©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a GC-rich Region Containing Sp1 Binding Site(s) as a Constitutive Responsive Element of the 2(I) Collagen Gene in Human Fibroblasts (*)

(Received for publication, October 7, 1994; and in revised form, December 7, 1994)

Takeshi Tamaki Kazunori Ohnishi Christoph Hartl E. Carwile LeRoy Maria Trojanowska (§)

From the Division of Rheumatology and Immunology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

To analyze regulatory elements in the human alpha2(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.


INTRODUCTION

Collagen type I, the most abundant mammalian collagen, consists of two alpha1(I) chains and one alpha2(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 (^1)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 TGFbeta 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 TGFbeta differs from mouse and may involve the Sp1 binding site(11) . Sp1 was also suggested in the TGFbeta stimulation of human COL1A1(12) , while a different responsive element has been proposed in TGFbeta 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.


MATERIALS AND METHODS

Cell Cultures

Newborn foreskin fibroblasts were obtained from the delivery suites of affiliated hospitals. Primary explant cultures were established in 150-cm^2 flasks in DMEM supplemented with 10% FCS and 2 mM glutamine. Monolayer cultures were maintained at 37 °C in 10% CO(2) in air. Fibroblasts in early passages were used for experiments.

Plasmid Constructions

A fragment of human COL1A2 (+58 to -772 bp relative to the transcription start site) was excised by XbaI and HindIII from the pMS-3.5/chloramphenicol acetyltransferase construct (16) (kindly provided by Dr. F. Ramirez). After filling in with Klenow polymerase the fragment was cloned into the EcoRV site of pGEM plasmid vector from Promega, excised with XbaI and HindIII, and subcloned in the proper orientation into ``basic chloramphenicol acetyltransferase vector'' from Promega. To generate deletion mutants, this construct was linearized by BglII and treated with Bal-31 nuclease, after which HindIII restriction sites were introduced at the deletion end points. The end points of the deletions were determined by nucleotide sequencing (Fig. 1). Substitution mutations were generated using the Clontech Transformer site-directed mutagenesis kit according to the manufacturer's protocol. Plasmids used in transient transfection assays were purified by a double CsCl gradient. At least two different plasmid preparations were used for each experiment.


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.



Transient Transfections and Chloramphenicol Acetyltransferase Assays

Human foreskin fibroblasts were grown to 90% confluence in 100-mm dishes in DMEM with 10% FCS. Monolayers were washed and cells transfected by the calcium phosphate technique (14) with 20 µg of various deletion or mutant promoter-chloramphenicol acetyltransferase constructs. In some experiments pSV-beta-galactosidase control vector (Promega) was co-transfected to normalize for transfection efficiency. After incubation overnight, the medium was replaced with DMEM containing 1% FCS. Incubation was then continued for 48 h. Cells were harvested in 0.25 M Tris-HCl, pH 8, and fractured by freeze-thawing. Extracts were normalized for protein contents as measured by Bio-Rad reagents and incubated with butyryl-CoA and [^14C]chloramphenicol for 90 min at 37 °C, an assay condition predetermined to be within the linear range of chloramphenicol acetyltransferase activity for these samples. Butyrated chloramphenicol was extracted using organic solvent (2:1 mixture of tetramethylpentadecane and xylene) and quantitated by scintillation counting. Each experiment was performed in duplicate.

DNase I Footprinting and DNA Mobility Shift Assays

Nuclear extracts were prepared according to Andrews and Faller(17) . Briefly, confluent cells from five 150-mm dishes were washed with phosphate-buffered saline and scraped into 1 ml of cold Buffer A (10 mM HEPES-KOH pH 7.9 at 4 °C, 1.5 mM MgCl(2), 10 mM KCl, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). The cells were allowed to swell on the ice for 10 min and then vortexed for 10 s. The tube was centrifuged for 2 min and the supernatant was discarded. The pellet was resuspended in 80 µl of cold Buffer C (20 mM HEPES-KOH pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl(2), 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min for high-salt extraction. Cellular debris was removed by centrifugation for 2 min at 4 °C, and the supernatant fraction was stored at -70 °C until use. The protein concentration of the extracts was determined using the Bio-Rad reagent.

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(2), 100 µM ZnCl(2), 4% glycerol, 10,000 cpm-labeled probe, 2 µg of poly(dI-dC)bulletpoly(dI-dC), and nuclear extracts containing 120 µg of protein. After subsequent addition of 5 µl of 10 mM MgCl(2), 5 mM CaCl(2), 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)bulletpoly(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 times 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.

