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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3286-3293
Detection of a Complex That Associates With the B Fibrinogen
G 455-A Polymorphism
By
Erika T. Brown and
Gerald M. Fuller
From the Laboratory of Medical Genetics and Department of Cell
Biology, University of Alabama at Birmingham, Birmingham, AL.
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ABSTRACT |
The promoter region of the B fibrinogen gene containing the
polymorphic site (G 455-A) shows an increase in
fibrinogen levels for individuals containing an adenine rather than a
guanine. Two methods were used to explore the possible functional role of this region. Electrophoretic mobility shift assays (EMSAs) were
performed using specific DNA probes containing base sequences pertinent
to the allelic site. Specific DNA binding proteins were detected and
their binding characteristics were determined. Secondly, we placed DNA
fragments containing different  455 nucleotide substitutions of the
B promoter upstream of a luciferase reporter gene and transfected
them into HepG2 cells to determine their effect on transactivation. An
adenine at position 455 resulted in greater luciferase activity than
when a guanine was present. UV cross-linking bound protein to the DNA
demonstrated a 47-kD protein binding preferentially to the site when a
guanine rather than an adenine was present at 455. We hypothesize
that a transactivation protein complex associates with the site, but
its association is stronger when guanine is present, thereby slowing
downstream B gene transcription. These data provide the first
molecular evidence that accounts for the increase in fibrinogen in
individuals carrying this allele.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
ELEVATED LEVELS OF fibrinogen are an
independent risk factor for the development of cardiovascular
diseases.1-9 Fibrinogen is one of the primary agents in
blood clot formation at sites of vascular damage. In instances of
physical trauma or infection, fibrinogen is transiently upregulated by
the cytokine interleukin-6 (IL-6).10-13 However, elevated
circulating fibrinogen levels are also associated with increased age,
gender, race, smoking, obesity, stress, elevated cholesterol, and
menopause.1 Structurally, fibrinogen is comprised of three
pairs of nonidentical polypeptide chains: A , B, and
 .10,12,13 The chains are encoded by three separate genes
clustered on chromosome 4q23-32.13 These genes are under
such stringent transcriptional control that if one chain is
overexpressed, the other two will upregulate; however, the molecular
basis of this coordinated signaling is not understood.14,15 The identified regulatory elements further complicate the understanding of the coordinated transcriptional control, because the promoter regions of the three chains are not completely identical. All three
genes have a type II IL-6 response element and TATA-like sequences;
however, only the chain has a USF site and the A and B chains
have C/EBP sites immediately proximal to the IL-6 response sequence
and, further downstream, HNF-1 sites.16-19 In addition to
these identified regulatory elements, there may be a number of other,
yet unidentified factors that are involved in regulating transcription
of each gene.
All three fibrinogen chains are essential for function and secretion of
the molecule; however, emphasis has been placed on the B chain for
two reasons. First, in studies performed to understand the order of
fibrinogen assembly, it was suggested that the B chain may be the
more prominent chain in monomer assembly, thereby marking it as the
nucleating chain.20-23 Second, additional experiments performed in human systems have shown that, when any one of the three
fibrinogen chain genes is transcriptionally upregulated, the result is
subsequent upregulation of the other two chains, with the largest
increase in total chain and fibrinogen production occurring when the
B chain is overexpressed.14,15,24,25 These observations
suggest that one of the determining factors of circulating fibrinogen
levels could be the rate at which the B chain gene is
transcriptionally regulated. It has been suggested that polymorphisms
found on the proximal promoter portion of the 5 flanking region
of the B gene may influence the interaction of transcription factors
that potentially bind to these polymorphic sites.26 To
date, a total of 11 polymorphisms have been found on the B
gene.27 Presently, no evidence has shown that there is a
direct correlation between B chain alleles and cardiovascular disease development; however, there is a direct correlation between B chain alleles and elevated circulating fibrinogen
levels.26,28-34 One polymorphism of interest is a
guanine-to-adenine (G  A) transition at the 455
position of the 5 flanking region of the B gene. Epidemiological evidence has shown that an adenine in the 455 position (the A allele) expresses increased levels of circulating fibrinogen.27-42 One previously reported study (abstract)
using 20-bp oligonucleotide probes implicated a protein binding within this region, but no additional information has been
reported.43
In this study, we have detected three complexes binding within the
region 468 to 439 of the B chain gene using
electrophoretic mobility shift assays (EMSAs). All three complexes bind
specifically to this site; however, one of the complexes preferentially
binds when a guanine is present in the 455 position rather than
an adenine. Functional analyses show that there is a 1.2- to 1.5-fold increase in B chain gene transcription when an adenine is in the
455 position, instead of a guanine. Mutation of the B chain gene sequence from 462 to 451 prevents binding of this
approximately 47-kD complex. These observations show a protein complex
that is allele specific and may potentially participate in B chain regulation by partially repressing B chain gene synthesis.
