From the Department of Cardiovascular Medicine, Henry
Wellcome Building for Genomic Medicine and ¶ Sir William Dunn
School of Pathology, University of Oxford, Oxford, United Kingdom
Received for publication, November 9, 2000
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ABSTRACT |
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Matrix metalloproteinase-2 (MMP-2) is an enzyme
with proteolytic activity against matrix and nonmatrix proteins,
particularly basement membrane constituents. Thus, any naturally
occurring genetic variants that directly affect gene expression and/or
protein function would be expected to impact on progression of
pathological processes involving tissue remodeling. We scanned a
2-kilobase pair promoter region and all 13 exons of the human
MMP-2 gene, from a panel of 32 individuals, and we
identified the position, nature, and relative allele frequencies of 15 variant loci as follows: 6 in the promoter, 1 in the 5'-untranslated
region, 6 in the coding region, 1 in intronic sequence, and 1 in the
3'-untranslated region. The majority of coding region polymorphisms
resulted in synonymous substitutions, whereas three promoter variants
(at The matrix metalloproteinases
(MMPs)1 constitute a family
of secreted and membrane-associated zinc-dependent
endopeptidases that are capable of selectively degrading a wide
spectrum of both extracellular matrix and nonmatrix proteins (1).
Currently upwards of 20 vertebrate MMPs have been reported that can be
categorized, by substrate specificity, to give the collagenases,
stromelysins, gelatinases, and membrane-type MMPs. The broad range of
substrates conveys a pivotal role for MMP involvement during both
normal physiological processes (e.g. embryonic development,
bone remodeling, angiogenesis, nerve growth, etc.) and pathological
states (e.g. arthritis, cancer, atherosclerosis, liver
fibrosis, etc.) (2). Accordingly, MMP activity is tightly coordinated
at several levels including transcriptional regulation, activation of
latent zymogen, and interaction with endogenous inhibitors (3).
MMP-2 (gelatinase A) has type IV collagenolytic activity and is
constitutively expressed by most connective tissue cells including endothelial cells, osteoblasts, fibroblasts, and myoblasts. The membrane-bound activation of pro-MMP-2 ensures that proteolytic activity, predominantly against components of the basement membrane, is
localized to discrete regions on the cell surface (4-6) thereby potentiating extracellular matrix remodeling as well as uniquely generating several different biologically active molecules including laminin, fibronectin, and monocyte chemoattractant protein-3 (7-10). Indeed, the majority of MMP-2 studies have focused on demonstrating an
essential role in promoting cell invasiveness during tumor angiogenesis, arthritis, and atherogenesis (11-15), as well as tumor
metastasis where levels of MMP-2 expression can be correlated with
tumor grade (16, 17). Not surprisingly, the design of specific and
selective inhibitors of MMP-2, for therapeutic intervention, remains an
intense focus of research (18).
The diseases in which a role for MMP-2 has been demonstrated are
characterized by varying individual susceptibility, implying the role
of genetic factors. Traditional linkage analysis methods for mapping
the genes of Mendelian disorders have not been as successful in the
studies of complex genetic diseases including coronary heart disease,
cancers, and arthritic disorders. Attention has therefore focused on
the rapid elucidation of a new class of genetic markers termed
single-nucleotide polymorphisms (SNPs). These are the most common types
of stable genetic variants estimated to occur, on average, every 1,000 bp and are therefore valuable markers in tests of association for
susceptibility, or resistance, to common and genetically complex
diseases (19-21) and pharmacogenetic traits (22). Indeed, the
emergence of the common disease-common variant hypothesis (21, 23) has
provided important examples of such associations, including the
APOE-4 allele in Alzheimer's disease (24) and the
CCR5 allele in HIV resistance (25). However, the power of
association-based studies can be compromised by multiple hypothesis
testing that is exacerbated by publication bias (26). This problem can
be overcome, in part, by distinguishing functional SNPs from neutral
counterparts across a candidate region thereby generating a panel of
robust, informative variants that are more likely to have an influence
in disease progression.
The specific predilection of MMP-2 for substrates important in basement
membrane integrity makes it a strong candidate for a number of
heritable traits including, for example, atherosclerosis (by allowing
immigration of smooth muscle cells and by facilitating plaque rupture).
Common variants that alter the amount of protein expressed, for
instance by affecting transcriptional regulation, or that subtly alter
the activity of the protein itself would be expected to have a
quantitative influence on disease activity. We have therefore
identified the nature and extent of genetic variation in the promoter
(~2 kb) and complete coding region of the human matrix
metalloproteinase-2 gene. We describe the in vitro
characterization of the entire panel of promoter polymorphisms, and we
identify one particular functional variant that alters MMP-2
promoter activity through allele-specific binding of the transcription
factor Sp1.
