©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the 5`-Flanking Region of the Gene for the Chain of Human Fibrinogen (*)

(Received for publication, August 9, 1995; and in revised form, September 7, 1995)

Jun Mizuguchi (§) Chao-Hong Hu Zhiyun Cao Keith R. Loeb Dominic W. Chung Earl W. Davie (¶)

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195-7350

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha- and beta-fibrinogen genes, the element in the gene for the chain of human fibrinogen was unaffected by the presence or elevated levels of the beta 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.


INTRODUCTION

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 alpha and beta 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 alpha and beta 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 alpha and beta 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) (^1)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 alpha and beta chains of human fibrinogen.


MATERIALS AND METHODS

Sequencing of the 5`-Flanking Region of the Human -Fibrinogen Gene

A recombinant phage, HI2, containing the 5`-flanking region of the gene for the human -fibrinogen gene (2) was digested with EcoRI, and fragments containing the 5`-flanking region were subcloned in pUC18. Sequential unidirectional deletions of the subcloned fragments were performed with the Exo III and mung bean nuclease method according to a protocol from Stratagene. DNA sequence was determined on overlapping plasmid templates by Sanger's dideoxy chain termination method (13) using Sequenase (U. S. Biochemical). The sequence was confirmed by independently sequencing the opposite strand.

Polymerase Chain Reaction

Segments of the 5`-flanking region of the human chain gene of defined lengths were either excised from the deletion plasmids prepared above or prepared by the polymerase chain reaction with specific oligonucleotide primers. The primers contained XbaI and HindIII restriction recognition sequences at the ends to facilitate cloning into pCAT-0 for reporter gene assays. The polymerase chain reactions were performed with Taq polymerase (Promega) in the presence of HI2 DNA as template and 0.2 µM oligonucleotide primers. The template was denatured at 94 °C for 1 min, the primers and template were annealed at 55 °C for 2 min, and DNA synthesis by Taq polymerase was carried out at 72 °C for 3 min. The cycle was repeated 30 times in an automated thermal cycler (Perkin-Elmer). The amplified DNA fragments were cloned directly into the vector pCRII (Invitrogen), and the sequence was verified by sequencing.

Cloning of Plasmids

DNA fragments were excised either from the pUC18 vector or from the pCRII vector by restriction enzymes and cloned into appropriate sites in pCAT-0 (Promega). The resultant plasmids were designated as pCAT-n, where n represents the length in nucleotides of the 5`-flanking sequence inserted in front of the chloramphenicol acetyltransferase (CAT) gene.

Cell Culture and Transfections

Human hepatoma cells (HepG2 and Hep3B) and HeLa cells were cultured in minimum essential medium containing 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, antibiotics (penicillin, streptomycin, and neomycin), and 10% fetal bovine serum. Human lung cells (IMR-90) were cultured in the same medium without sodium pyruvate. Chinese hamster ovary cells (CHO) were cultured in Ham's F12 medium. Cells were incubated in a 5% CO(2) atmosphere at 37 °C.

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 pSVbeta(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 pSVbeta, was transfected into HepG2, HeLa, and IMR-90 cells by the calcium phosphate method described above. In other coexpression studies, the plasmid pMSV-C/EPBbeta or pMSV-C/EPB, containing cDNAs coding for either the C/EBPbeta or C/EBP isoforms(17) , were generously provided by Dr. Steven L. McKnight and used in a similar manner.

CAT Levels and beta-Galactosidase Assays

The level of CAT antigen in extracts of transfected cells was determined by an enzyme-linked immunosorbent assay with CAT-specific antibodies according to a protocol from Boehringer Mannheim using known amounts of CAT as standards. beta-Galactosidase activity was determined by the colorimetric method of Herbomel et al.(18) . For each series of transfection experiments, the CAT levels were normalized to the beta-galactosidase levels to correct for differences in cell number and transfection efficiency. The plasmid, pSV2-CAT, in which the CAT gene is under the control of a SV40 promoter and enhancer and the promoterless plasmid pCAT-0 served as positive and negative controls. All transfection studies were repeated at least 3 times, and the standard deviation of CAT expression determinations were below 12%.