Furthermore, binding of this complex may not only affect the amount of
B chain that is produced, but also total circulating fibrinogen
levels, given the coordinated transcriptional control of all three
chains.
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MATERIALS AND METHODS |
Cell culture.
The human hepatocarcinoma cell line, HepG2 (ATCC, Rockville, MD), was
used and maintained in Dulbecco's modified Eagle's medium (DMEM; Cellgro, Herndon, VA, and Sigma, St Louis, MO),
1× nonessential amino acids (Cellgro), 1 mmol/L sodium pyruvate,
10% fetal bovine serum (Sigma), and 1× antibiotic/antimycotic
(ciprofloxacin [Pentex, Kankakee, IL], and later
penicillin/streptomycin [Cellgro] and nystatin [GIBCO-BRL, Grand
Island, NY]). Cells were incubated at 37°C under 5%
CO2.
Plasmid constructs and functional assays.
Constructs of the B gene from 636 to +16 were derived by
polymerase chain reaction (PCR) amplification. Primers for the constructs were designed to facilitate subcloning of the region from
623 to +9 into the pGL2 basic luciferase vector (Promega, Madison, WI), which uses the luciferase gene as its reporter. HepG2 DNA
is heterozygous for both alleles; therefore, the amplified A allele
construct was isolated by digesting G allele fragments with the
restriction enzyme Hae III and then subcloned into the pGL2
vector, and the G allele was obtained by performing site-directed mutagenesis by the procedure provided by the manufacturer (Clontech, Palo Alto, CA) on the A allele construct in the pGL2 vector. The subcloned G and A allele constructs were sequenced (Amersham, Arlington
Heights, IL) for verification of alleles and to ensure that no
mutations were introduced during PCR amplification. The vectors plus
construct were transfected into HepG2 cells using the calcium phosphate
transfection method.44 Transfected cells were stimulated
with 50 ng/mL of recombinant human IL-6 (rhIL-6; Promega)
for 16 hours. The cells were harvested, lysates were prepared, and
luciferase production was quantitated using a luciferase assay system
kit (Promega) and a luminometer (Biorad, Hercules, CA).
Nuclear extract preparation.