Isolation of the Human MMP-2 Promoter--
The Human Genome
Walking kit (CLONTECH) was used to obtain
additional MMP-2 5'-flanking sequence to that previously published (27). A primer complementary to the sequence +51/+72 relative to the
major transcription start site (28) was used in combination with an
adaptor primer in primary PCRs using five human genomic libraries as
templates. A secondary PCR, using a nested MMP-2-specific primer
( Primer Design for Scanning the MMP-2 Gene--
Eleven
overlapping PCR amplicons were designed to analyze the promoter
sequence (1900 bp), whereas 25 PCR fragments were generated to scan the
5'-UTR, coding region, and 3'-UTR (see Table I). To address the extent
of genetic variation at splice site junctions, scanning primers were
designed based on sequence information obtained by amplifying between
exons, using oligonucleotides based on the genomic organization
(28).
Scanning the MMP-2 Gene--
Denaturing high performance liquid
chromatography (DHPLC) was used to scan the MMP-2 gene for
sequence variation, using the WAVETM DNA Fragment Analysis System
(Transgenomic), as previously described (29). Optimal PCR conditions
for faithful amplification were derived for each amplicon; reactions
were performed in 50-µl volumes containing 20 ng of genomic DNA, 50 mM potassium chloride, 25-pmol primers, 200 µM dNTPs, 1 mM MgCl2, and 2.5 units of PfuTurbo polymerase (Stratagene). Genomic DNA was
amplified from a panel of 32 unrelated healthy Caucasian subjects. PCR
products (45 µl) were denatured at 95 °C for 4 min and allowed to
reanneal to form homo- and heteroduplexes by cooling to 25 °C over a
50-min period. 10 µl of each PCR product was applied to the
WAVETM machine using varying column temperatures
(57-69 °C), as predetermined by the Transgenomic Analysis Software
(according to the predicted melting characteristics of each amplicon).
Individual PCR products, which displayed heteroduplex signaling
patterns, were purified with QIAquickTM purification columns (Qiagen),
and both strands were sequenced on an ABI 377.
Human MMP-2 Promoter Constructs--
Luciferase reporter
plasmids were constructed by cloning 3 concatenated copies of the
adjacent 24 nucleotides, flanking a variant, upstream of a previously
characterized minimal promoter region of the human translation
initiation factor EIF-4AI gene (30). KpnI sites
were introduced at the 5'- and 3'-ends of concatamers to facilitate
cloning. The sequences of the forward oligonucleotides used for
concatenation are shown with variant(s) in lowercase: Cell Lines and Transient Transfections--
The RAW264.7, A10,
and 293 cell lines were obtained from the Sir William Dunn School of
Pathology Cell Bank, and reagents were purchased from Life
Technologies, Inc. Cells were grown in RPMI 1640 (RAW264.7) or
Dulbecco's modified Eagle's medium (A10 and 293) supplemented with
10% (v/v) heat-inactivated fetal calf serum, 2 mM
glutamine, and antibiotics at 37 °C and 5% CO2 in a
humidified incubator. For transient transfection experiments, 5 × 104 cells were plated in 10-mm 24-multiwell plates and
grown to 60-70% confluence. Transfection was carried out using
FuGENETM 6 Transfection Reagent (Roche Molecular Biochemicals)
according to the manufacturer's protocol. Cells were cotransfected
with 0.5 µg of reporter plasmid and 0.1 µg of
pcDNA3- Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts were prepared as described previously (31). EMSAs were
performed using the following double-stranded oligonucleotides as
probes: Isolation of the Human MMP-2 Promoter--
To generate additional
sequence data for variant analysis, an extended promoter region of the
human MMP-2 gene was isolated. Nested MMP-2-specific primers
were used with adaptor primers to amplify the promoter from five
adaptor-ligated human genomic libraries. The DraI,
SspI, and ScaI libraries yielded products of 1.7, 4.1, and 6.0 kb, respectively, that were cloned and analyzed by
restriction enzyme mapping. The DNA sequence extending 1900 bp upstream
of the major transcription start site (28) was determined, and 11 overlapping amplicons were designed for DHPLC analysis.
Determination of Flanking Intronic Sequence--
Additional
intronic sequence was generated to that available (28) by sequencing
introns, isolated as PCR products, and the information obtained was
used to design appropriate primers for DHPLC analysis to evaluate the
extent of genetic variation at MMP-2 splice-site junctions.
Identification of Novel Genetic Variants in the MMP-2
Gene--
Thirty four different PCR primer pairs, encompassing the
promoter sequence (1900 bp), 5'-UTR, complete coding region, and 3'-UTR, were designed to assess the nature and extent of nucleotide variation in the human MMP-2 gene (see Table
I). Amplicons were generated from a panel
of 32 unrelated healthy Caucasian individuals. This number was chosen
to provide >90% power to detect polymorphisms with an allele
frequency of >5% (21), on the basis that common variants were the
principal target of this screen. PCR products were applied to the DHPLC
column and subjected to partial heat denaturation, over a 6.8-min
interval, to produce sequence-specific chromatograms. Elution profiles
were compared with each other, with double or multiple peak patterns
indicating the presence of polymorphic site(s) (Fig.
1). The nature and location of variants were determined by dye-terminator sequencing.
By using this approach, a total of 15 novel sequence variants were
identified in the MMP-2 gene. All variants were single base
substitutions, comprising seven transversions and eight transitions distributed throughout the gene with six variants in the promoter, one
in the 5'-UTR, six in the coding region, one in intervening sequence,
and one in the 3'-UTR (Fig. 2).