Electrophoretic Mobility Shift Assays

Nuclear extracts from HepG2 cells and NIH3T3 fibroblasts were prepared according to the method of Wegenka et al.(19) . Probes for binding and mobility shift studies were labeled by fill-in synthesis of recessed 3` ends employing the Klenow fragment of DNA polymerase I and [alpha-P]dCTP. Binding of the labeled probe with nuclear proteins or USF was performed with 1.5 ng of duplex labeled probe (20,000 cpm) and 10 µg of nuclear protein or 1 ng of purified recombinant USF (kindly provided by Drs. Frank Brunel and Robert Roeder) in the presence of 20 mM Tris-HCl, pH 7.9, 0.1 M KCl, 0.2 mM EDTA, 5 mM dithiothreitol, and 10% glycerol for 20 min at room temperature. Reaction mixtures were analyzed on a 5% polyacrylamide gel containing 7 mM Tris-HCl, pH 7.9, 3 mM sodium acetate and 1 mM EDTA. In competition studies, excess unlabeled oligonucleotide probe and a probe containing an unrelated sequence (a C/EBP binding site) at 200-fold molar excess were used. In other studies, antibodies against USF from a diluted rabbit antiserum (provided by Drs. Frank Brunel and Robert Roeder) were added to the reaction mixture simultaneously with the labeled probe.


RESULTS

5`-Flanking Sequence of the Human -Fibrinogen Gene

In a previous characterization of the human -fibrinogen gene, the sequence of 1747 base pairs of the 5`-flanking region prior to the transcription initiation site was established(2) . In the present study, sequence determination on both strands was extended to an EcoRI site 3937 bp from the site of transcription initiation (Fig. 1), establishing an additional 2190 bases of sequence of this region of the gene coding for the chain of human fibrinogen. In this sequence determination, two discrepancies with the previously published sequence have been identified: insertion of a T at -751 bp, and the sequence CTGA replaces TGAA at -1717 bp.


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.



CAT Reporter Gene Assays

Progressive deletions of the 5`-flanking region of the gene for the chain of human fibrinogen were constructed and cloned in front of the chloramphenicol acetyltransferase (CAT) reporter gene in the promoterless plasmid pCAT-0. These constructs were transfected into human hepatoma HepG2 cells, and transient expression of the CAT gene with and without IL-6 was measured. The sequence from -1 to -47 bp showed little promoter activity, whereas the sequence from -47 to -224 bp supported significant transcription activity, indicating the presence of a promoter (Fig. 2A). This transcription activity was further increased by sequences from -258 to -299 bp indicating the presence of an additional positive element in this region. The region from -1 to -299 bp does not contain sequences that respond to IL-6 since the expression of CAT with IL-6 stimulation was comparable to that without IL-6 stimulation, and the ratio of CAT expression levels with and without IL-6 stimulation remains relatively constant (ratio approx 1.3). The sequence between -299 and -407 bp reduced the expression of CAT by approximately 60% compared to the pCAT-299, indicating the presence of a negative element in this region. This region also contained an apparent IL-6 responsive element that increased the expression of CAT 1.9-fold when stimulated with IL-6. The sequence between -715 and -954 bp resulted in a decrease in transcription activity in the presence or absence of IL-6 stimulation indicating the presence of yet another negative element. The sequence from -954 to -3936 bp caused a gradual decrease in transcription activity, and no particularly significant regulatory element was evident. These results suggested that sequences close to the transcription initiation site play a significant role in the regulation of the gene coding for the chain of human fibrinogen. The sequence from -47 to -299 bp contained the promoter and other positive elements that support the expression of the gene for the chain in an IL-6-independent manner, while transcriptional activity was further modulated by sequences from -299 to -407 bp. These latter sequences include a negative element as well as an IL-6 responsive element (see below).


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.

