(Received for publication, October 7, 1994; and in revised form, December 7, 1994)
From the
To analyze regulatory elements in the human 2(I) collagen
gene (COL1A2) promoter, a series of deletion mutants from
-323 to -186 base pairs was tested in transient
transfection assays in human fibroblasts. A strong positive responsive
element was mapped to a GC-rich region located between base pairs
-303 and -271. This region contains three binding sites
(GC-boxes) resembling recognition sites for the transcription factor
Sp1. Substitution mutations in the GC-boxes abolished binding to the
GC-rich region in gel shift analyses and resulted in 90% reduction of
promoter activity in transient transfection assays. We demonstrated
that transcription factor Sp1 is essential for binding based on the
following observations. 1) Sp1 consensus binding site alone competes by
binding to the GC-rich region in the DNase I protection assay; 2) both
Sp1 consensus binding site and Sp1 antibodies prevent the formation of
a DNA-protein complex in the mobility shift assay; 3) anti-Sp1
antibodies recognize a component of the complex competed for by Sp1
consensus binding site.
Collagen type I, the most abundant mammalian collagen, consists
of two 1(I) chains and one
2(I) chain, which are coordinately
expressed(1, 2) . The expression of type I collagen is
strictly regulated during development (2) and is
tissuespecific(3) . Excessive deposition of type I collagen is
characteristic of many fibrotic disorders (4) and most likely
results from transcriptional activation of collagen genes in response
to cytokines and other factors present in fibrotic lesion.
Transcriptional regulation of type I collagen genes has been studied
in several species, including mouse, human, rat, and
chicken(1, 2, 5, 6) . Previous
studies showed that a 350-bp ()sequence in the 5` regulatory
region of the mouse COL1A2 gene was sufficient to direct
tissue-specific expression of reporter genes in transgenic
mice(7) . Several regulatory elements and cognate transcription
factors have been characterized within this promoter region, including
CBF (CCAAT binding factor)(8) , a member of the CTF/NF1 family
involved in stimulation of this promoter by TGF
in murine
cells(9) , as well as a binding site for a transcription
repressor termed IF1(10) . Recent studies of the human COL1A2 promoter have indicated that regulation of human COL1A2 promoter by TGF
differs from mouse and may involve
the Sp1 binding site(11) . Sp1 was also suggested in the
TGF
stimulation of human COL1A1(12) , while a
different responsive element has been proposed in TGF
regulation
of rat COL1A1 promoter(13) . Thus, it appears that
regulation of collagen type I genes at the transcription level may
differ between COL1A1 and COL1A2 genes and
may be species specific. Because of the species specificity in the
regulation of collagen genes, elucidation of the mechanism of basal
collagen transcription and its modulation by cytokines in human
fibroblasts is essential in order to understand matrix gene regulation
in human fibrosis.
We have previously demonstrated that human COL1A2 promoter exhibited higher transcription activity in scleroderma fibroblasts than in healthy skin fibroblasts; the sequence involved in the up-regulation of COL1A2 promoter in scleroderma fibroblast mapped between -376 and -108 bp(14) . Subsequent analyses of this promoter region have demonstrated that a GC-rich responsive element containing the putative Sp1 binding site is important for basal promoter activity (15) . In the present study, we dissect the functional elements of this GC-rich region, providing further information on the control of basal transcription of the human COL1A2 gene.
Figure 1: Schematic representation of COL1A2 promoter. Positions of the restriction sites are shown. The location of the promoter fragment from bp -353 to bp -234 used as a probe in footprinting and gel shift experiments is depicted as a whiteline. Three potential Sp1 recognition sites (GC-boxes) are depicted as boxes and given subsequent numbers. The nucleotide sequence of the GC-rich region bp -330 to bp -261 is shown underneath. End points of the deletion constructs are marked by arrows. Sequences corresponding to GC-boxes are underlined. Substitution mutations in GC-boxes are shown underneath, with mutated nucleotides shown in boldface.