Analysis of Variants in the MMP-2 Coding Region, Introns, and
3'-UTR--
The majority of coding region variants (Fig.
2A) results in synonymous substitutions; however, the G
We identified one intronic sequence variant in intron 5, located 11 bp
downstream of the intron start site. This does not map to known
splice-site junction consensus sequences and is therefore unlikely to
affect mRNA splicing patterns. We also identified a common A Analysis of Variants in the MMP-2 Promoter and 5'-UTR--
Six
variants were identified in the promoter region (four transversions
and two transitions) that were evenly distributed across a ~2-kb
interval with the exception of two T Generation of Reporter Gene Constructs to Measure Differences in
Allelic Expression between MMP-2 Promoter Variants--
To distinguish
functional variants from nonfunctional neutral counterparts, we
generated a panel of reporter gene constructs that were used to measure
differences in allelic expression, in the context of the regulatory
region in which the variant was located. Specifically, four reporter
gene constructs were made per variant, in which three concatenated
copies of the 24-bp nucleotide region flanking a variant were cloned
immediately upstream, and in both orientations, of the human EIF-4AI
minimal promoter (30) driving the expression of a luciferase reporter
gene (see Fig. 3). The resultant
constructs were used to transfect transiently several different cell
lines including epithelial cells (293), macrophages (RAW264.7), and
smooth muscle cells (A10), using a pcDNA3-
Prioritization of those variants most likely to be functional was based
on previous studies that proposed a minimum threshold of a 2-fold
difference in allelic expression as being a plausible indicator of
functionality (38, 39). Based on these guidelines the
In contrast, the Sp1 consensus sequence (CCACC) spanning the
Curiously the
Taken together, as expected, these results suggest that the majority of
variants characterized in this study are nonfunctional neutral SNPs,
whereas the Allele-specific Binding of Nuclear Protein at the The Transcription Factor Sp1 Binds to the Sp1 Functions as an Activator of MMP-2 Promoter Activity in an
Allele-specific Manner--
To determine the allele-specific effects
of Sp1 binding upon native promoter activity, two luciferase reporter
gene constructs were generated by PCR, spanning The common disease-common variant hypothesis takes account of the
observation that the human population has relatively limited genetic
diversity, such that common variants may contribute significantly to
genetic risk for common disease (21, 23). The human matrix metalloproteinase-2 gene (MMP-2) possesses proteolytic
activity against type IV collagen, a major component of the basement
membrane, and is therefore implicated in an extensive array of
pathologies including atherogenesis, arthritis, and tumor growth and
metastasis (11-17). The present investigation was designed to search
for naturally occurring genetic variation in the MMP-2 gene,
to analyze the functional effects of promoter variants on gene
expression and, thereby, to generate an informative panel of
polymorphisms to test for possible association with a variety of
clinical phenotypes.
MMP-2 spans 17 kb and contains 13 exons encoding a 72-kDa protein (28).
We scanned ~3.1 kb of MMP-2 transcribed sequence, 1.9 kb of promoter
sequence, and ~1 kb of intronic sequence (Fig. 1 and Table I).
Fifteen novel single base substitutions were identified as being
distributed throughout the gene with six variants in the promoter (1 SNP/317 bp), one in the 5'-UTR (1 SNP/280 bp), six in the coding region
(1 SNP/328 bp), one in intronic sequence (1 SNP/1000 bp), and one in
the 3'-UTR (1 SNP/799 bp; Fig. 2). Collectively, these data represent a
medium level of sequence diversity, reflecting sequence conservation
through natural selection during evolution, which is similar to
previous reports assessing the extent of molecular variation at other
loci (40, 41). In concordance with these studies, we found that
variants in the coding region were much more prevalent at synonymous
than nonsynonymous sites, with only one SNP causing an amino acid
substitution (from glycine to serine at codon 456). This highly
conserved amino acid is situated in the hinge region of the protein,
which links the catalytic domain to the hemopexin-like domain. The
physical constraints imposed on glycine by this proline-rich linker
region suggest that substitution by a larger hydrophilic amino acid may
have a deleterious effect upon the juxtaposition of the two domains thereby disrupting normal enzymatic activity (32). We are currently undertaking in vitro studies to address this issue.
Predicting the functional consequences of the synonymous substitutions
is more difficult due to the limited number of studies addressing the
contribution of such variants to structural diversity of mRNA. However, a recent investigation demonstrated that single nucleotide variation at synonymous sites can give rise to allele-specific mRNA
folds that ultimately possess different biological functions (42).
Accordingly, it remains possible that some synonymous SNPs may not be
silent neutral variants.