Nuclear extracts were obtained from HepG2 cells by performing a
modification of the published protocol.45 Cells were washed twice with cold phosphate-buffered saline (PBS), scraped from the
plates, resuspended in PBS, and pelleted at 1,500 rpm for 5 minutes at
4°C. The pellets were resuspended in 5 mL of buffer A (10 mmol/L
HEPES, pH 7.6, 15 mmol/L KCl, 0.15 mmol/L spermine, 0.5 mmol/L
spermidine, 2 mmol/L EDTA, pH 8.0, 2.4 mol/L sucrose, 0.5 mmol/L
dithiothreitol [DTT], 0.5 mmol/L phenylmethyl sulfonyl fluoride [PMSF], and 1% trasylol aprotinin [15
mg/mL]) and, using a tight pestle, homogenized with 20 to 25 strokes
in a Dounce homogenizer. The cell homogenate volume was increased to 14 mL with buffer A, divided into four 3.5-mL volumes, and placed over 1.3 mL of buffer B (10 mmol/L HEPES, pH 7.6, 15 mmol/L KCl, 0.15 mmol/L
spermine, 0.5 mmol/L spermidine, 2 mmol/L EDTA, pH 8.0, 2.0 mol/L
sucrose, 10% glycerol, 0.5 mmol/L DTT, 0.5 mmol/L PMSF, and 1%
trasylol aprotinin [15 mg/mL]) to form a sucrose gradient. The
samples were centrifuged at 27,400 rpm for 2 hours at 2°C. The
supernatants were removed and the pellets were pooled and resuspended
in 200 mL of extraction buffer (0.4 mol/L KCl, 20 mmol/L HEPES, pH 7.0, 20% glycerol, 2 mmol/L DTT, 1 mmol/L benzamidine, 0.2 mmol/L PMSF, 0.2 mmol/L EDTA, pH 8.0, 1 mmol/L sodium fluoride, 1 mmol/L sodium
diphosphate, and 0.1 mmol/L sodium orthovanadate) and centrifuged at
30,000 rpm for 1 hour at 0°C to pellet the chromatin. The
supernatant was removed from the chromatin pellet, aliquoted, and
stored at 80°C for use in EMSAs.
EMSAs.
Oligonucleotides of 34 bp were synthesized by Integrated DNA
Technologies, Inc (Coralville, IA) and radiolabeled with
[-32P] TTP (0.4 ng/ , ~100,000 cpm/; Amersham)
by the Klenow fragment of DNA Polymerase I (Boehringer Mannheim,
Indianapolis, IN). Nuclear extracts were thawed on ice before being
incorporated into the binding reaction. Protein concentrations of the
nuclear extracts were determined by using a commercially available
protein assay kit (Pierce, Rockford, IL). For the binding reactions, 20 µg of nuclear extract was added to binding buffer (10 mmol/L Tris, pH 7.5, 1 mmol/L DTT, 100 mmol/L KCl, 1 mmol/L EDTA, 0.2 mmol/L PMSF, 1 mg/mL bovine serum albumin [BSA], 5% glycerol), and 1 mmol/L DTT and
preincubated at room temperature for 10 minutes with 1 µg of poly
dI/dC · poly dI/dC in a reaction volume of 20 µL. Radiolabeled probe was then added to the binding reactions and incubation at room
temperature continued for 20 minutes. The competition assays were
performed using unlabeled probes combined with radiolabeled probes
added to the binding reaction together to prevent proteins from binding
to one probe before the other was introduced. The binding reactions
were loaded onto 7% nondenaturing polyacrylamide gels and run in
0.5× TBE at 500 V for 2.5 to 3 hours. Gels were placed on Whatman
3 paper (Whatman, Maidstone, UK), dried, and exposed to
autoradiograph film for 8 to 24 hours.
UV cross-linking assay.
UV cross-linking analysis was performed according to the published
procedure46 and by using binding assays as described for
the EMSAs; however, whole cell extract instead of nuclear extract was
used in the binding reaction. The whole cell extract was obtained by
using a modification of the published procedure.47 At
4°C, the cells were washed twice with cold PBS, scraped from the
plates with 500 mL of PBS, and pelleted at 1,500 rpm for 1 minute, and
the supernatants were discarded. Two volumes of extraction buffer (same
used for nuclear extract), approximately 250 µL, was added, the
pellets were vortexed, and the samples were put in an ethanol/dry-ice
bath for 2 to 3 minutes. The contents were allowed to thaw on ice and
vigorously mixed and vortexed, followed by centrifugation for 15 minutes at 13,000 rpm in the cold. The supernatants were removed and
aliquoted, and the pellets were discarded. After the binding reactions
were completed, the samples were covered with plastic wrap and exposed
to UV light at 254 nm for 5 minutes at approximately 5 cm from the UV
light source. The samples were then resolved on a 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel.
