(Received for publication, August 9, 1995; and in revised form, September 7, 1995)
From the
The 5`-flanking region of the gene coding for the chain of
human fibrinogen was isolated, sequenced, and characterized. The
principal site of transcription initiation was determined by primer
extension analysis and the RNase protection assay and shown to be at an
adenine residue located 55 nucleotides upstream from the initiator
methionine codon, or 13,399 nucleotides downstream from the
polyadenylation site of the gene coding for the
chain. Transient
expression of constructs containing sequentially deleted 5`-flanking
sequences of the
chain gene fused to the chloramphenicol
acetyltransferase reporter gene showed that the promoter was
liver-specific and inducible by interleukin 6 (IL-6). The shortest DNA
fragment with significant promoter activity and full response to IL-6
stimulation encompassed the region from -217 to +1 base
pairs (bp). Although six potential IL-6 responsive sequences homologous
to the type II IL-6 responsive element were present, a single sequence
of CTGGGA localized from -122 to -127 bp was shown to be a
functional element in IL-6 induction. A hepatocyte nuclear factor 1
(HNF-1) binding site, present from -47 to -59 bp, in
combination with other upstream elements, was essential for
liver-specific expression of the gene. A functional CCAAT/enhancer
binding protein site (C/EBP, -134 to -142 bp) was also
identified within 217 bp from the transcription initiation site. An
additional positive element (-1393 to -1133 bp) and a
negative element (-1133 to -749 bp) were also found in the
upstream region of the
-fibrinogen gene.
Fibrinogen is a plasma glycoprotein that participates in two
crucial events in hemostasis. It is an adhesive protein essential for
platelet aggregation, and it is converted from soluble fibrinogen to
fibrin that polymerizes to form an insoluble fibrin clot at the final
stage of the blood coagulation cascade(1) . Fibrinogen is
synthesized in hepatic parenchymal cells and is secreted into the blood
as a heterodimer consisting of two , two
, and two
polypeptides linked by 29 disulfide bonds. Recent studies have shown
that high circulating levels of fibrinogen are associated with an
increased risk of myocardial infarction and stroke, suggesting that
fibrinogen can be an independent and important cardiovascular risk
factor(2, 3) . Therefore, studies on the regulation of
fibrinogen synthesis may provide an explanation for the high expression
levels in these individuals.
The three chains of human fibrinogen
are encoded by three distinct single-copy genes, the sequence of which
has been determined(4) . They are closely linked in a region of
approximately 50 kb ()of DNA located on chromosome
4q23-q32(5) . The three genes are arranged in the order of
-
-
, with the gene for the
chain transcribed in the
opposite direction. Studies in rats indicate that the expression of the
three chains of fibrinogen is under coordinate regulation at the
transcriptional level (6, 7) . Recently, the
constitutive expression of the human
chain has been shown to be
mediated by hepatocyte nuclear factor 1 (HNF-1) (8, 9, 10) . This transcription factor is
also important in the expression of the rat
and
chains(11) .
As an acute-phase protein, the level of
fibrinogen in circulation rises in response to trauma and inflammation.
Massive defibrination and exposure to fibrinogen degradation products,
fragments D and E, lead to a 4-7-fold increase in fibrinogen
synthesis in the liver (12) that is apparently mediated by
interleukin 6 (IL-6)(13) . Response to IL-6 stimulation in the
human fibrinogen chain gene depends primarily on an IL-6
responsive element with the consensus sequence CTGGG/AA. This element
also occurs in the promoter regions of other type II acute-phase
proteins, such as
-macroglobulin and
T-kininogen(14, 15) .
In order to further
understand the regulation and coordinate expression of the three chains
of human fibrinogen, it is essential to study the transcriptional
regulation of the and
chains in greater detail. In the
present report, the sequence of the 5`-flanking region of the gene
coding for the
chain of human fibrinogen has been determined and
several regulatory elements, including a HNF-1 site, an IL-6-responsive
element, and a C/EBP binding site, have been identified within 200 bp
of the transcriptional initiation site. An additional positive and a
negative element were also identified in the upstream region of the
5`-flanking sequence.
A fragment which overlaps the human - and
-fibrinogen genes was obtained by PCR with primers from the
extreme 3`-flanking sequence of the
chain gene and exon 1 of the
human
-fibrinogen gene using human genomic DNA as templates. The
primers were designed with flanking EcoRI recognition
sequences, and the PCR product was cloned into the EcoRI site
of pUC18. Recombinant plasmids containing the PCR product cloned in
both directions were mapped by restriction endonuclease digestions and
were unidirectionally deleted by the ExoIII-mung bean nuclease
method after cleavage with SphI and BamHI according
to a protocol provided by Stratagene. The sequence was determined on
both strands by the dideoxy chain termination method of Sanger et
al.(17) .
Figure 1:
Linkage of the human
- and
-fibrinogen genes. The solid bars represent
the structural genes. The arrows represent the direction of
transcription. The shaded bars represent sequences contained
in the recombinant
phage and the PCR
product.
Figure 2:
Intergenic sequence between the genes for
the and
chains of human fibrinogen. Transcription
initiation sites for the
chain gene determined by primer
extension and RNase protection are underlined, and the major
initiation site is designated as +1 and double-underlined. The upstream sequence is negatively
numbered relative to the transcription initiation site. Sequence
numbers in parentheses refer to the numbering previously used in
the
chain gene(4) . Regulatory sequences established by
reporter gene studies and mutation studies are double-underlined.
Figure 3: Primer extension analysis. Lanes 1 and 2, primer extension products with 50 µg and 25 µg of RNA from HepG2 cells; lanes 3 and 4, primer extension product with 50 µg and 25 µg of RNA from NIH3T3 cells; lanes G, A, T, and C, DNA sequence ladder for size comparison.
Figure 4:
Expression of CAT gene in different types
of cells. Constructs containing varying lengths of the 5`-flanking
sequence were transfected into human hepatoma HepG2, human cervical
carcinoma HeLa, and rat fibroblasts NIH3T3 cells. CAT expression was
normalized to -galactosidase activity and was expressed as a
percentage of the positive control pSV2-CAT
(100%).
Although the sequence
up to -73 bp was unable to support transcription by itself, this
region contains a sequence that is necessary for efficient
transcription in combination with other upstream regulatory elements. A
sequence homologous to the hepatocyte nuclear factor 1 (HNF-1) site was
identified at -47 to -59 bp (GCCAATGATTAAC). The lack of
apparent independent promoter activity at this site may be attributable
to an imperfect match with the palindromic consensus HNF-1 sequence.
However, the importance of this sequence in the overall transcription
process is shown by the construct pCAT-217 mHNF1 in which mutations in
this sequence (ATTAAC
to
AGGGAC) abolished transcription supported by sequences upstream of this
region. These results confirm that the HNF-1 core-like sequence,
together with sequences upstream of -73 bp are necessary for
transcription of the
-fibrinogen gene.
Figure 5:
Binding of nuclear proteins to the HNF-1
core-like sequence. DNA-protein complexes were formed by incubating
end-labeled duplex oligonucleotide h-HNF1 with nuclear extracts
from HepG2 cells prepared with and without prior IL-6 stimulation.
Competition was performed with 10-fold and 100-fold excesses of
unlabeled duplex oligonucleotides with a HNF-1 consensus sequence (HNF1) and a shortened HNF-1 sequence (neh
-HNF1). F indicates the position of free
probes; nucleotides shown in lowercase were introduced for 3`
fill-in labeling.
Figure 6:
Expression of CAT gene in transfected
HepG2 cells in the absence and presence of IL-6. A, deletion
constructs containing varying lengths of the 5`-flanking sequence of
the gene for the chain of human fibrinogen were transfected into
HepG2. IL-6 when used was added to a final concentration of 30
units/ml. CAT expression was normalized to
-galactosidase activity
and was expressed as a percentage of the positive control pSV2-CAT
(100%). B, pCAT-749m1 contains mutations of CTGGGA to CTCTAG
at -127 bp, pCAT-749m2 contains mutations of CTGGAA to CTCTAG at
-228 bp, and pCAT-749m1m2 is a double mutant with sequences
changed at the -127-bp and -228-bp sites. CAT expression
levels were expressed as percentages of the uninduced wild-type
construct pCAT-749.
Sequence-specific interaction of nucleotides containing IL-6
responsive elements with nuclear proteins was studied by mobility shift
assays. In these studies, labeled duplex oligonucleotides containing
sequences around the functional IL-6 site (-127 bp), a
nonfunctional site (-228 bp), and a mutant IL-6 site
(-127m1) were incubated with nuclear proteins prepared from HepG2
cells with and without prior stimulation with IL-6. As shown in Fig. 7, all three oligonucleotides formed multiple complexes
with HepG2 nuclear proteins. The difference in the pattern produced by
a functional and a nonfunctional IL-6 responsive element is shown in
the relative intensity of the bands rather than a distinct appearance
or disappearance of bands. Thus, all three oligonucleotides formed
complexes A and C. Competition with a core IL-6 consensus sequence
(acute phase response element) reduced the abundance of complex A. In
the case of the functional IL-6 sequence (-127 bp), this
reduction was accompanied by an increase in complex C, whereas with the
mutant IL-6 sequence (-127m1) and the nonfunctional sequence
(-228 bp), no accompanying increase in complex C was observed.
Similar studies were also performed with two additional nonfunctional
IL-6 sequences from the fibrinogen chain gene, the results are
comparable to the nonfunctional site at -228 bp (data not shown),
and only quantitative differences in the ratio of the complexes were
observed. Nuclear proteins prepared from IL-6 stimulated HepG2 cells
gave rise to an additional complex (Band B) with all three
oligonucleotides. Formation of complex B was also abolished by
competition with the core IL-6 consensus sequence. The formation of
this complex is attributed to the presence of novel protein or a
post-translationally modified protein in IL-6 stimulated HepG2 cells.
These binding studies do not show readily the difference between a
functional and a nonfunctional IL-6 site and suggest that sequences
surrounding these sites may have a contribution.
Figure 7: Mobility shift assays with IL-6 elements. Complexes were formed with duplex oligonucleotides with nuclear extracts prepared from HepG2 cells with and without prior IL-6 stimulation. Probe IL-127 contains the functional IL-6 sequence, probe mIL-127 contains a mutated IL-6 sequence, and probe IL-228 contains a nonfunctional IL-6 sequence. In competition studies, a sequence containing an IL-6 consensus sequence (acute-phase response element, APRE) was used. Bands A-D represent DNA-protein complexes; band F indicates the position of the free probes; nucleotides shown in lowercase were introduced into the duplex for 3` end-labeling.
Figure 8:
CAT expression by reporter constructs.
pCAT-217mC/EBP contains a mutation of ATTGAGCAA
to ACCTAGCAA in the
C/EBP site of construct pCAT-217. A, effect of mutation on
IL-6 stimulation. B, effect of co-expression of C/EBP
and
C/EBP
isoforms. Control, cotransfection with plasmid
Bluescript. Uninduced pCAT-217 expression levels were used as reference
(100%).
Studies on the assembly of fibrinogen showed that only fully
assembled six-chain fibrinogen molecules were secreted (29, 30, 31) and in HepG2 cells the
chain is present in limiting amounts. However, disproportionate
chain synthesis may be attributable to, in part, variations in the
origin of cell lines and culture conditions in vitro.
Preliminary studies with another human hepatoma cell line, HuH7, showed
that the
chain and not the
chain is limiting. (
)Therefore, studies on the regulation of the
- and
-fibrinogen genes are equally important, particularly in an
acute-phase response, in which the increase in total fibrinogen
synthesis far exceeds the amount that a compensatory increase in
chain synthesis alone can account for. A concerted increase in the
synthesis of all three chains is necessary to account for the observed
increase. To better understand the regulation of fibrinogen synthesis,
we have focused on the
chain gene in the present studies.
The
intergenic sequence between the genes for the and
chain of
human fibrinogen present in the phage
HI
13 is incomplete
since there is no sequence overlap between its 3` end and exon 1 of the
chain gene. The polymerase chain reaction was used to obtain the
overlap between these two genes and establish the intergenic distance
to be 13,399 bp, about 3 kb longer than previously
reported(5) . Furthermore, as shown in reporter gene studies,
this newly isolated 3-kb fragment contains elements that are
functionally important in the transcriptional regulation of the human
-fibrinogen gene.
Significant sequence identity (68%) was
observed between the 5`-flanking region of the human and rat
chain gene extending to about 221 bp upstream from the transcription
initiation site. Sequence conservation suggests that this region may
contain functionally conserved regulatory elements. The transcription
initiation sites determined by primer extension analyses and RNase
protection assays showed one major and several minor sites.
Transcription initiation appears to occur at conserved sites in the rat
and human
-fibrinogen genes. Both transcription initiation sites
are associated with a potential upstream TATA-like sequence (
TTTAA
). There are potential
CCAAT-like sequences in the promoters of the human
,
, and
chain genes, but the CCAAT sequence from -58 to -54
bp in the
chain gene is not likely to be functional since it
overlaps with the HNF-1 binding site and is not conserved in the rat
chain gene.
No significant homology for at least 3 kb was
observed in the 5`-flanking sequences of the three human fibrinogen
genes and the three genes appear to be regulated independently.
However, several potential common cis-acting elements were
noted in the promoters for the human and
chain genes. Both
genes contain HNF-1, IL-6, and C/EBP sites that apparently contribute
to the overall transcription of the two genes. The HNF-1 binding sites
in the promoters of the human
and
chain gene appears to
mediate constitutive liver-specific expression to differing
extents(8, 33) . The functional importance of the
HNF-1 sequence in the human
chain promoter is further confirmed
by mutation studies.
Fibrinogen is up-regulated by IL-6 during the
acute-phase response. The expression level of the human fibrinogen
chain was stimulated 2- to 3-fold in HepG2 cells by IL-6, which
is comparable in magnitude to the stimulation observed in
vivo(13) . This is in contrast to the 5- to 10-fold
induction reported for the IL-6-responsive element in the human
chain gene(10) . Our present studies on six potential IL-6
responsive sequences in the
-fibrinogen gene show that sequences
adjacent to the consensus sequence, e.g. the C/EBP site, may
determine which sequence would in fact be functional and the magnitude
of the response. This interpretation is in agreement with observations
on the IL-6 responsive element in the human
chain gene that an
adjacent C/EBP binding site may be modulating and further increasing
the magnitude of the IL-6 response(8, 36) . Mobility
shift assays show that multiple protein-DNA complexes are formed by the
IL-6 responsive sequence with nuclear proteins suggesting the
involvement of several proteins. One protein that binds to the IL-6
consensus sequence, designated as the acute phase response factor, also
known as Stat-3, has been cloned and was found to be ubiquitously
expressed(34, 37) . However, Stat-3 does not bind to
the IL-6 consensus sequence of the rat
-fibrinogen gene,
suggesting that the IL-6 response may involve a novel transcription
factor(38) .
In preliminary studies, dexamethasone elicited
a small but reproducible increase in CAT reporter gene expression in
HepG2 cells from the first 217 bp of the 5`-flanking sequence of the
-fibrinogen gene (data not shown). However, this region does not
contain sequences homologous to a typical glucocorticoid responsive
element, and HepG2 cells have been shown to contain limiting amounts of
glucocorticoid receptor(35) . Further studies are necessary to
define the effect and mechanism of dexamethasone regulation of
-fibrinogen gene expression.
Since the level of circulating fibrinogen that correlates with a high risk of myocardial infarction and stroke does not exceed the normal range by a 2-fold increase, subtle functional differences in promoters and enhancers in each of the three fibrinogen genes may be crucial in determining the level of fibrinogen in circulation. These considerations further emphasize the necessity for detailed characterization of the regulatory elements in each of the three fibrinogen genes.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36478[GenBank].