Likewise, SNPs that affect gene regulation are equally important in
disease risk, but it is much more difficult to segregate such variants
from among the much larger pool of neutral counterparts by inspection
alone, given our limited knowledge of DNA regulatory regions. To
circumvent this problem we functionally characterized the entire panel
of promoter polymorphisms, extending over a 2-kb interval, identified
in this study. Limited functional analyses of the MMP-2
promoter have been performed previously, and although sequence analysis
demonstrates the presence of multiple potential cis-acting
elements, few have been characterized. We therefore generated a panel
of constructs that allowed direct comparison of allelic expression in
the context of the regulatory element in which the variant was located
(Fig. 3). Data base analysis confirmed that three variants, at Our biological experiments did, however, demonstrate that the common C
We believe that the MMP-2 In summary, we have described the nature and extent of nucleotide
variation in the human MMP-2 gene by identifying 15 novel SNPs distributed throughout the coding and noncoding regions. We have
characterized an unreported functional promoter polymorphism that
produces an allele-specific effect on expression through abolishing
binding of the transcription factor Sp1. We believe this systematic
approach of characterizing functional variants in the regulatory
regions of important candidate genes will facilitate the rapid
elucidation of common disease susceptibility, or resistance, variants.
These studies indicate that the MMP21306,
790, and +220) mapped onto cis-acting
elements. We functionally characterized all promoter variants by
transient transfection experiments with 293, RAW264.7, and A10 cells.
The common C
T transition at
1306 (allele frequency 0.26),
which disrupts an Sp1-type promoter site (CCACC box), displayed a
strikingly lower promoter activity with the T allele. Electrophoretic
mobility shift assays confirmed that these differences in allelic
expression were attributable to abolition of Sp1 binding. These data
suggest that this common functional genetic variant influences
MMP-2 gene transcription in an allele-specific manner and
is therefore an important candidate to test for association in a wide
spectrum of pathologies for which a role for MMP-2 is implicated,
including atherogenesis and tumor invasion and metastasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
13/+10 relative to the transcription start site), was performed, and
products from the DraI, SspI, and ScaI
libraries were gel-purified and cloned into pBluescript II SK(
)
(Stratagene). Sequence extending 1900 bp upstream of the transcription
start site was obtained by sequencing several independent clones on an
ABI 377 automated sequencing apparatus with cycle sequencing according
to the manufacturer's instructions (PerkinElmer Life Sciences).
1575G,
5'-AAGACATAATCgTGACCTCCAATG-3';
1575A,
5'-AAGACATAATCaTGACCTCCAATG-3';
1306C,
5'-ACCCAGCACTCcACCTCTTTAGCT-3';
1306T,
5'-ACCCAGCACTCtACCTCTTTAGCT-3';
955C, 5'-TGCCATGGCATTTATAcACTGCCA-3';
955A, 5'-TGCCATGGCATTTATAaACTGCCA-3';
790T/
787T,
5'-GTGACCTCTATCtTAtTAAACCAG-3';
790G/
787T,
5'-GTGACCTCTATCgTAtTAAACCAG-3';
790T/
787G,
5'-GTGACCTCTATCtTAgTAAACCAG-3'; 790G/
787G,
5'-GTGACCTCTATCgTAgTAAACCAG-3';
168G, 5'-AGGCGGCCGGGgAAAAGAGGTGGA-3';
168T, 5'-AGGCGGCCGGGtAAAAGAGGTGGA-3'; +220G,
5'-ACCAGGCGGCGAgGCGGCCACACG-3'; and +220C,
5'-ACCAGGCGGCGAcGCGGCCACACG-3'. Additionally, a PCR-based
approach was used to generate a construct encompassing
1691 to +10 of
the human MMP-2 promoter. 5'-HindIII and
3'-XhoI cloning sites were included in the forward and
reverse primers, respectively. To obtain the
1691/+10 DNA fragment,
the oligonucleotide sequence
1691/
1671
(5'-GTCAAAGCTTA-AAACTGACTCTGGAAAGTCA-3') and
11/+10
(5'-GTCACTCGAGTCTGGATGCAGCGGAAACAAG-3') were used in the PCR in
combination with PfuTurbo polymerase to ensure high fidelity
amplification. The PCR product was digested with HindIII and
XhoI and ligated into an appropriately digested pGL3-Basic vector. The resulting construct was designated as p1306T because sequence analysis demonstrated that it contains a thymine at the
1306
polymorphic site. Subsequently, p1306T acted as a template to generate
a construct with a thymine to cytosine point mutation at the
1306
position, assigned p1306C, using the QuikChangeTM
Site-directed Mutagenesis Kit (Stratagene), according to the manufacturer's instructions. All constructs used in this study were
restriction-mapped and sequenced to confirm their authenticity.
-galactosidase expression vector (30) to standardize for
transfection efficiency. Cells were incubated for 24 h, washed
twice in phosphate-buffered saline, and harvested by the addition of
100 µl of lysis buffer (Luciferase Assay System, Promega). Luciferase
levels were quantified using a Luminoskan Ascent Luminometer
(Labsystems), and
-galactosidase activities were measured using a
commercially available ELISA kit (Roche Molecular Biochemicals). All
experiments were carried out in triplicate and independently performed
at least three times. Results were expressed as a ratio of luciferase
activity to
-galactosidase activity, and statistical levels of
significance, for comparison between transfections, were determined by
the Student's t test. Mammalian expression vectors
containing wild-type and mutant murine GATA-1 were a kind gift from Dr.
Sjaak Philipsen (Erasmus University, Rotterdam, The Netherlands).