For isolation of DNA-bound complex III, whole cell extract (as
described) was used in the EMSA binding reaction and allowed to bind
onto a 34-bp internally labeled oligonucleotide probe. The binding
reactions were scaled up 5×, UV irradiated for 10 minutes, and
treated with DNAse I (Promega; published procedure).46 The
reactions were run on a 4% nondenaturing gel 2× thicker than the
previous EMSAs; the gel was not dried and was exposed to autoradiograph film overnight. The band corresponding to DNA-bound complex III was cut
from the gel, eluted (published procedure),48 and run on a
10% SDS-PAGE gel.
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RESULTS |
In vitro model of G/A allelic differences in B
fibrinogen expression.
Epidemiological studies have indicated that individuals heterozygous or
homozygous for an adenine in the 455 position of the B chain
gene have higher circulating fibrinogen levels than their homozygous G
(guanine at 455) counterparts. To observe if the alleles
affected transcriptional activity, the functionality of the promoter
was assessed when the A allele (adenine in the 455 position) or
the G allele (guanine in the 455 position) is present.
Constructs of the region of the B chain gene from 636 to +16,
containing all of the known proximal promoter elements required for
B chain transcription (HNF-1 site, C/EBP consensus site, and IL-6
response element), were synthesized by PCR amplification from HepG2 DNA
and subcloned into the pGL2 basic luciferase vector, containing an
adenine or guanine in the 455 position
(Fig 1A). After transfection into HepG2
cells and 16 hours of stimulation with rhIL-6, luciferase activity was
quantitated (Fig 1B). A series of 10 independent luciferase experiments
were performed and the increase in luciferase activity over basal level
readings was averaged. Activity of the pGL2 vector only (control) was
low, as expected, with an average luciferase activity of 1.1- ± 0.2-fold. There was approximately 1.4-fold greater luciferase activity
from the pGL2/A-455 construct over the pGL2/G-455 construct, with
average increases in luciferase activity after IL-6 stimulation of 3.2- ± 1.2-fold for pGL2/G-455 and 4.8- ± 1.4-fold for pGL2/A-455; the difference in luciferase activity between both alleles was statistically significant (P = .048). The pGL2/A-455 construct had higher luciferase activity than the pGL2/G-455 construct, between
the range of 1.2- to 1.5-fold in the independent experiments. The
results of these functional studies show that, in these in vitro
conditions, the A allele is associated with increased B chain
transcription. These results coincide with the epidemiological evidence, correlating the A allele with higher levels of circulating fibrinogen.
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| Fig 1.
The A allele shows greater promoter functionality in
response to IL-6. (A) The sequence of the B chain gene from 623
to +9 was subcloned into the pGL2 basic luciferase vector, upstream
from the luciferase gene. This portion of the B chain gene contains
all of the known proximal promoter elements required for induction of
B chain gene transcription: HNF-1 (hepatocyte nuclear factor-1),
79 to 91; C/EBP site (CAAT-enhancer binding protein site), 124
to 132; IL-6 RE (IL-6 response element), 137 to
142,16 and, in addition, the 455 F/S (455
nucleotide and its proximal 5 and 3 flanking sites). (B)
After stimulation of HepG2 cells transfected with either pGL2 vector
only (control), pGL2/G-455 (guanine at 455 of the B construct),
and pGL2/A-455 (adenine at 455 of the B construct) with IL-6 for
16 hours, the luciferase activity was quantitated and the fold increase
in luciferase activity over basal level was statistically averaged for
10 independent experiments. The activity for the pGL2 vector only was
1.1- ± 0.2-fold, for pGL2/G-455 was 3.5- ± 1.2-fold, and for
pGL2/A-455 was 4.8- ± 1.4-fold increase over basal level activity.
Statistical analyses using a two-sample t-test for independent
samples showed that luciferase activity under the control of the
pGL2/A-455 construct was significantly different from pGL2/G-455
activity (P = .048).
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Detection of DNA binding proteins and assessment of binding
specificity.
The increase in luciferase activity under control of the A allele
implies that a transactivation factor(s) binds to this site, resulting
in an increase in B gene transcription. Furthermore, the presence of
a particular nucleotide in the 455 position must affect the
binding of the transactivating factor(s) by altering its binding
affinity, which could affect B transcription.
