(Received for publication, March 22, 1995; and in revised form, July 13, 1995)
From the
The human coagulation protease factor VII plays a pivotal role
in the initiation of the coagulation cascade by both the extrinsic and
the intrinsic pathway. Although the gene, encoding factor VII, is
expressed predominantly in the liver, the mechanisms underlying this
tissue-specific expression have not been elucidated. In this study, we
have analyzed the contribution of 5 kilobases upstream of the ATG
translational initiation codon upon hepatic factor VII gene
transcription. Transient transfection assays of a set of nested
deletions in both liver and non-liver cell lines, HepG2 and HeLa
respectively, indicate that several regions are involved in
liver-specific expression. A slight negative effect on factor VII
promoter activity in HepG2 cells is mediated by sequences upstream of
position -1212. DNase I protection experiments reveal six
footprints, FPVII1 through FPVII6, within the proximal 714 base pairs
but a minimal promoter of 165 base pairs containing only FPVII3-6
is sufficient to confer liver-specific expression in HepG2 cells.
Interestingly, FPVII6, at position -14 to +10 on the sense
strand, would indicate that an as yet unknown transcription factor
covers the ATG translational initiation codon. Gel retardation
experiments show that the liver-enriched transcription factor HNF-4
binds specifically to footprint FPVII4 at position -71 to
-49. Furthermore, a T A transversion, that in the HNF-4
binding site of factor IX causes a severe bleeding disorder, was
introduced into the HNF-4-binding site of factor VII and reduced
promoter activity by 20-50%. Coordinate HNF-4-mediated regulation
of several blood protease genes as well as genes involved in lipid
metabolism might account for the positive correlation of these factors
with increased risk of occlusive heart diseases.
Factor VII is a vitamin K-dependent protease of the coagulation cascade present in plasma in trace amounts (1) which, when complexed with tissue factor and calcium ions, can cleave both factor X and factor IX initiating the extrinsic pathway and the intrinsic pathway of blood coagulation, which results finally in the formation of a fibrin clot.
The human factor VII gene is a single copy gene on
chromosome 13q34, 3 kb ()upstream of the coagulation
protease factor X gene(2, 3) , which consists of eight
exons spread over 12 kb of genomic DNA, which produces a 2.4-kb mRNA
encoding a mature protein of 254 amino acids(4) .
A positive
correlation between high plasma factor VII antigen levels (FVIIag),
procoagulant activity (FVIIc), and an increased risk of coronary heart
disease has been demonstrated(5) . Although both increased
FVIIag and FVIIc levels are associated with other risk parameters such
as higher age, use of oral contraceptives, and plasma triglyceride and
cholesterol (see (6) and references therein), the picture is
complicated as both FVIIag and FVIIc are strongly correlated with
plasma triglyceride levels(7) , while the correlation between
cholesterol and FVIIag is rather weak. More detailed studies emphasized
the strong correlation between triglycerides present in very low
density lipoprotein and low density lipoprotein particles and FVIIag
levels(8, 9, 10) . Intriguingly, males
heterozygous for an Arg to Gln
polymorphism
found in about 20% of the caucasian population have FVIIag
concentrations reduced by 20-25%, which presumably reduces the
risk of coronary heart disease and thrombosis by lowering the
proportion of factor VII molecules being in the activated
state(11, 12) .
From these prospective studies it is possible that a drug that interferes with factor VII expression levels could be an antithrombotic, and the first step toward such a drug would be to understand the regulatory mechanisms of factor VII expression. As only 522 nucleotides of the factor VII 5`-flanking region have been published(4) , we have cloned and sequenced a further 4291 bp and functionally characterized this sequence in reporter gene assays. We have identified cis-acting regulatory sequences in the promoter by DNase I footprinting analysis, and we show that HNF-4 is a positive regulator of factor VII expression.
Figure 1: Partial nucleotide sequence of the human factor VII 5`-flanking region. 613 nucleotides out of 4813 nucleotides cloned and sequenced are shown. The most 5`-nucleotide at position -523 previously published is indicated by an asterisk. Additional nucleotides not present in the published sequence are shown in bold. Restriction sites important for cloning purposes are shown above the sequence. The amino acid sequence of the first exon is shown.