1306C, 5'-ACCCAGCACTCCACCTCTTTAGCT-3';
1306T,
5'-ACCCAGCACTCTACCTCTTTAGCT-3'; and Sp1cons,
5'-ATTCGATCGGGGCGGGGCGAGC-3'. 0.25 ng of
32P-end-labeled oligonucleotide (>30,000 cpm) was
incubated for 20 min at room temperature with 10 µg of nuclear
extract in a 20-µl reaction volume containing 40 mM Hepes
(pH 7.9), 12.5% glycerol, 1 mM EDTA, 2 µg of
poly(dI-dC), 0.1 µg of bovine serum albumin, and 0.2 µg of sheared
salmon sperm. For competition experiments, a 50-100-fold molar excess
of unlabeled double-stranded oligonucleotide containing either cold
probe, an Sp1-mutant consensus site
(5'-ATTCGATCGGTTCGGGGCGAGC-3'), or a nonspecific oligonucleotide
(5'-GAGCGCATACTGACTATCGGAGAC-3') was preincubated for 10 min at
room temperature with the nuclear extracts prior to the addition of
labeled probe. For supershift experiments, antibodies raised against
Sp1 (sc-59X), AP-2 (sc-184X), RANTES (sc-1410), and control rabbit IgG
were purchased, with Sp1-blocking peptide (sc-59P), from Santa Cruz
Biotechnology. Antibody (2 µg), or antibody-blocking peptide complex,
was incubated with nuclear extracts at 4 °C for 30 min, followed by
an additional incubation for 20 min at room temperature with labeled
oligonucleotide. Samples were run on a nondenaturing 7% polyacrylamide
gel in 0.25× TBE, at 250 V for 2-3 h. Dried gels were exposed
overnight to Kodak X-Omat film at
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Primers used to amplify regions of the human MMP-2 gene for DHPLC
analysis
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Fig. 1.
DHPLC analysis of the MMP-2
gene. Chromatograms produced by DHPLC analysis of exon 9 for
three different individuals (A-C). Nucleotide variation is
detected by high resolution separation of heteroduplexes, which form in
PCR samples having internal sequence variation, from homoduplex
counterparts. For example, individual A displays a single
peak of homoduplex DNA, whereas individuals B and
C exhibit multiple peaks caused by heteroduplex generation
during PCR amplification. Sequence analysis identifies the nature and
location of variants, individual B (G A, nt 1660),
individual C (G
A, nt 1646), thereby illustrating the
specificity and sensitivity of this technique. The x axis
displays column retention time in minutes; the y axis is a
measure of absorbance (converted to microvolts); the common deflection
peak at 0.5 min is due to residual dNTPs and unincorporated primers
from the PCR.
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Fig. 2.
Genetic variants in the MMP-2
gene. Summary of the location, nature, and minor allele
frequencies of single base substitutions identified in the 5'-UTR,
coding region, intronic sequence, and 3'-UTR (A) and the
promoter region of the human MMP-2 gene from a panel of 32 unrelated healthy Caucasian individuals (B). Nucleotide
positions are referenced relative to the major transcription start site
(28), and amino acids are designated by the standard 1-letter
code.
A transition at cDNA position 1646 causes a nonconservative amino
acid change from glycine to serine (G456S). This amino acid is situated
in the hinge region of the protein, which links the catalytic domain to
the hemopexin-like domain, and is believed to be important in the
targeting of substrates (32). Clustal alignment revealed that
Gly-456 is conserved across species (chicken, rabbit, rat, and
mouse) demonstrating the importance of this small neutral residue and
signifying the potential impact that a substitution to a larger
hydrophilic amino acid may have upon enzymatic activity/substrate specificity. We are currently undertaking in vitro studies
to address possible functional effects of this variant.
C
transversion in the 3'-UTR of the gene (nt 2523). Sequence analysis
established that this variant does not lie within, or in close
proximity to, adenylate/uridylate-rich elements that are known to be
bound by families of proteins implicated in the regulation of mRNA
stability (33).
G transversions at
790 and
787 (Fig. 2B). Sequence analysis demonstrated an error in
the published sequence (27) with the AG dinucleotide reported at
position
1569 actually being the G
A polymorphism at
1575
(allowing for the different transcription start sites mapped in these
studies). To determine whether variants created or abolished potential
cis-acting elements, we used the TRANSFAC data base (34) to
identify polymorphisms that might have an effect on transcription
through altering the binding of transcription factors (see Table
II). Interestingly, three variants mapped
onto regions displaying 100% homology to previously reported consensus sequences as follows: stimulating protein 1 (Sp1) at
1306 (35), an
inverted GATA-1 site at
790 (36), and a cell
cycle-dependent element (CDE) in the 5'-UTR, at +220 (37).
We therefore considered the +220 variant as being potentially integral
to the regulatory mechanisms imposed by the MMP-2 promoter
and included it in our subsequent analyses.
Mapping of MMP-2 promoter variants to potential cis-acting sequences
using the TRANSFAC database (34)
-galactosidase
expression vector to standardize for transfection efficiency. Data were
presented as the fold increase in allelic expression relative to empty
vector (pEIF-4AI) (see Table III).
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Fig. 3.
Cloning strategy used to compare allelic
expression of MMP-2 promoter variants. Schematic
of constructs used to measure allelic expression in transient
transfection experiments, as shown for the 1306 variant. Each
construct contains three concatenated 24-bp DNA oligonucleotides
flanking the C or T allele. Concatenated oligonucleotides were cloned
in both directions for each variant to verify that differences in
allelic expression were independent of orientation and position of the
regulatory element. Allelic expression, standardized for transfection
efficiency, was measured as fold increase relative to the pEIF-4AI
construct.
Transient transfection data demonstrating differences in allelic
expression between MMP-2 promoter variants
1575G/A and
955C/A variants were classified as neutral as they consistently
produced similar levels of luciferase activity for both alleles in all
cell lines studied; the
168G/T polymorphism produced allelic
differences in some cell lines, but these were below the 2-fold
threshold. Thus, these transfection data correlated with our prior data
base analysis confirming that these variants do not map to known
cis-acting elements (Table II). Additionally the +220G/C
variant, mapped to a CDE consensus sequence (CGCGG), exhibited varying
luciferase activities that were beneath the 2-fold threshold for
differences in allelic expression.
1306
polymorphic site displayed allele-specific transcriptional effects as
1306C transfectants expressed at least 2-fold higher luciferase
activity than cells transfected with
1306T constructs: 1.71 ± 0.25 versus 0.57 ± 0.09, p < 0.05 (
1306C and
1306T, respectively, RAW264.7). This effect was observed
in several other cell lines (data not shown) and collectively provided
strong supportive data correlating reduced expression from the T allele
with the abolition of the Sp1 consensus site. A similar association
between
790 allelic expression and an inverted GATA-1 element
(CTATCT) could not, however, be inferred as comparable luciferase
levels were measured between these constructs as follows: 6.12 ± 0.22 versus 6.85 ± 0.09, p < 0.05 (
790T/
787T and 790G/
787T, A10). Indeed, similar results were
attained when cells were cotransfected with a GATA-1 expression vector
(data not shown).
787G allele, downstream of the GATA-1 site, did appear
to regulate transcription in a cell type-specific manner. For example,
790T/
787G transfectants in 293 cells showed a 4.3-fold reduction in
promoter activity compared with the wild-type construct (
790T/
787T)
versus a 1.2-fold reduction in A10 cells (p < 0.05-0.001). However, the fact that this variant does not map to
known cis-acting elements coupled with the nondetection of
nuclear protein(s) binding to this region by EMSAs (data not shown)
leaves the explanations of these differences unclear.
1306 C
T transition may influence MMP-2
promoter activity in an allele-specific manner.
1306
Polymorphic Site of the MMP-2 Promoter--
We performed EMSAs to
investigate whether differences in allelic expression between the
1306C and
1306T allele were attributable to the differential
binding of nuclear protein(s). In these assays, two oligonucleotide
probes corresponding to the sequence from
1317 to
1294 in the
MMP-2 promoter, with either a T or C at the
1306
polymorphic site (Fig. 4A),
were 32P-labeled and allowed to interact with crude nuclear
extracts prepared from different cell lines including smooth muscle
cells (A10), epithelial cells (293), and monocytic leukemia cells
(U937) (Fig. 4, B-D, respectively). Two DNA-protein
complexes (designated I and II) were consistently
detected with the
1306C probe, but not the
1306T probe, in these
assays, irrespective of cell type. To determine the sequence
specificity of these DNA-protein complexes, competition experiments
were performed. Both bands were competed with 50- and 100-fold excess
of unlabeled
1306C probe (lanes 10 and 11) but
not by 50- or 100-fold excess of unlabeled
1306T probe (lanes
12 and 13). Furthermore, the specificity of these bands
was confirmed by addition of 100-fold excess of nonspecific competitor
(lane 14). In contrast, a third DNA-protein complex (Fig.
4C, designated III) was confirmed to be
nonspecific using these controls. These assays clearly demonstrated the
ability of the
1306C allele, not the
1306T allele, to bind
specifically nuclear protein(s).
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Fig. 4.
Electrophoretic mobility shift assays:
allele-specific binding of nuclear protein(s) at the 1306
position. A, sequences of oligonucleotide probes used
to investigate allele-specific functionality (
1306 variant is shown
in bold, and putative Sp1 consensus is
underlined). 32P-Labeled T or C allele-specific
probes corresponding to the sequence from
1317 to
1294 of the
MMP-2 promoter were incubated with nuclear extracts prepared
from A10 (B), 293 (C), and U937 (D)
cells prior to polyacrylamide gel electrophoresis. Lanes 1 and 8, probes alone; lanes 2 and 9,
probes with nuclear extracts in absence of competitor; lanes
3-7 and 10-14, probes with nuclear extracts in the
presence of various unlabeled competitors, as indicated
below the bottom autoradiograph. The bands
representing DNA-protein complexes are indicated: I and
II, specific; III, nonspecific.
