(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 characterized for its promoter activity. Reporter
gene studies using chloramphenicol acetyltransferase as the indicator
along with mutations in the DNA showed that a TATA-like sequence
(-20 to -23 base pairs (bp)), a CAAT-like sequence
(-54 to -57 bp), and an upstream stimulatory factor (USF)
binding site (-66 to -77 bp) constitute a minimal promoter
that mediates liver-specific expression of the gene. Electrophoretic
gel mobility assays and antibody binding studies confirmed the
interaction of USF with the binding site. An IL-6 responsive element
with a sequence of CTGGAA located at -301 to -306 bp was
shown to be a functional element in the IL-6 response. In contrast to
the IL-6 responsive elements in the human
- and
-fibrinogen
genes, the element in the gene for the
chain of human fibrinogen
was unaffected by the presence or elevated levels of the
or
isoforms of the CCAAT/enhancer binding proteins. A negative element
with sequence homology to several silencer elements was also identified
in the region of -348 to -390 bp of the gene for the
chain of human fibrinogen. A comparison of the regulatory elements in
the genes coding for all three chains in fibrinogen is also presented.
The gene coding for the chain of human fibrinogen is a
single-copy gene located at chromosome 4q23-q32(1) . It is
approximately 8.5 kilobases in length and consists of 10
exons(2) . The
chain is expressed primarily in the liver (3) and is assembled into functional fibrinogen molecules
together with the
and
chains. Extrahepatic transcripts of
the
chain have been observed in bone marrow, lung, and
brain(4) . Expression in megakaryocytes, however, is
controversial. Recent studies using purified and cultured
megakaryocytes have shown that mRNA for all three fibrinogen chains
were undetectable by the sensitive method of reverse
transcriptase-polymerase chain reaction. This suggested that
transcripts observed in bone marrow were most likely derived from
granulocytes(5) . In the absence of the
and
chain,
extrahepatic expression of the
chain does not lead to assembly
and secretion of functional fibrinogen. Thus, the fate of the
translated
chain in extrahepatic tissues in the absence of the
and
chains remains unclear.
The transcript for the
chain of human fibrinogen is differentially processed in liver into two
forms of mature mRNA, which code for two forms of the
chain,
called the
and
` chains. These chains occur in a ratio of
about 9:1, respectively, in humans(6) . The two chains result
from alternative utilization of a polyadenylation site in the ninth
intron of the gene, thus removing the 3` acceptor site of this intron
as well as the following 10th exon sequences. This enables the
immediate 5` end of the 9th intron to serve as an extension of the
preceding exon. Translation of this form of mRNA leads to the
replacement of the last 4 amino acids of the more abundant
chain
by a sequence of 20 amino acids in the
` variant
form(7, 8) . Recombinant fibrinogen molecules
containing two
` variant chains have been produced in baby hamster
kidney cells (9) and were unable to support platelet
aggregation. These experiments further demonstrate the importance of
the carboxyl end of the regular
chain in this
reaction(10) .
Previous studies showed that the regulation
of the gene for the chain of rat fibrinogen involved an Sp1 site,
a CAAT-binding site, and the upstream stimulatory factor (USF) (
)binding site for the adenovirus major late promoter Ad-ML,
also known as MLTF(11, 12) . A comparison of the
5`-flanking sequence of the genes for the human and rat
chain
indicates that the USF site is conserved, while the GC-rich Sp1 site is
no longer present in the human gene. In this paper, the role of the
binding sites for USF, IL-6, and other elements in the regulation of
the gene coding for the
chain of human fibrinogen is reported,
and these data are compared with the regulatory elements in the genes
coding for the
and
chains of human fibrinogen.
DNA transfections were performed by the calcium phosphate
coprecipitation method of Graham and van der Eb(14) . Cells
were cultured to 50-60% confluence in 10-cm culture dishes and
transfected with 15 µg of CAT reporter plasmid and 5 µg of
pSV(15) , which served as an internal control. The cells
were exposed to the calcium phosphate-DNA coprecipitate for 4 h, and
the cells were shocked with 15% glycerol in minimum essential medium
for 2 min. The cells were then cultured in fresh medium. After 24 h,
interleukin 6 (IL-6, Boehringer Mannheim), when needed, was added to
the medium to a final concentration of 30 units/ml. The cells were
cultured for an additional 24 h, washed, and harvested. Cell extracts
were prepared according to a procedure provided by Boehringer Mannheim.
In coexpression studies, 5 µg of the recombinant plasmid pCX-USF
containing a cDNA coding for the 43-kDa isoform of human USF under the
control of human cytomegalovirus promoter (16) (kindly provided
by Drs. Frank Brunel and Robert Roeder, Rockefeller University),
together with 15 µg of a CAT reporter plasmid and 5 µg of
pSV, was transfected into HepG2, HeLa, and IMR-90 cells by the
calcium phosphate method described above. In other coexpression
studies, the plasmid pMSV-C/EPB
or pMSV-C/EPB
, containing
cDNAs coding for either the C/EBP
or C/EBP
isoforms(17) , were generously provided by Dr. Steven L.