To detect the presence of DNA binding proteins, EMSAs were performed.
The EMSAs were performed using 34-bp oligonucleotide probes, having the
sequence from 468 to 439 of the B chain gene with
either a guanine (G-455 probe, representing the G-allele) or an adenine
(A-455 probe, representing the A-allele) in the 455 position
(Fig 2), that were allowed to bind with
HepG2 nuclear extract. The results showed three constitutively bound
complexes, designated complex I, complex II, and complex III on both
A-455 and G-455 probes, present before and after IL-6 stimulation (data not shown), which is evident with the other B chain gene response elements.18,19
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| Fig 2.
Presence of complexes binding onto the 455 flanking
region. To detect complexes that may potentially bind onto the 455
nucleotide and its proximal 5 and 3 flanking regions, two
34-bp oligonucleotide probes were used in EMSA analysis having the
sequence of the B chain from 468 to 439. The probes contained
either a guanine (G-455) or an adenine (A-455) in the 455 position.
EMSA results resolved on 7% nondenaturing gels show three complexes
binding onto the radiolabeled probes when either an adenine (A-455),
representing the A allele (A, lanes 1 through 5), or a guanine (G-455),
representing the G allele (B, lanes 6 through 10), is present. To
assess the specificity of the bound complexes, both probes were
competed with increasing concentrations of unlabeled probe: 0-fold,
10-fold (4 ng), 100-fold (40 ng), 250-fold (100 ng), and 500-fold (200 ng) excess the radiolabeled probe concentration. Both competition
assays, radiolabeled A-455 versus unlabeled A-455 (lanes 1 through 5)
and radiolabeled G-455 versus unlabeled G-455 (lanes 6 through 10),
show that all three complexes are binding onto both probes
specifically; however, complexes II and III appear to bind more
specifically.
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To determine if these protein complexes were specific, a series of
competition assays were performed in which the radiolabeled probes were
competed against their unlabeled sequences, radiolabeled A-455 versus
unlabeled A-455 (Fig 2, lanes 1 through 5) and radiolabeled G-455
versus unlabeled G-455 (Fig 2, lanes 6 through 10). The unlabeled
competitors were competed against the labeled probes at concentrations
of 10-fold (4 ng), 100-fold (40 ng), 250-fold (100 ng), and 500-fold
(200 ng) the concentration of the radiolabeled probes. The results show
that all three complexes are binding to both probes specifically;
however, complexes II and III are competed away at lower concentrations
of unlabeled competitor, implying a more specific binding affinity to
this site. A 1,000-fold excess of cold competitor was required to
compete away complex I (data not shown).
Detection of preferential allelic binding.
Three protein complexes have been shown to bind within the region
468 to 439. Furthermore, the three complexes are able to
bind specifically to this site regardless of whether an
adenine or a guanine is in the 455 position. It was of interest
to determine whether these complexes may bind preferentially to either
one of the two nucleotides. As a result, a set of cross-competition EMSAs were performed in which radiolabeled A-455 was competed against
unlabeled G-455 (Fig 3A, lanes 1 through 5)
and radiolabeled G-455 was competed against unlabeled A-455 (Fig 3A,
lanes 6 through 10) at the same concentrations used in the previous
competition assays. The competition assay involving radiolabeled A-455
versus unlabeled G-455 showed complexes II and III being competed away, as in the previous competition assays; however, complex I was not
competed away to the same extent as the other two complexes, which is
also comparable to the previous competition assays. In the radiolabeled
G-455 versus unlabeled A-455 cross-competition assay, again as in the
three previous competition assays, complex II was competed away and, to
a lesser extent, complex I. Conversely, complex III was unable to be
completely competed away from the radiolabeled G-455 probe by unlabeled
A-455 competitor. In fact, at 1,000-fold unlabeled A-455 competitor,
complex III is still present (data not shown). These results suggest
that one of the three complexes, complex III, preferentially binds
within the region of interest when a guanine rather than an adenine is
present.