Starting from this construct a 1.4-kb KpnI/BglII promoter fragment and ExoIII/S1 deletion derivatives were ligated into the KpnI/BamHI sites of the promoterless luciferase vector pGL2-basic (pLUC-1.6/-34, pLUC-1.2/-34, pLUC-474/-34, see Fig. 3).
Figure 3: Factor VII promoter deletion analysis by luciferase reporter gene assay. Left, schematic drawing of the factor VII promoter sequences used in transient transfection experiments. The 5`-most nucleotide of each factor VII promoter derivative either generated by restriction enzyme digests or exonucleaseIII/S1 treatment is indicated by its position number with reference to the translational initiation codon. Factor VII promoter sequences are shown as a single line. Double lines represent vector sequences. Plasmids named by the extension ``Leyden'' differ from their progenitor plasmids by a single base exchange in the putative HNF-4-binding site as indicated by an A. Internal deletions in plasmids pLUC-474d1 through d3 are indicated by gray boxes and position numbers. Right, relative luciferase expression in percent of pLUC-3.9/-34 expression as means ± standard deviation of four to eight independent transfections with at least two DNA preparations in HepG2 and HeLa cells, respectively. ND, not determined.
Longer promoter constructs were obtained by inserting the original KpnI promoter fragment or an ExoIII/S1 deletion derivative into the single KpnI site in plasmid pLUC-1.6/-34 giving pLUC-3.9/-34 and pLUC-2.5/-34, respectively (Fig. 3). The plasmids pLUC-981/-34 and pLUC-712/-34 are ApaI and SmaI deletion derivatives of pLUC-1.2/-34, respectively.
To generate the plasmids pLUC-237/-34 and pLUC-165/-34, the PstI or NsiI sites in the progenitor plasmid pLUC-474/-34 were treated with T4 DNA-polymerase and fused to the SmaI site of the vector. In pLUC-46/-34 the single NcoI site was filled up by Klenow-polymerase and fused to SmaI. Construct pLUC-2.5/+40 was generated by blunt end fusion of a PCR product amplified from promoter and luciferase parts of pLUC-712/+131 and replacing the promoter-luciferase EcoRI fragment in pLUC-2.5/-34. Plasmid pLUC-712/+131 itself originates from inserting the originally cloned SmaI fragment into the filled-in HindIII site of pGL2-basic. All PCR fragments were sequenced to rule out amplification artifacts.
The plasmids pLUC-474/-34``Leyden'' and pLUC-165/-34``Leyden'' were generated by replacing the StyI-NcoI fragments containing the putative HNF-4 site by a double-stranded oligonucleotide that contained the Leyden-specific point mutation (Table 1). The mutation was also introduced into plasmid pLUC-1.6/-34 by replacing the EcoRI fragment that contained the HNF-4-binding site by the corresponding fragment from pLUC-474/-34``Leyden.''
All plasmid derivatives containing the SV40 enhancer were generated by replacing the BamHI-HindIII vector fragment, which contains luciferase gene followed by the SV40 T antigen intron and polyadenylation signal, with the identical part from pGL2-control (Promega), that also contains the 250-bp SV40 enhancer downstream of the polyadenylation signal.
COS-7 cells were transfected with
Lipofectin Reagent (Life Technologies Inc.) following the
manufacturer's protocol. Transient transfections into HepG2 and
HeLa cells were performed by the calcium phosphate coprecipitation
technique as described by Ausubel et al.(15) .
Plasmid DNAs were isolated with DNA purification columns purchased from
Qiagen. Cultured cells, replated 24 h before transfection to about 70%
confluence, were cotransfected with 0.48 pmol of luciferase plasmid (e.g. 3 µg of pLUC-3.9/-34) and 1.5 µg of pCMV
used as an internal control. The precipitate was removed 16 h later,
and the cells were exposed for 3 min to 0.4 ml of 10% glycerol in
medium, washed three times with phosphate-buffered saline, and grown in
fresh medium for 48 h. In HeLa cell transfections the glycerol shock
was omitted. Cells were harvested using 150 µl of reporter lysis
buffer (Promega) according to the manufacturer's recommendations.