TATA-like and CAAT-like Sequences

The sequence within the first 64 bp of the gene for the chain of human fibrinogen, although capable of supporting transcription of the CAT gene, did not show high promoter activity. Within this region, two motifs were identified by comparison with known regulatory sequences: a TATA-like sequence (TAAA) located at -20 to -23 bp and a CAAT-like sequence (CCAT) located at -54 to -57 bp. To test whether these two sequences may play a role in transcription, mutations were constructed in each of these regions. Accordingly, the CCAT sequence in pCAT-64 was changed to GGGT (designated as mCAAT-64), and this reduced the transcriptional activity of pCAT-64 by 75%. Similarly, the TAAA sequence was mutated to ACCA (MTATA-224) to assess its functional properties. Because of the inherently low activity of pCAT-64, the wild type pCAT-224 construct was used. Transcription activity supported by the mutant form was reduced by 50%. Taken together, these mutational studies suggest that although the CCAT and the TAAA sequence can support very low transcription activity by themselves, they play a functional role and are necessary for efficient transcription in conjunction with adjacent upstream sequences.

USF Binding Site

The first sequence in the 5`-flanking region that was able to support a significant expression of the CAT reporter gene was associated with the sequence from -64 to -85 bp. This region contained the sequence GGCCCCGTGATC (-66 to -77 bp), which is 83% (10 out of 12 residues) identical with the upstream stimulatory factor binding site (USF binding site) also known as the adenovirus major late promoter, or MLTF(20, 21) . To test whether this sequence supports liver-specific transcription, reporter constructs containing this sequence were transfected into HepG2, Hep3B, CHO, and HeLa cells, and the expression of the CAT reporter gene was compared. Expression was consistently highest in HepG2 cells, reduced to about 50% in Hep3B cells, very low in CHO cells (3%), and undetectable in HeLa cells (data not shown). Since USF was initially isolated from HeLa cells, and since the two hepatoma cell lines gave varying levels of reporter gene expression, inherent differences in cells superimpose on differences in tissue specificity. Although USF is ubiquitous, it is present in varying amounts in different cells, and its level may directly influence the expression level of a gene under the control of an USF binding site. To assess the effect of different USF levels on expression, reporter constructs were cotransfected into HepG2 cells with a cDNA coding for human USF. As shown in Fig. 3, coexpression of USF in HepG2 cells further increased the expression of CAT by about 7-fold. These results suggest that in HepG2 cells, and by inference in Hep3B cells as well, the expression of the gene for the chain is limited by the level of USF. However, expression in HeLa cells and IMR-90 lung cells compared to HepG2 cells was much lower but did increase with an increase of abundance of USF. These data suggest that another mechanism(s) exists to account for the limited expression of the gene for the chain of fibrinogen in IMR-90 and HeLa cells.


Figure 3: Effect of coexpressed USF on CAT reporter expression in human fetal lung fibroblasts (IMR-90), hepatoma (HepG2), and HeLa cells.



Electrophoretic Mobility Shift Assays

Binding of protein(s) to the putative USF site in the -fibrinogen gene was studied in mobility shift assays in polyacrylamide gels. A labeled duplex oligonucleotide containing the putative USF recognition sequence from the gene for the chain was incubated with a preparation of purified recombinant USF. The protein-DNA complex migrated as a single band with a retarded mobility in a polyacrylamide gel (lane 2, Fig. 4). Nuclear proteins from HepG2 cells formed two apparent complexes, one of which shows a retarded mobility identical with that of purified USF (lane 3). A mixture of HepG2 nuclear proteins and recombinant USF produced the same two bands as HepG2 nuclear extract alone, except that the USF-specific band was much more intense (lane 4). The formation of the USF-specific complex was abolished by the presence of a 200-fold molar excess of unlabeled oligonucleotide probe identical in sequence with the labeled probe (lane 5). An oligonucleotide with an unrelated sequence was unable to compete and abolish complex formation (lane 6), indicating that the formation of the complex was dependent on a sequence-specific interaction of the protein with the probe. Competition with an oligonucleotide with the adenovirus major late promoter sequence (20, 21) was equally effective in abolishing complex formation (data not shown). Antibodies against USF (1:40 and 1:400 dilution of a rabbit antiserum) recognized and bound to the USF-DNA complex, and this antibody-bound complex exhibited a further retarded mobility in the polyacrylamide gel (lanes 7 and 8). The presence of excess antibodies against USF (lane 8) caused a further retardation of the entire USF-specific band which contained complexes formed by both purified USF and HepG2 nuclear proteins. Retardation of the entire band indicated that the protein in HepG2 extract was in fact USF and was recognized by the specific antibody to USF. The further retarded band was abolished upon competition with excess unlabeled probe but remained unchanged with a probe with an unrelated sequence (lanes 9 and 10). Nuclear extracts from fibroblasts (NIH3T3 cells, lane 11) and HeLa cells (data not shown) form a trace of the USF-specific complex, which is in agreement with the interpretation that low CAT reporter gene expression in these cells was attributable, in part, to a low level of USF.