Oligonucleotides used as probes, competitors, or polymerase
chain reaction primers were purchased from OPERON-Technologies, except
for Sp1 and egr consensus oligonucleotides, which were
purchased from Santa Cruz Biotechnology. Radioactive probes were
generated by [-
P]ATP end labeling (Sp1
consensus probe) or by polymerase chain reaction (130-mer wild type and
mutated) using [
-
P]ATP end-labeled primers
on coding or non-coding strands. For DNase I footprinting, the binding
reaction was performed for 30 min at room temperature in 50 µl of
10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 mM dithiothreitol, 1 mM MgCl
, 100 µM ZnCl
, 4% glycerol, 10,000 cpm-labeled probe, 2 µg
of poly(dI-dC)
poly(dI-dC), and nuclear extracts containing 120
µg of protein. After subsequent addition of 5 µl of 10 mM MgCl
, 5 mM CaCl
, and incubation
for 1 min at room temperature, 0.02 (for conditions without nuclear
extract) to 3 units of DNase I (purchased from Boehringer Mannheim) was
added. Digestion with DNase I continued for 1 min at room temperature
and was terminated by 140 µl of 192 mM sodium acetate, 32
mM EDTA, 0.14% SDS, and 64 µg/ml yeast RNA. After
phenol/chloroform extraction and subsequent precipitation with ethanol,
digested probe was dissolved in 3 µl of 95% formamide containing 10
mM EDTA, 0.3% bromphenol blue, and 0.3% xylene cyanol and was
electrophoresed on 8% polyacrylamide/urea gel along with Maxam-Gilbert
G + A sequencing reactions as size markers.
For DNA mobility
shift assay, the binding reaction was performed for 30 min in
12-20 µl of binding buffer containing 10,000 cpm-labeled
probe, 2 µg of poly(dI-dC)poly(dI-dC), and nuclear extracts
containing 3-5 µg of protein. In some assays double-stranded
competitors were added. In assays with antibodies, nuclear extracts
were preincubated with appropriate antibodies for 1 h on ice before
binding reactions. Anti-Sp1 antibody was purchased from Santa Cruz
Biotechnology. Rabbit IgG was purchased from Sigma. Separation of free
radiolabeled DNA from DNA-protein complexes was carried out on a 5%
nondenaturing polyacrylamide gel. Electrophoresis was carried out in
0.5
Tris borate electrophoresis buffer at 200 V at 4 °C.
Autoradiography was performed by overnight exposure to Kodak X-OMAT
XAR2 film with intensifying screens at -70 °C.
Figure 2: Functional activity of the COL1A2 promoter in human fibroblasts. Plasmids containing various lengths of COL1A2 promoter sequences cloned upstream from the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into newborn foreskin fibroblasts as described under ``Materials and Methods.'' A diagram on the left shows the deletion end points in relation to the GC-boxes. The bargraph on the right shows the promoter activity of each deletion construct relative to the -323 promoter, which was arbitrarily set at 100. The means ± S.E. for separate experiments are shown at right. The number of experiments used to calculate the mean is shown in parentheses. Asterisks indicate statistically significant results (p < 0.0001, Mann-Whitney U test).
Figure 3: Footprinting analysis of the COL1A2 promoter. 5`-End-labeled fragments containing the wild type (WT) or mutated (all GC-boxes mutated as described in the text) promoter region between -353 and -234 were used as probes in the binding reaction containing nuclear extract (120 µg/lane) from human foreskin fibroblast. A, coding strand; B, noncoding strand. Lanes1 and 9, GA reaction of wild type DNA fragment; lanes2 and 10, wild type DNA without nuclear extract; lanes3 and 11, wild type DNA with nuclear extract; lanes4 and 12, mutated DNA with nuclear extract; lanes5 and 13, mutated DNA without nuclear extract; lanes6 and 14, GA reaction of mutated DNA fragment; lane7, 5000 molar excess of cold Sp1 consensus sequence added; lane8, 5000 molar excess of cold 39-mer added. Protected regions are shown diagrammatically, with the limits of each footprint and position of GC-boxes as indicated.
To further analyze the DNA binding proteins, we employed DNA mobility shift assays. We observed several DNA-protein complexes (Fig. 4, lane1). Formation of the largest complexes consisting of three bands was abolished by an excess of non-radioactive probe, indicating specific binding (lane2). The formation of these complexes can also be abolished with an excess of the 39-mer (lane3). We then tested if these complexes contain protein factors that are related to any known GC-rich sequence binding proteins, such as Sp1, Egr, or AP-2. We observed that competition with AP-2 or Egr binding site oligonucleotides did not exert any effects on binding (data not shown); however, one of the complexes (middle band) can be efficiently competed off by an excess of Sp1 consensus sequence (Fig. 4, lane4). Taken together, the results from the DNase I protection and mobility shift assays suggest that Sp1 related factor(s) contribute to the binding to the GC-rich region and that this complex is represented by the ``middle'' band in the mobility shift assay.