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| Fig 3.
Complex III preferentially binds to the G allele. (A) In
the cross-competition assays, performed to detect preferential allelic
binding of the complexes, when radiolabeled A-455 probe is competed
against increasing concentrations of unlabeled G-455 probe (lanes 1 through 5), the three complexes are able to be competed away from the
radiolabeled A-455 probe, with competition patterns similar to the
results in Fig 2. Complex III is not competed away from radiolabed
G-455 by increasing concentrations of A-455 unlabeled competitor (lanes
6 through 10), even at 1,000-fold cold competitor (data not shown), as
seen in the three previous competition assays, implying that complex
III preferentially binds to the G allele or a guanine in the 455
position. (B) Decrease in band intensity of complex III at increasing
concentrations of cold competitor was analyzed by scanning densitometer
analysis to observe the strength with which the unlabeled competitiors
competed against the radiolabeled probes. Results for all of the
competition assays were comparable with the exception of the labeled
G-455 probe versus unlabled A-455 probe competition assay. The
unlabeled A-455 competitor was unable to competitively bind with
complex III in the presence of labeled G-455 at the same rate as the
other competition assays, also showing that complex III preferentally
binds to the G allele, rather than to the A allele. The assays were
resolved on 7% nondenaturing gels. ( ), Labeled A-455 probe versus
unlabeled A-455 probe; ( ), labeled A-455 probe versus unlabeled
G-455 probe; ( ), labeled G-455 probe versus unlabeled G-455 probe;
( ), labeled G-455 probe versus unlabeled A-455 probe.
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To estimate the binding kinetics occurring in the competition assays,
the decrease in band intensity for complex III with increasing
concentrations of unlabeled competitor was established by using
densitometer scanning analysis. The results were plotted with decrease
in band intensity of complex III against increasing concentrations of
unlabeled competitor (Fig 3B). The plots show similar patterns of
competition for radiolabeled A-455 versus unlabeled A-455, radiolabeled
G-455 versus unlabeled G-455, and radiolabeled A-455 versus unlabeled
G-455. However, the plot of radiolabeled G-455 versus unlabeled A-455
shows that the unlabeled A-455 competitor is unable to compete complex
III away from the radiolabeled G-455 probe to the extent that it is
competed away in the other three competition assays.
Importance of the B fibrinogen 455 nucleotide.
The EMSAs performed used probes that were representative of the G and A
alleles, both of which are purines. It was of interest to observe how a
pyrimidine, or even the absence of a nucleotide, in the 455
position would affect binding of the complexes, especially complex III.
Another series of competition assays involving EMSAs were performed
using two additional probes again containing the sequence from
439 to 468, denoted as T-455 (thymine in the 455 position) and N-455 (absence of a nucleotide in the 455
position; Fig 4). Binding of all three
complexes was evident on the T-455 and N-455 probes, just as on the
G-455 and A-455 probes. To assess the binding specificity of these
complexes on the two additional probes, the radiolabeled A-455, T-455,
N-455, and G-455 probes were competed against 500-fold unlabeled G-455,
A-455, T-455, and N-455 competitors (Fig 4). In the competition assays
involving radiolabeled A-455, T-455, and N-455, all three complexes
were competed away, with complexes II and III being competed away to a
greater extent than complex I. However, the only probe that was able to
compete complex III away from radiolabeled G-455 probe (window no. 4)
was itself, unlabeled G-455.
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| Fig 4.
A guanine in the 455 position is important for
preferential binding of complex III. In addition to G-455 and A-455
oligonucleotide probes being used in the EMSA analyses, two additional
probes with the sequence from 468 to 439 with the following
changes, T-455 (thymine in 455 position) and N-455 (no nucleotide in
455 position), were used to determine the effect that the nucleotide
in the 455 position has on the binding patterns of the complexes.