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.



IL-6 Responsive Element

Preliminary deletion studies indicate that a sequence(s) which conferred IL-6 responsiveness was located from -299 bp to -407 bp (Fig. 2A). To further localize the IL-6 responsive sequence(s), synthetic oligonucleotide cassettes containing sequences from -299 to -407 bp were inserted in front of pCAT-299, and the response to IL-6 stimulation was measured. Insertion of the sequence from -300 to -342 bp led to a 2.4-fold increase in CAT gene expression when stimulated with IL-6. This region contains the sequence CTGGAA (-301 to -306 bp), which was homologous to the core element of the type II IL-6 responsive element. These were previously shown to mediate IL-6 response in the promoter for the beta chain of fibrinogen (22) and the rat alpha(2)-macroglobulin promoter (23) . Accordingly, mutations in this putative IL-6 responsive element were introduced (CTGGAA to CTCTAG), and these changes essentially abolished the IL-6 response. These results show that the sequence present from -301 to -306 bp was a functional IL-6 responsive element in the gene for the chain of human fibrinogen. In other studies, the constructs pCAT-190, pCAT-224, pCAT-407, and pCAT-2847 were cotransfected with a cDNA coding for either C/EBPbeta or C/EBP. The expression level of CAT reporter gene was unaffected by the elevated level of C/EBPbeta (Fig. 5). There is a slight increase in CAT expression with the pCAT-407 construct in the presence of increased C/EBP. This increase is minimal compared to the greater than 2.5-fold increase that is attributed to a C/EBP element adjacent to the IL-6 site in the gene for the alpha chain of human fibrinogen (24) .


Figure 5: Effect of coexpression of C/EBPbeta and C/EBP isoforms on CAT expression by pCAT constructs.



Negative Element

Oligonucleotide cassettes were also used to further define the sequence between -299 and -407 bp that mediated the negative transcriptional effect. As shown in Fig. 6, maximal reduction of transcriptional activity was associated with the sequence between -348 and -390 bp, and this effect was independent of sequence orientation and unaffected by IL-6 stimulation. The negative element from -348 to -390 bp was also inserted in a construct in which the CAT gene was under the control of the SV40 promoter and enhancer (pSV2-CAT). This element was unable to reduce the expression of CAT (data not shown) indicating that its effect may be position-specific or promoter-specific.


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.




DISCUSSION

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 alpha chain(24) , and for the beta 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 alpha, beta, and chain of human fibrinogen. Locations of regulatory sequences in the genes for the alpha and beta chains are taken from Refs. 22, 24, 25.



The genes for the alpha and beta 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 beta chains, but HuH7 cells contain limiting quantities of alpha chains. (^2)The limiting fibrinogen chain, either alpha or beta 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 alpha and beta 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 alpha 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/EBPbeta 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 beta- 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 beta-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.


FOOTNOTES

*
This work was supported in part by Grant HL16919 of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U36503[GenBank].

§
Supported in part by a grant from the Chemo-Sero Therapeutic Research Institute, Kumamoto, Japan. Present address: Development Section, Third Production Dept., The Chemo-Sero-Therapeutic Research Institute, 668 Okubo Shimizu, Kumamoto 860, Japan.

To whom correspondence should be addressed: Dept. of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195-7350.

(^1)
The abbreviations used are: USF, upstream stimulatory factor; bp, base pair(s); CAT, chloramphenicol acetyltransferase; IL, interleukin.

(^2)
S. Huang and E. W. Davie, personal communication.


ACKNOWLEDGEMENTS

We thank Carol H. Miao for helpful suggestions and Jeff E. Harris for his expert technical assistance.


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