Induction experiments with transfected HepG2 cells were started 24 h
after glycerol shock. Cells were harvested after incubation for 0.5, 1,
3, 6, 12, and 24 h with either one, or combinations, of the following
substances: 100 units/ml IL6, 100 units/ml IL1-, 1 µM dexamethasone, 100 nM phorbol 12-myristate 13-acetate,
and 10 µM forskolin.
DNase I
footprints were performed in a total volume of 50 µl, containing 20
mM HEPES, pH 7.5, 30 mM KCl, 4 mM
MgCl, 0.5 mM EDTA, 0.5 mM dithiothreitol,
4% glycerol, and 1.5 µg of poly(dI-dC)
poly(dI-dC). Nuclear
extracts containing 10-60 µg of total protein were incubated
for 15 min at room temperature. One to two ng of end-labeled fragments
(1-2
10
counts/min) were added and incubated
for another 15 min at room temperature. Limited digestion was achieved
by adding 5 µl of Ca
/Mg
solution (final concentration 1 mM MgCl
, 0.5
mM CaCl
) and 0.33-1 units of freshly diluted
DNase I for 1 min at room temperature. The reaction was stopped by 140
µl of DNase I stop buffer (192 mM sodium acetate, 32
mM EDTA, 0.14% SDS, and 64 µg/ml yeast RNA). The DNA was
treated with phenol, ethanol precipitated, and analyzed on 6%
polyacrylamide, 7 M urea sequencing gels.
The 5`-flanking sequence has neither a typical TATA nor CAAT box as found in promoters of the other coagulation proteins: factor IX (17) , X(3, 18) , XII(19) , and prothrombin(20, 21) .
A homology search of the 4813-bp 5`-flanking sequence against the EMBL databank identified three Alu repeats at position -4742 to -4440, -2739 to -2518, and -942 to -642. Sequence alignment of the factor VII and factor X promoters revealed a similarity of 86% for a small 37-bp element located in factor VII promoter at position -2340 to -2304 and in the factor X promoter in the same orientation at position -520 to -485. The functional relevance, if any, of this element is unclear as deletion of these sequences in the factor X or factor VII promoters did not alter reporter gene expression significantly (see Fig. 3and Refs. 3, 18).
Figure 2:
Tissue distribution of factor VII
transcripts determined by a multiple tissue Northern blot. The multiple
tissue Northern blot purchased from Clontech was hybridized with a
randomly labeled factor VII cDNA fragment corresponding to codons
-60 to +152 (panel A) and with a
-actin-specific probe (panel B), respectively. Lane
1, heart; 2, brain; 3, placenta; 4,
lung; 5, liver; 6, skeletal muscle; 7,
kidney; 8, pancreas. A strong signal corresponding to a
transcript of the correct size is visible in liver (lane
5).
Deletion of sequences from -4813 down to position -1601 did not significantly alter luciferase expression, but a longer deletion to position -1212 doubled luciferase expression (Fig. 3). Further truncation of the promoter down to position -165 retained the high promoter activity of pLUC-981/-34, but a longer deletion to position -46 drastically reduced promoter activity down to values seen with the promoterless control vector. These results suggested that the first 165 bp upstream from the translational initiation codon are sufficient to confer expression in hepatocytes. Activity was restored to 73% on plasmid pLUC-474d3/-34 by sequences from -474 to -355, although the presence or absence of this fragment on the longer constructs pLUC-474/-34, pLUC-237/-34, and pLUC-165/-34 had no impact on expression.
The findpatterns search for cis-acting regulatory sequences did not identify any further putative binding sites for liver-enriched transcription factors, but when repeated with more degenerated consensus sequences a potential binding site for the liver-enriched transcription factor HNF-4 at position -67 to -56 was seen.