1306 C Allele but Not
the T Allele--
To determine the identity of the nuclear protein(s)
that bind, in an allele-specific manner, to the MMP-2
promoter sequence at the
1306 site, we performed additional EMSAs to
investigate the observation that an Sp1 consensus sequence (CCACC) is
abolished by the presence of a T at the
1306 site;
1307C(C/T)ACC
1303. We therefore performed
EMSAs using two oligonucleotide probes comprising the previously
described
1306C probe and an Sp1 consensus probe
(5'-ATTCGATCGGGGCGGGGCGAGC-3'). As seen in Fig.
5A, 32P-labeled
Sp1 consensus probe forms two specific DNA-protein complexes (lane 2) identical to those observed with the
1306C probe
(lane 9), each being eliminated by excess unlabeled probe
(lane 3, and lanes 10 and 14,
respectively). Lanes 6 and 13 demonstrate the specificity of these DNA-protein complexes by competition experiments with 100-fold excess of unlabeled mutated Sp1 consensus probe (5'-ATTCGATCGGTTCGGGGCGAGC-3'). Furthermore, the ability of the C
allele, but not the T allele, to compete specifically for Sp1 binding
was confirmed by additional competition experiments using 100-fold
excess of unlabeled
1306T probe (lanes 4 and
12), 50-or 100-fold excess of unlabeled
1306C probe
(lanes 5 and 7), and 100-fold excess of unlabeled
Sp1 consensus probe (lane 11). Supershift assays were then
performed to confirm Sp1 binding using the Sp1 consensus probe (Fig.
5B, lanes 1-6) or
1306C probe (Fig.
5B, lanes 7-12) in either the absence
(lanes 1 and 7) or presence of different
antibodies (lanes 2-6 and lanes 8-12,
respectively). Both DNA-protein complexes were successfully
supershifted with anti-Sp1 antibody (lanes 2 and
8). In contrast, preincubation of Sp1 antibody with an Sp1
antibody-specific blocking peptide abrogated the formation of a
supershift complex (lanes 3 and 9). The
specificity of the Sp1 supershift was further confirmed using a variety
of isotype-matched antibodies (lanes 4-6 and lanes
10-12, respectively). The results obtained from these EMSAs were
reproduced using several different nuclear extracts as well as
recombinant Sp1 protein (data not shown).
View larger version (59K):
[in a new window]
Fig. 5.
Electrophoretic mobility shift assays
demonstrate that Sp1 can bind to the 1306C allele but not the
1306T
allele. A, 32P-labeled Sp1 consensus probe
(Sp1cons; lanes 1-7) or
1306C probe
(lanes 8-14) were incubated in the absence (lanes
1 and 8) or presence of A10 nuclear extracts
(lanes 2-7 and lanes 9-14), electrophoresed,
and visualized by autoradiography. Competition experiments with
100-fold excess of unlabeled Sp1 consensus probe (lanes 3 and 11), unlabeled
1306T probe (lanes 4 and
12), unlabeled
1306C probe (lanes 5 and
10), and unlabeled mutated Sp1 consensus probe (Sp1mut;
lanes 6 and 13) were performed as shown.
Lanes 7 and 14 depict competition experiments
with a 50-fold excess of unlabeled
1306C probe. The bands
representing specific DNA-protein complexes (I and
II) are indicated. B, probes were prepared as
above and incubated with 293 nuclear extracts either in the absence
(lanes 1 and 7) or presence of antibodies to Sp1
(lanes 2 and 8), AP-2 (lanes 4 and
10), RANTES (lanes 5 and 11), or
rabbit IgG (lanes 6 and 12). Lanes 3 and 9 represent anti-Sp1 antibody preincubated with anti-Sp1
antibody-specific blocking peptide prior to addition of nuclear
extracts. Sp1 antibody supershifted both bands of the DNA-protein
complex as indicated by the arrow. Nonspecific AP-2, RANTES,
or rabbit IgG had no effect.
1691 to +10 of the
MMP-2 promoter sequence, with either a T or C at the
1306
polymorphic site (Fig. 6A) and
used to transfect transiently epithelial cells (293) and macrophages
(RAW264.7). As shown in Fig. 6B, reporter gene expression driven by the C allelic MMP-2 promoter was ~1.6-fold
greater than reporter gene expression directed by the T allelic
counterpart in epithelial cells (4.0 ± 0.53 versus
2.45 ± 0.31, p < 0.001) and ~1.4-fold greater
in macrophages (4.27 ± 0.29 versus 2.98 ± 0.37, p < 0.01) emphasizing the biological significance of
this common variant, on promoter activity, through recruitment of
Sp1.
View larger version (13K):
[in a new window]
Fig. 6.
Transient reporter gene expression assays
with constructs containing full-length MMP-2
promoter. A, schematic presentation of reporter
gene constructs, used in transient transfections, containing a 2-kb
MMP-2 promoter with the only difference between the two
constructs being a T or C at the 1306 polymorphic site. B,
epithelial cells (293) and macrophages (RAW264.7) were transiently
transfected as described under "Experimental Procedures."