The radiolabeled probes (A-455, T-455, N-455, and G-455) were competed
against all four unlabeled probes at a concentration of 500-fold excess
labeled probe. Evident on the 7% nondenaturing gel, complex III was
completely competed away in all of these competition assays, except the
radiolabeled G-455 competition assay (window no. 4), thus providing
more evidence supporting the allelic specficity of complex III to the
G-allele.
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The results of these assays show that all three complexes are able to
bind within the region of interest when an adenine, a guanine, or even
a pyrimidine, such as thymine, or no nucleotide is present in the
455 position. These data further indicate that complex III
appears to preferentially bind within the region of interest when a
guanine is in the 455 position.
Analysis of complex III.
To explore in more detail the importance of the nucleotide sequence
surrounding the 455 nucleotide, we constructed a series of
mutations in the 5 and 3 flanking regions. EMSAs were
performed using probes with the sequence of the B chain gene from
468 to 439 having the G allele, but hexanucleotide
sequences from 462 to 457 (GM-2), 456 to
451 (GM-1), and 450 to 445 (GM-3) were mutated by
changing the purines to pyrimidines and the pyrimidines to purines
(Fig 5A). Probes GM1 and GM2 show marked
alteration of binding of all three complexes; however, GM3 shows no
hindrance in complex binding. The results of the EMSAs showed that
mutation of the sequence from 462 to 451 affected the
binding of all three complexes (Fig 5B). These findings indicate that
this sequence, which immediately flanks the 455 nucleotide, is
important for recognition and binding of all three complexes,
especially complex III.
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| Fig 5.
Analysis of complex formation. (A) To determine the
effect of sequence disruption on formation of the complexes, EMSA
analyses were performed using three new oligonucleotide probes in which
three hexanucleotide sequences, 462 to 457 (probe GM-2), 456
to 451 (probe GM-1), and 450 to 445 (probe GM-3), were mutated
by changing the purines to pyrimidines and the pyrimidines to purines.
(B) The results show that all three complexes, especially complex III,
were unable to sufficiently bind when the sequence from 462 to
451 was mutated. (C) UV cross-linking analysis of complex III was
performed to determine the specificity of its binding onto the G-455
probe and to approximate its molecular weight. All samples were binding
reactions containing whole cell extract, were irradiated for 5 minutes
(unless indicated otherwise), and were resolved on a 10% SDS-PAGE gel.
Lane 1, no protein extract; lane 2, no irradiation; lane 3, protein
extract (nonreduced); lane 4, protein extract (reduced); lane 5, addition of 100-fold unlabeled G-455 probe; lane 6, no extract, instead
BSA (50 µg); lane 7, a probe with the same sequence as G-455 (Fig
2A), but internally labeled on the bottom strand with
[-32P] dCTP, was used to verify the molecular weight
of complex III, obtained from previous UV cross-linking analyses. After
UV irradiation, DNAse I treatment, and isolation of the DNA-bound
complex III band from a 4% nondenaturing gel, resolution on a 10%
SDS-PAGE gel still shows that the approximate molecular weight of
complex III is 47 kD. The results show that complex III is a protein of
approximately 47 kD that specifically binds to the G-455 probe,
containing the sequence from 468 to 439 of the B chain gene,
flanking the 455 nucleotide.
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UV cross-linking assays were performed to approximate the molecular
mass of complex III (Fig 5C). For this assessment, whole cell extract
was used in the binding assays along with the G-455 probe, because
complexes I and II are not as enriched as they are in nuclear extract
and complex III becomes the most prominent band (data not shown). This
was advantageous in avoiding a partial purification step. After 5 minutes of irradiation (or indicated otherwise), results show that, in
lane 1, in which no protein extract has been added to the binding
reaction, there is no band present, indicating that there are no
extraneous proteins in the binding reaction that would bind to the
G-455 probe. Lane 2 shows the absence of band formation before
irradiation. Both lanes 3 and 4, representing nonreduced and reduced
protein, respectively, show a prominent band representing complex III,
migrating at approximately 47 kD. Lane 5 shows a decrease in band
intensity upon the addition of 100-fold unlabeled G-455 probe,
indicating that the complex binds specifically to the G-455 probe and
is able to be competed away from the radiolabeled probe by its
unlabeled sequence as a competitor. In lanes 3 through 5, in addition
to complex III, there are higher molecular weight complexes present
that have been observed in all of the EMSAs; the importance of these
complexes is presently being investigated. Lane 6, consisting of the
binding reaction with 50 µg of BSA instead of protein extract, shows
no band formation, confirming that the DNA-protein interaction between complex III and the G-455 probe is specific. To exclude the possibility that the oligonuceotide probe may alter the molecular weight, the
DNA-bound complex III was UV irradiated, treated with DNAse I, run, and
cut out of the nondenaturing gel. The eluted DNA-protein complex was
subsequently run on a 10% SDS-PAGE gel. The complex continued to
migrate at approximately 47 kD (lane 7). These results suggest that the
protein comprising complex III is approximately 47 kD and specifically
recognizes and binds to the DNA sequence of the G-455 probe.