Figure 4:
EMSA of the HNF-4-binding site with crude
nuclear extracts. A, a double-stranded end-labeled
oligonucleotide, with nucleotides -77 to -43 containing the
HNF-4-binding site, was incubated with crude nuclear extracts from
HepG2 cells. Lane 1, free oligonucleotide (F) without
added nuclear extract; lanes 2-4, incubated with 10, 30,
and 60 µg of HepG2 nuclear extract, respectively (C indicates the proteinDNA complex); lanes 5 and 6: incubation with 30 µg of extract and competition by 20-
and 400-fold molar excess of unlabeled wt oligonucleotide; lane
7, competition by 400-fold molar excess of unlabeled
Leyden-specific oligonucleotide; lane 8, free Leyden-specific
oligonucleotide; lane 9, with 30 µg of HepG2 nuclear
extracts; lanes 10 and 11, competition by 400-fold
molar excess of mutated and wt oligonucleotide, respectively. B, the free HNF-4 oligonucleotide (lane 1, F) was retarded by incubation with 10 µg of HepG2 nuclear
extracts (lane 2, C) and supershifted by 1 and 2
µl of undiluted HNF-4 antiserum, respectively (lanes 3 and 4, S); lanes 5, 7, and 9,
the same oligonucleotide with HeLa, L132, and COS-7 cell nuclear
extracts, respectively; lanes 6, 8, and 10,
after incubation with HNF-4 antiserum. A supershift of the
protein
DNA complex did not occur.
To prove unambiguously that the protein binding is indeed HNF-4, we repeated the EMSA assays in the presence or absence of an antiserum raised against HNF-4(26) . The slower migrating complex obtained with HepG2 nuclear extracts (Fig. 4B, lane 2, C) was supershifted upon addition of the HNF-4 antiserum (lanes 3 and 4, S). In contrast, the slower migrating complexes obtained with crude nuclear extracts from HeLa, L132, and COS-7 cells (lanes 5, 7, and 9) that could be competed for by cold oligonucleotide (data not shown) were not recognized by the anti HNF-4 antiserum (lanes 6, 8, and 10).
To further investigate the functional role of the HNF-4 site in HepG2 cell experiments, we replaced the StyI/NcoI fragment containing the wt-binding site in plasmid pLUC-165/-34 and pLUC-474/-34 by an oligonucleotide that contains the Leyden-specific point mutation (Table 1). Out of the longer resulting plasmid pLUC-474/-34``Leyden,'' we subcloned the mutated putative HNF-4-binding site into pLUC-1.6/-34 and assayed all three constructs by transient transfection assays. This point mutation reduced the promoter activity to about 80-50% of the particular wt plasmid in HepG2 cells (Fig. 3) which confirms the functional importance of the HNF-4-binding site for liver-specific expression.
Figure 5:
DNase I protection assay of the factor VII
promoter antisense strand. Prior to DNase I digestion the labeled
fragment (U lane) was incubated either with bovine serum
albumin (B lane) or with 10, 20, 30, 40, and 60 µg of
HepG2 nuclear extract, respectively. G/A denotes G- and
A-specific Maxam-Gilbert sequencing reaction. Footprints FPVII3-6
protected from DNase I digestion are indicated by squares. The
antisense strand (shown here) of promoter fragment -314 to
+129 was labeled by filling in the EcoRI site with
[-
P]dATP. Incorporation of
[
-
P]dCTP into the AvaI end of this
fragment labeled the sense strand which gave similar results.
Footprints FPVII1 and 2 were identified on antisense and sense strands
of promoter sequences from position -714 to -315 (data not
shown).
Figure 6: Electrophoretic mobility shift assay of sequences from position -20 to +12. The end-labeled double-stranded oligonucleotide comprising nucleotides -20 to +12. Free oligonucleotide (lane 1, F) migrated at a slower rate after incubation with 10 and 30 µg of HepG2 nuclear extract (lanes 2 and 3, C). A 400-fold molar excess of unlabeled oligonucleotide efficiently competed for binding (lane 4). In contrast, the HNF-4 oligonucleotide fails to compete for binding (lane 5). In COS-7 nuclear extracts a distinct but different binding activity is present (lane 6).