Luciferase and
-galactosidase levels were determined in triplicate
and standardized for transfection efficiency; fold increase was
determined by defining the activity of the empty pGL3 Basic vector as
1. Data shown are mean fold increase ± S.D. from eight
independent experiments. **, p < 0.01; ***,
p < 0.001.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1306,
790, and +220, mapped perfectly to consensus sequences for Sp1,
GATA-1 and CDE, respectively (Table II). Transient transfection studies
were performed in multiple cell types based on the applicability of
each to different disease phenotypes. By using selection criteria based
on previous proposals (38, 39), we demonstrated that the majority of
variants assessed in this manner were nonfunctional, comprising those
that did not map to known consensus site(s) as well as some that did,
namely variants at
790 (GATA-1) and +220 (CDE) (Table III). These
results may be explained, in part, by the nature of the consensus
element itself. The
790 variant maps to a degenerate site outside of the core GATA-1 consensus region, whereas the CDE is typically found in
cell cycle regulator genes in association with a contiguous element,
absent in the MMP-2 promoter, to form a bipartite repressor regulating gene expression. The apparent nonfunctionality of the
790
and +220 transversions are important observations demonstrating the
inherent problems that exist in predicting which SNPs in noncoding DNA
will be functional.
T transition at position
1306, which interrupts an Sp1 site, is
indeed functional. Transient transfection experiments showed that the
1306C allele increased promoter activity in two different luciferase
reporter gene constructs, one in the context of the Sp1 regulatory
element and the other in the background of the native MMP-2
promoter. Both showed that reporter gene expression was between
~1.4-2-fold higher with the C allele than the T allele (Fig. 6 and
Table III). We performed EMSAs to determine whether disparity in
allelic expression was attributable to the differential binding of
nuclear protein(s) (Fig. 4). Two DNA-protein complexes were detected as
binding to the C allele, but not the T allele, and additional
competition experiments combined with supershift analysis identified
the protein binding to this region as Sp1 (Fig. 5). Sp1 is a
ubiquitously expressed transcription factor that binds to GC/GT-rich
elements and regulates a variety of genes in a constitutive or
inducible manner (43, 44). One such motif, the CCACC box, has been
shown to be essential for Sp1 binding and promoter function in several
genes by invariably activating transcription (35, 45, 46). Sp1 is a
multifunctional protein that can directly interact with the basal
transcription complex, as recently shown for the MMP-2
proximal promoter (47), or alternatively function as a more general
transcription factor and play an important role in directing
tissue-specific expression (48, 49). Its ability to bend DNA (50) and
self-associate to loop out intervening promoter regions (51) enables it
to interact with other transcription factors, such as NF-
B (52), to
exert a synergistic effect essential for modulating gene activation.
Clearly any variant that abolishes Sp1 binding, such as the
MMP-2
1306 polymorphism, has the potential to affect the
level and specificity of gene transcription. Indeed these results
highlight the importance of the combinatorial nature of transcription
factors in adjusting MMP-2 expression profiles and imply that this
regulatory mechanism may be more important than previously suggested
(53), thereby introducing a further level of control ahead of proenzyme activation.
1306 polymorphism is a strong
candidate variant for direct allelic association studies, based on
several observations. First, although these findings do not preclude
the possibility that functional variants exist elsewhere in the
MMP-2 promoter, the functional effect of the
MMP-2
1306 polymorphism on gene expression through Sp1
binding is clear. Second, the identification of several nonfunctional
variants reduces the need for multiple hypothesis testing thereby
reducing the risk of false positive association. Third, the high allele
frequency of this variant (0.26) makes it more informative for
association analysis, particularly in family-based tests, and also
increases the power to detect linkage disequilibrium within the region. Interestingly, a T
C polymorphism in the human CYP17
gene that creates an Sp1 site (CCAC(T/C)) has been shown to be
associated with polycystic ovary syndrome (54), breast cancer (55, 56), and prostate cancer (57).
1306 polymorphism will be informative in tests of association in a wide spectrum of
pathologies in which a role for MMP-2 is implicated. Such studies will
test the robustness of the common disease-common variant hypothesis and
improve our understanding of SNP involvement in complex genetic disease.
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ACKNOWLEDGEMENTS |
---|
We thank A. Henney for initial support of this project, and we thank C. Whatling, C. Quinn, and D. O'Reilly for invaluable technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by British Heart Foundation Grant RG/1995008.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ298926.
§ Recipient of a British Heart Foundation Studentship Grant FS/97051.
British Heart Foundation Basic Science Lecturer and supported
by Grant BS/99003.
** To whom correspondence should be addressed: Dept. of Cardiovascular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, UK. Tel.: 44-0-1865-220257; Fax: 44-0-1865-768844; E-mail: hugh.watkins@cardiov.ox.ac.uk.
Published, JBC Papers in Press, December 12, 2000 DOI 10.1074/jbc.M010242200
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ABBREVIATIONS |
---|
The abbreviations used are: MMPs, matrix metalloproteinases; SNPs, single-nucleotide polymorphisms; PCR, polymerase chain reaction; DHPLC, denaturing high performance liquid chromatography; 5'-UTR, 5'-untranslated region; 3'-UTR, 3'-untranslated region; Sp1, stimulating protein 1; CDE, cell cycle-dependent element; kb, kilobase pair; bp, base pair; nt, nucleotide; EMSAs, electrophoretic mobility shift assays.
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