McKnight and used in a similar manner.
Figure 1:
Sequence of the
5`-flanking region of the human -fibrinogen gene. The
transcription initiation site is designates as +1. Regulatory
sequences established by reporter gene studies and mutation studies are double-underlined. The region containing the negative element
is underlined.
Figure 2: Expression of 5` deletion CAT constructs in HepG2 cells in the presence (shaded bar) and absence (open bar) of IL-6 stimulation.
The first 224 bp was further studied with additional deletion constructs that contain stepwise deletions of 20 to 50 bp. In these studies, the -224 bp construct, pCAT-224, was used as the reference. As shown in Fig. 2B, sequences in three regions showed significant reproducible effects in transcription: from -1 to -47 bp, from -64 to -85 bp, and from -140 to -224 bp. None of the constructs within this region, however, showed a significant response to IL-6 stimulation.
Figure 3: Effect of coexpressed USF on CAT reporter expression in human fetal lung fibroblasts (IMR-90), hepatoma (HepG2), and HeLa cells.
Figure 4:
Electrophoretic mobility shifts of
DNA-protein complexes. The labeled probe was a duplex oligonucleotide
spanning -58 to -87 bp from the human -fibrinogen
gene. The unrelated oligonucleotide contains a C/EBP binding site. The
position of the USF-specific band is marked by an arrowhead. Lane 1, control; lane 2, 1 ng of purified recombinant
USF; lane 3, 10 µg of HepG2 nuclear protein; lane
4, USF and HepG2 nuclear protein; lane 5, USF, HepG2
nuclear protein, and 200-fold molar excess of unlabeled probe; lane
6, competition with the unrelated oligonucleotide; lane
7, USF, HepG2 nuclear protein, and 1 µl of a 400-fold diluted
antiserum; lane 8, recombinant USF, HepG2 nuclear protein, and
1 µl of a 40-fold diluted antieserum; lane 9, USF, HepG2
nuclear protein, 1 µl of 40-fold diluted antiserum, and 200-fold
molar excess of unlabeled probe; lane 10, USF, HepG2 nuclear
protein, 1 µl of a 40-fold diluted antiserum, and 200-fold molar
excess of unrelated oligonucleotide; lane 11, fibroblast
(NIH3T3) nuclear protein; lane 12, USF and NIH3T3 nuclear
protein; lane 13, USF, NIH3T3 nuclear protein, and 200-fold
excess of unlabeled probe; lane 14, fibroblast nuclear protein
and 200-fold excess of unrelated
oligonucleotide.
Figure 5:
Effect of coexpression of C/EBP and
C/EBP
isoforms on CAT expression by pCAT
constructs.
Figure 6:
Reduction in CAT reporter expression by
upstream sequences in the -fibrinogen gene. Overlapping cassette
oligonucleotides CS1 to CS5 were inserted in front of pCAT-229 either
in the normal (F) or reverse (R)
orientation.
The three genes of fibrinogen have evolved from a common
ancestor, and studies reported in this investigation of the gene for
the chain, together with those in the gene for the
chain(24) , and for the
chain of human
fibrinogen(22, 25) , make it possible to compare the
mechanisms of regulation in this gene family. A schematic
representation of established regulatory sites is summarized in Fig. 7. Common features as well as differences were noted. All
three fibrinogen genes apparently contain TATA-like sequences that
presumably facilitate the choice of transcription initiation sites by
RNA polymerase II.
Figure 7:
Schematic representation of the locations
of regulatory elements in the genes for the ,
, and
chain of human fibrinogen. Locations of regulatory sequences in the
genes for the
and
chains are taken from Refs. 22, 24,
25.
The genes for the and
chains are
apparently expressed in a liver-specific manner by a HNF1-dependent
mechanism(22, 24, 26) . In contrast,
liver-specific expression of the gene for the
chain is apparently
mediated by USF. Neither HNF1 nor USF is found exclusively in the liver
and, by themselves, cannot account for liver-specific expression.
Coexpression of USF in HepG2 cells, mobility shift, and antibody
studies show that the abundance of USF clearly plays a role in
determining the level of liver-specific expression for the
chain.
However, expression of CAT reporter activity under the regulation of
the USF site in IMR-90 and HeLa cells remains inefficient despite the
presence of an increased abundance of USF, suggesting the presence of
other limiting factors. Recent studies on HNF1 show that tissue
specificity is apparently mediated by the presence of a cofactor or
accessory protein, designated as dimerization cofactor of HNF-1
(DCoH)(27) . Similarly, the B cell-specific expression of
immunoglobulin genes mediated by the octamer motif and octamer-binding
proteins Oct-1 and Oct-2 apparently involves the participation of a B
cell-specific coactivator, Oct-binding factor 1 (OBF-1)(28) .
By analogy, efficient expression of USF-regulated genes may involve the
presence of specific cofactors in the liver. Experiments are in
progress to explore this possibility.
The inherent difference in the
promoter activity of the USF and HNF1 sites may account for the
apparent unbalanced synthesis of the three fibrinogen chains. Thus,
HepG2 cells in culture produce limiting quantities of chains, but
HuH7 cells contain limiting quantities of
chains. (
)The limiting fibrinogen chain, either
or
chain, appears to be regulated by a HNF1-related mechanism. This
inherent difference in the properties of the promoters may also account
for the extrahepatic transcription of the
chain gene detected in
bone marrow, lung, and brain(4) .
Our observation that the
USF binding site in the gene for the chain of human fibrinogen
can support liver-specific expression is in marked contrast to results
reported by Morgan et al.(11) who showed that no
tissue- or species-specific transcription control elements were present
within the first 847 bp of the 5`-flanking sequence of the gene for the
chain in rat fibrinogen. The discrepancy may be attributed in
part to the likelihood that the 847-bp segment of the rat gene used in
the latter study included negative or silencer sequences that would
reduce liver-specific expression to a level that tissue specificity
might not be evident. Alternatively, this discrepancy may also be
attributed to inherent differences between the human and rat genes.
Thus, the Sp1 site in the gene for the
chain of rat fibrinogen
(-46 to -51 bp) immediately next to the CAAT binding site
has been mutated and is no longer present in the comparable region of
the human gene. Changes elsewhere in the 5`-flanking sequence may also
be responsible for the observed differences.
Results in Fig. 2, A and B, suggest the presence of weak positive elements between -140 and -224 bp and from -258 to -299 bp. Sequence comparison suggests this effect may be attributed to the presence of a sequence (-188 bp to -201 bp) that is essentially identical (13/14) to the NF1 consensus sequence(29) . No apparent recognition sequence for transcriptional factors was identified in the region from -258 to -299 bp. Additional studies are necessary to define the sequences and the nuclear proteins involved.
Current evidence indicates that
the IL-6 response in the three fibrinogen genes is attributable to the
presence of a single element with sequence identity to the consensus
sequence of the type II IL-6 response sequence CTGGAA. Despite multiple
occurrences of sequence matching, this consensus sequence in the
5`-flanking region of each of the three fibrinogen genes, only one such
sequence in each gene has been shown to be functional. No correlation
of the activity of the IL-6 responsive element with its distance from
either the HNF1, USF, or the transcription initiation site was
observed. However, a unique difference between the IL-6 responsive
element in the gene for the chain of fibrinogen and those in the
genes for the
and
chains was observed. In the latter two
genes, the IL-6 responsive elements were associated with an adjacent
C/EBP site, and, in the case of the gene for the
chain, mutations
in both the IL-6 responsive element and the C/EBP site abolished the
IL-6 response. However, in the gene for the
chain, the IL-6
responsive element was not associated with a C/EBP site, and elevated
levels of C/EBP
alone showed a slight effect on the expression of
the
-fibrinogen gene. The fact that only C/EBP
but not
C/EBP
was able to elicit this slight increase and that this region
does not contain a consensus C/EBP binding site suggest that the effect
may be indirectly related to the overexpression of C/EBP
. The
difference in the magnitude of response and apparent independence from
C/EBP remained a distinguishing feature of the IL-6 responsive element
in the gene for the
chain.
The negative element identified in
the gene for the chain appeared to involve the sequence in the
region of -333 to -407 bp. Maximal activity involved a
distended region that centers around and contains the motif AGGA
(-359 to -362 bp and -372 to -375 bp). This
motif was similar in sequence to the silencer element in the human
globin, gastrin, factor IX, the mouse c-myc, rat growth
hormone, and chicken vimentin genes, which contain the sequence AGGA or
its inverse complement TCCT (summarized in (30) ). It is clear
from the overlapping cassette studies that sequences immediately
surrounding the AGGA motifs are also important. So far, our results
indicate that this element in the
chain for fibrinogen was
functional in both orientations in conjunction with the native USF
promoter but unable to reduce the expression of the reporter gene under
the control of the SV40 viral promoter and enhancer. A comparison of
the sequences in the
- and
-fibrinogen genes which show
apparent silencer activities reveals little extended homology. However,
a short conserved sequence (CTTAA) is present in both genes. In the
-fibrinogen gene, this conserved sequence is part of the C/EBP
consensus sequence and is unlikely to mediate the silencer or negative
response. Further studies are needed to clarify the sequences involved
and how this negative element modulates the liver-specific expression
of the gene for the
chain of human fibrinogen.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36503[GenBank].