None of the promoter-reporter fusion discussed above contained this cis-acting site. To test the functional importance of this site, we constructed the new plasmid pLUC-2.5/+40, where the translational initiation codon of the luciferase gene was fused to position +40 in the factor VII coding region. In transient transfection experiments, luciferase expression from construct pLUC-2.5/+40 was 1.9-fold higher than from plasmid pLUC-2.5/-34 (Fig. 7). Furthermore, when the first factor VII exon and part of the first intron up to position +131 were included in plasmid pLUC-712/+131 reporter gene expression was about 2.7 times higher than from plasmid pLUC-712/-34. These experiments suggest that sequences downstream of the putative HNF-4-binding site and the first exon are also involved in expression of factor VII.
Figure 7: Factor VII promoter constructs including 5`-untranslated leader sequences and coding region. Left, schematic drawing of the factor VII promoter sequences used in transient transfection experiments. Factor VII promoter sequences are shown as single line. Double lines represent vector sequences. The position numbers of the 5`-most and 3`-most nucleotides are given above the line. Right, relative luciferase expression values in HepG2 and HeLa cells relative to expression of pLUC-3.9/-34 (see Fig. 3).
During the past few years, a number of studies on promoters of coagulation proteases like prothrombin(20, 21, 28) , factor IX(27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) , and factor X (3, 18) have been published. The regulation of factor IX gene expression is of particular interest, since the Leyden phenotype of the severe bleeding disorder hemophilia B is caused by several point mutations within the promoter region, of which a number map in the HNF-4-binding site, reducing the affinity of HNF-4 for the promoter. Both the factor IX and the factor X promoter are positively regulated by the liver-specific transcription factor HNF-4.
In this work we show that measurable amounts of factor VII transcripts are present only in liver suggesting that regulation takes place at the transcriptional rather than at the translational level (Fig. 2), and we have defined promoter elements involved in liver-specific expression of the coagulation factor VII present in the 4813 bp of factor VII 5`-flanking sequence.
A number of studies show that triglyceride and cholesterol levels in plasma are positively correlated with the risk for coronary heart diseases and thrombosis (5) and the plasma levels of lipoproteins and activity of vitamin K-dependent coagulation proteases are positively correlated with lipid concentrations in plasma(8, 9, 10) . The apolipoprotein genes AI, CIII, and AIV form a gene cluster, and, since coregulation has been shown, it has been hypothesized that apoCIII, AI, AII, AIV, and E have evolved from a common ancestor(40, 41) . Interestingly, the genes encoding factor VII and factor X are also clustered and separated by only 2823 nucleotides(3) . The significant similarity of vitamin K-dependent blood coagulation proteins suggests that the genes could have also evolved from a common ancestral gene and that they might be regulated by a common mechanism. The close correlation of FVIIag level and activity of factor IX, factor X, and prothrombin with the lipoprotein metabolism tempted us to compare the factor VII promoter and promoters of apolipoprotein genes and coagulation proteases factor IX and X with respect to common regulatory mechanisms.
In our experiments the proximal 165 bp upstream from the ATG translational initiation codon were sufficient for liver-specific expression (Fig. 3), and DNase I protection assays on the proximal 714-bp promoter fragment revealed a set of six footprints of which four map in the first 145 nucleotides (Fig. 5). Removal of the protein-binding sites VII1 and 2 at a distance from 380 to 450 from the translational initiation codon had no effect on the transcriptional activity (compare pLUC-474/-34 to pLUC-237/-34). However, the presence of these binding sites might account for the higher promoter activity in plasmid pLUC-474d3/-34 as compared to pLUC-46/-34 (Fig. 3).
Including sequences up to position +40 in the promoter-luciferase construct pLUC-2.5/+40 raised expression levels to about two times the values obtained with the plasmid pLUC-2.5/-34 (Fig. 7). The effect is cell line specific as almost no expression from this plasmid is detectable in HeLa cells. As demonstrated by the strong footprint FPVII6 in DNase I protection experiments (Fig. 5) and by EMSA experiments (Fig. 6), an as yet unidentified regulatory protein binds specifically to this sequence which covers the ATG translational initiation codon.