Shift-Western Blotting

Shift-Western blotting was performed based on the protocol of Demczuk et al.(18) with modification. First, the DNA mobility shift assay was performed as described above. The binding reaction was duplicated, one for autoradiography and one for blotting, except that the binding reaction subsequently used for blotting contained a 100-fold excess of probe. The transfer of protein-DNA complexes to nitrocellulose filter was performed in 25 mM Tris-HCl, 192 mM glycine, 20% methanol in a typical Western blotting apparatus at 30 V overnight at 4 °C. For protein detection, the blotted membrane was blocked for 60 min in Tris-buffered saline with 0.1% Tween 20 and 5% skim milk powder, and subsequent manipulations were done in the absence of skim milk powder. Primary antibodies were applied at a dilution of 1:500, and enhanced chemiluminescence protein detection was done as described by Amersham with anti-rabbit peroxidaseconjugated antibodies from Amersham Corp.


RESULTS

Functional Analysis of COL1A2 Promoter

To further characterize cis-regulatory elements in the human COL1A2 -376 to -108 promoter region we generated a series of progressive 5` deletions linked to chloramphenicol acetyltransferase reporter gene (Fig. 1). These constructs were tested in transient transfection assays in human newborn foreskin fibroblasts (Fig. 2). While deletion up to -299 and -289 did not affect promoter activity, a substantial decrease was seen with deletion at -264. Further deletion to -186 maintained the similar low levels of promoter activity. These findings are consistent with previously reported deletion analysis of the human COL1A2 promoter (16) . Thus the promoter region between -289 and -264 is sufficient to direct significant constitutive transcriptional activity. This promoter segment contains a GC-rich motif (GC-box) that resembles binding sites for transcription factor Sp1. Two additional, almost identical GC-boxes are located upstream from this segment, i.e. between -303 and -288 (Fig. 1).


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).



Factors Binding to GC-rich Region of COL1A2 Promoter

In order to identify the trans-acting factors that interact with this GC-rich region, a DNA fragment from -353 to -234 (130-mer) was used in DNase I footprint assays with human fibroblast nuclear extracts. A broad 60-bp protected region that included the three GC-boxes and an additional 10-bp upstream region was observed in both coding and non-coding strands (Fig. 3, lanes2, 3 and 10, 11). To assess the role of the GC-boxes in the DNA-protein interactions in this region we introduced substitution mutations into all GC-boxes in a manner reported to abolish Sp1 binding (Fig. 1, (19) ). This resulted in a loss of DNase I protection as compared with wild type DNA. The 10-bp element upstream of the GC-boxes remained partially protected (Fig. 3, lanes4, 5 and 12, 13). DNase I protection was also abolished in the presence of an excess of a 39-mer promoter fragment (-306 to -268) that encompasses only the GC-rich region (Fig. 3, lane8) or in the presence of an excess of Sp1 consensus sequence (Fig. 3, lane7).


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.



The Factor Binding to GC-rich Region Is Related to Sp1

We then asked whether the binding proteins are immunologically related to Sp1. First, the binding reaction was performed in the presence of Sp1 antibody or an equal amount of control antibody (Fig. 5A, lanes1-3). In agreement with the above gel shift analysis, anti-Sp1 antibody blocked formation of only one of the DNA-protein complexes (middle band); the same amount of antibody completely inhibited binding to consensus Sp1 oligonucleotide (Fig. 5A, lanes 4-6). The presence of Sp1 in the ``middle'' complex was further confirmed by direct analysis of the DNA-protein complexes by Western blotting with anti-Sp1 antibody (Fig. 5B). These data provide further evidence that Sp1 is at least one of the binding proteins that interact with the GC-rich region of human COL1A2 promoter.


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.



Effect of Substitution Mutations in GC-boxes on Promoter Function

To analyze the contribution of the GC-boxes to the COL1A2 promoter activity we introduced substitution mutations into each GC-box individually and in combination using a -353 promoter construct. GC boxes were mutated in the manner reported to abolish Sp1 binding (GGGCGG was changed into GTTCGG as shown in Fig. 1). The promoter constructs carrying various substitution mutations were analyzed by transient transfection assays (Table 1). In agreement with deletion analysis (Fig. 2) single mutations in the first or second GC-box did not have significant effects on the promoter activity, while mutation in the third GC-box decreased promoter activity by about 30%. A further decrease in the promoter activity was observed with a double mutant in the second and third GC-boxes (about 50%), and almost total loss of the promoter activity (about 90%) was observed with all three GC-boxes mutated. Promoter activity of the triple mutant corresponds to the activity seen with deletion that removes the GC-rich region at -264.



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.




DISCUSSION

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.


FOOTNOTES

*
This work was supported by the RGK Foundation and by a grant from the United Scleroderma Foundation collaborative funding efforts. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Rheumatology and Immunology, Dept. of Medicine, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel.: 803-792-2000; Fax: 803-792-7121.

(^1)
The abbreviations used are: bp, base pair(s); COL1A2, the gene coding for human collagen pro-alpha2 type I chain; TGFbeta, transforming growth factor-beta; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.