| |
DISCUSSION |
The G-to-A transition at the 455 position of the 5
flanking portion of the B gene is an allele that has been associated with varying levels of circulating fibrinogen. Individuals that are
homozygous for the G allele (guanine at 455) have the lowest circulating levels in comparison to heterozygous and homozygous A
allele (adenine at 455) individuals. The epidemiological
evidence for these findings provides results that are generally
statistically significant; however, the increase in circulating
fibrinogen levels with regard to the presence of A alleles is not
dramatic.27-42 Presently, it is not known how such a slight
increase in circulating fibrinogen levels can potentially have
injurious physiological consequences. To better understand the possible
connection between increased fibrinogen levels and regulation of the
B gene, we performed molecular analyses to observe transcriptional
activities occurring at the 455 site and proximal flanking
regions.
We detected three complexes that bind onto the region of the B gene
from 468 to 439. The three complexes are able to
specifically bind to this region whether an adenine (the A allele), a
guanine (the G allele), a pyrimidine, or no nucleotide is present in
the 455 position. One of those three complexes, complex III,
preferentially binds to the G allele, which is associated with the
lower circulating fibrinogen levels. These data imply that the peculiar
DNA-protein interaction that is occurring, with regard to complex III,
is of a quantitative rather than of a qualitative nature, meaning that
the presence of a particular nucleotide does not completely abolish
binding, but the strength of the interaction is affected.
Information from functional analyses showed that, under in vitro
conditions, the A allele is correlated with greater luciferase activity, implying that the A allele is somehow associated with increased B chain transcription. The results of the functional assays coincide with the evidence provided in the epidemiological studies that connect the A allele with higher circulating fibrinogen levels. From these observations, it can be inferred that the detected complex, complex III, may have potential repressor-like functions in
the transcriptional regulation of the B chain gene. Furthermore, its
partial repressive activities may be altered if it is not able to
sufficiently bind to its recognition sequence if not conserved, as in
the case of the A allele being present. If this occurs, B chain
transcription may be elevated, even slightly, resulting in increased
levels of circulating fibrinogen. The existence of a negative
regulatory element associated with this chain is not without precedent.
Other studies have shown repressive elements associated with the human
A, , and a more proximal portion of the B
chain.16,17,19
Complex III was the only complex identified that showed preferential
allelic binding. Analysis of this protein complex suggested that it has
a molecular weight of approximately 47 kD. Furthermore, its recognition
sequence appears to reside within the region of 462 to
451.
The data shown in this study provide the initial steps in identifying a
differential element in B chain regulation that may be one of the
factors causing increased amounts of circulating fibrinogen.
Furthermore, an increase in fibrinogen, in conjunction with other
genetic or external factors, may be one of the many predisposing
components in cardiovascular disease development.
| |
FOOTNOTES |
Submitted February 24, 1998;
accepted June 30, 1998.
Supported in part by the GAANN Fellowship (to E.T.B.).
Address reprint requests to Gerald M. Fuller, PhD, University of
Alabama at Birmingham, Department of Cell Biology, 1918 University Blvd, MCLM 680, Birmingham, AL 35294-0005.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract