experiments utilizing
keratinocyte nuclear extracts (not shown).
[View Larger Version of this Image (66K GIF file)]
512 and
505 of the human COL7A1 promoter.
Toward this end, supershift experiments were performed with polyclonal
antiserum specific for either Sp1, AP-2, or c-Jun. As shown in Fig.
7B, the two major complexes observed with the WT
oligonucleotide were replaced by a unique supershifted complex when
using the polyclonal anti-Sp1 antiserum (Fig. 7B, lane
2 versus lane 1). In contrast, no supershift was observed when
using specific antibodies directed against AP-2 (Fig. 7B,
lane 3) or c-Jun (Fig. 7B, lane 4).
Similarly, the Sp1 antibody supershifted all three complexes formed
with oligonucleotide I (Fig. 7B, lane 6 versus lane 5), whereas neither AP-2 nor c-Jun antibodies had an
effect on the electrophoretic mobility of the DNA-protein complexes
(Fig. 7B, lanes 7 and 8,
respectively). These data indicate that all three complexes are
immunologically related to Sp1.
512 and
505 of
the COL7A1 promoter.
512 and
505 of
COL7A1. Also, these data indicate that integrity of box A is required for Sp1 binding.
Fig. 8.
Recombinant Sp1 binds the COL7A1
promoter fragment containing box A. Probes WT, I, and
ImA and a radiolabeled consensus Sp1 oligonucleotide were
used in parallel gel mobility shift assays with human recombinant Sp1
as described under "Materials and Methods." Samples were separated
on a 4% acrylamide gel under nondenaturing conditions. Note that Sp1
binds all but ImA probes.
[View Larger Version of this Image (91K GIF file)]
524 COL7A1
promoter/CAT construct was co-transfected into Drosophila
SL2 cells in the presence of either pPacSp1 or pPac0 (see "Materials
and Methods"). As shown in Fig. 9, the
COL7A1 promoter had negligible basal activity in
Sp1-deficient SL2 cells. In contrast, the promoter activity was highly
induced in response to Sp1 expression, demonstrating that Sp1 is a
transcription factor essential for high expression of the
COL7A1 gene.
Fig. 9.
Expression of Sp1 in Sp1-deficient
Drosophila SL2 cells induces COL7A1 promoter
activity. 524 COL7A1 promoter/CAT plasmid construct
was transfected in Drosophila SL2 cells with either empty
pPac0 or pPacSp1 expression vector. CAT activity, representing the
promoter activity, was determined 48 h later using
[14C]chloramphenicol as a substrate as described under
"Materials and Methods." A representative experiment is shown.
AC, acetylated chloramphenicol; C, unacetylated
chloramphenicol.
[View Larger Version of this Image (73K GIF file)]
505, away
from the transcription start site, and may not be involved in the
initiation of the transcription. In this context, three other Sp1 sites
(GGGCGGG) are located at positions
150,
121, and
25 upstream of
the transcription initiation site (13). The fact that the
COL7A1 promoter not only lacks a typical TATA element but
also contains a Sp1 motif at a position usually occupied by the TATA
box, around position
20, supports the notion that Sp1 may be involved
in the initiation of the transcription of TATA-less promoter genes.
342 and
271 of the human
COL1A2 gene have been suggested to play a role in
transforming growth factor
-mediated up-regulation of the promoter
activity (41). However, we have recently excluded these Sp1 sites from growth factor responsiveness, although they are essential for basal
activity of the promoter (42), the latter observation being in
agreement with another recent study (43). Also, it should be noted that
Sp1 was recently shown to be essential for the basal expression of
other extracellular matrix genes, including syndecan-1 (44), COL1A1
(45), COL6A1 (46), and the collagen/laminin receptor
2 integrin
(47), and may therefore be an important factor in repair processes,
such as wound healing.
deletion constructs of the
COL7A1 promoter, we have identified a functional region
within the human COL7A1 promoter necessary for relatively
high level of expression of the gene in both fibroblasts and
keratinocytes. This region located between nucleotides
524 and
456
contains a functional GT box that binds Sp1 between residues
512 and
505. Disruption of the nucleotide sequence of this 8-bp element
prevents the binding of Sp1 and reduces the promoter activity by
~70%. In addition, recombinant expression of Sp1 in Sp1-deficient
Drosophila SL2 cells results in high expression of the
COL7A1 promoter. Collectively, these results provide the
first in-depth characterization of the transcription mechanisms
regulating the expression of COL7A1. Also, our study
provides further evidence that Sp1 is an essential transcription factor
for the expression of extracellular matrix genes.
*
This work was supported in part by National Institutes of
Health Grants RO1-AR41439 and T32-AR07651 (to J. U.) and a Research Career Development Award from the Dermatology Foundation (to A. M.).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: Dept. of Dermatology
and Cutaneous Biology, Jefferson Medical College, Thomas Jefferson
University, 233 South 10th St., Rm. 430, Philadelphia, PA 19107. Tel.:
215-503-5775; Fax: 215-923-9354; E-mail:
mauviel1{at}jeflin.tju.edu.
1
The abbreviations used are: CAT, chloramphenicol
acetyltransferase; WT, wild type; bp, base pair(s).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.