Figure 4: DNA mobility shift assay with competing unlabeled DNA. Nuclear extract (3-5 µg/lane) prepared from human foreskin fibroblasts was incubated with 5`-end-labeled COL1A2 promoter fragment from bp -353 to bp -234 (130-mer) in the absence (lane1) or presence (lanes2-4) of various unlabeled competitor oligonucleotides (200-fold molar excess). Lane2, 130-mer added; lane3, 39-mer (bp -306 to bp -268) added; lane4, Sp1 consensus (ATTCGATCGGGGCGGGGCGAGC) added.
Figure 5: Evidence that Sp1 binds to GC-rich region. Panel A, nuclear extract from human foreskin fibroblasts was preincubated with 5 µg of rabbit anti-Sp1 antibody (lanes2 and 5) or with 5 µg of rabbit IgG (lanes3 and 6) and used in a binding reaction with a 130-mer probe (lanes 1-3) or consensus Sp1 probe (lanes 4-6). Panel B, nuclear extract from human foreskin fibroblasts was incubated with a 130-mer probe (lanes1 and 3) or with a Sp1 consensus probe (lanes2 and 4). Lane5 contains extract only. After electrophoresis on nondenaturing polyacrylamide gel, autoradiography was performed for lanes1 and 2. Lanes3-5 were transferred to nitrocellulose filter, incubated with rabbit anti-Sp1 antibody and developed using ECL detection system.
In parallel experiments the effects of these mutations on DNA-protein binding were analyzed using DNA mobility shift assays. As shown in Fig. 6, single box mutations (lanes 2-4) had little effect on binding. When two GC boxes were mutated (lane5), a substantial decrease of binding was observed, and almost complete loss of binding occurred when all of the three GC boxes were mutated (lane6).
Figure 6: Effects of substitution mutations in GC-boxes on DNA-protein complex formation. Nuclear extract from human foreskin fibroblasts was incubated with wild type (WT) 130-mer probe (lane1) or probes carrying mutations in GC-boxes. Lane2, first GC box mutant; lane3, second GC-box mutant; lane4, third GC-box mutant; lane5, double mutant in the second and third GC-boxes; lane6, triple mutant in the first, second, and third GC-boxes.
In this study we characterized a GC-rich region of human COL1A2 promoter that directs significant promoter activity in human fibroblasts. In this region we identified three binding sites (GC-boxes) that closely resemble recognition sites for transcription factor Sp1. Based on transient transfection studies with the substitution mutants in GC-boxes (Table 1) we demonstrated that each of these sites contributed to the activity of COL1A2 promoter, and together they accounted for most (90%) of the promoter activity in vivo. It also appears that the proximal site (third GC-box) contributes more to the promoter activity than does either the first or second GC-box.
There are complex DNA-protein interactions within this DNA segment characterized by the broad protected area in the DNase I footprinting analysis and by several complexes identified by electrophoretic mobility shift assay. We were able to identify directly one of the components of this multiprotein complex as transcription factor Sp1, which is essential for binding to this GC-rich region. This is based on the following observations. 1) The Sp1 consensus binding site alone can compete off the binding to the GC-rich region in the DNase I protection assay (Fig. 3). 2) Both the Sp1 consensus binding site and Sp1 antibody can prevent the formation of one of the specific DNA-protein complexes in the mobility shift assay ( Fig. 4and Fig. 5A). 3) In a direct demonstration of the presence of Sp1 in the DNA-protein complex, the antibody recognizes specifically the ``middle band'' that can be competed off by the Sp1 consensus binding site (Fig. 5B).
Sp1, an ubiquitous transcription factor, has been shown to regulate many viral and cellular promoters(20) . The role of Sp1 in regulating collagen type I transcription has previously been investigated. It has been shown that Sp1 binding within the first intron of human COL1A1 gene has a modest inhibitory effect on transcription activity in transient transfection assays into chicken tendon fibroblasts(21) . In a different experimental system using mouse COL1A1 promoter, two sets of overlapping binding sites for Sp1 and NF1 have been identified(22) . Depending on the cell type used for transfection assays, opposite effects of Sp1 were observed. When assays were performed in NIH 3T3 cells that have high endogenous levels of Sp1 and NF1, additional amounts of Sp1 introduced via expression vector seemed to have a slight inhibitory effect on COL1A1 transcription levels, while NF1 had stimulatory effects(22) . However, in Drosophila Schneider L2 cells that lack endogenous Sp1 or NF1, plasmid-expressed Sp1 was a very potent transactivator of collagen gene, while NF1 inhibited this transactivation(23) . Based on these experiments, the role of Sp1 in regulating constitutive expression of COL1A1 promoters is presently unclear. In our experimental system using human COL1A2 promoter and human fibroblasts, we demonstrated that Sp1 is a part of the protein complex that acts as a strong transactivator of COL1A2 promoter.