Inclusion of the entire factor VII exon I and the 5`-terminal part of the first intron in plasmid pLUC-712/+131 has a similar effect; plasmid pLUC-712/-34 and pLUC-712/+131 differ 2.7-fold (Fig. 7). This effect is slightly more pronounced than in the plasmid pair pLUC-2.5/-34 and pLUC-2.5/+40 which argues for an additional regulatory function located within exon I or the 5`-end of intron I. This is similar to transcriptional regulation of von Willebrand factor which has a positive regulatory region located within the first exon (42) and in apoB where HNF-1 and C/EBP bind to an enhancer in the second intron (43) .
C/EBP- and
related factors that recognize the same binding sites as C/EBP-
play an important role in expression of apolipoproteins AI (44, 45, 46) , AII(47, 48) ,
B(49, 50, 51) , and coagulation factor
IX(32, 33, 38) , and we anticipated a similar
function of C/EBP-
at position -690 to -682 at the
putative C/EBP-
/NF-IL6-binding site in the factor VII expression.
However, the DNase I protection assay performed on the factor VII
promoter did not show a footprint over the putative C/EBP-binding site
(see below), and in transient transfections deletion of this binding
site in plasmid pLUC-474/-34 does not alter expression when compared to
pLUC-712/-34 (Fig. 3) and probably does not mediate acute phase
response of factor VII expression by C/EBP-
as examined by
induction and cotransfection experiments (data not shown). One
explanation for the lack of this interaction could be the very low
C/EBP concentration in HepG2 cells, which also accounts for the weak
factor IX expression in HepG2 cells(32) .
In order to prove the functional
importance of the HNF-4-binding site for factor VII expression, we
introduced the Leyden-specific -20 T to A mutation (ACTTTG
ACTTAG; Table 1and Table 2) into the HNF-4-binding site of
the factor VII promoter which lowered promoter activity in HepG2 cells
by 20-50% (Fig. 3) and reduced the binding of HNF-4 in
crude extracts (Fig. 4). The only slight promoter activity
reduction of about 20% seen in pLUC-1.6/-34``Leyden'' and
pLUC-474/-34``Leyden'' as compared to the 50% reduction in
pLUC-165/-34``Leyden'' could be caused by the positive effect
of promoter sequence -474 to -355 which also restores
promoter activity in plasmid pLUC-474d3/-34 (Fig. 3). The
identical point mutation introduced into the HNF-4-binding site of the
factor X promoter reduced activity by 80.8%, whereas the totally random
mutagenesis of ACTTTG to GACAAT reduced factor X promoter activity by
82.8%(21) . The apolipoprotein genes apoAI(55) ,
CIII(56, 57) , and AIV (41, 58) as
well as AII (56) and B (51, 56) are also
positively regulated by HNF-4. Mutagenesis of the HNF-4-binding site in
the apoB and apoCIII promoter elements BA1 and CIIIB reduced activity
of reporter gene constructs by 98.5 and 92%, and deletion of the
elements A1D and AIIJ from the apoAI and apoAII promoters reduced
promoter activity by 50% and 30% (56) which is in the range of
the effect seen in the factor VII promoter. Since all apolipoprotein
genes and the majority of coagulation protease genes are positively
regulated by HNF-4 we hypothesize that the HNF-4 concentration is one
possible link causing the correlation between lipid metabolism and
vitamin K-dependent coagulation proteases. Although the positive effect
of HNF-4 in apolipoprotein expression is dependent on additional
factors as shown by the synergism between HNF-4 and C/EBP binding to
overlapping sites in the apoB promoter (51) and the requirement
for additional transcription factors as suggested for the apoAII and
apoCIII promoter, this dependence can be overcome in the apoCIII
promoter by high level expression of HNF-4(56) . One or more of
the strong footprints in the factor VII promoter could also be involved
in mediating HNF-4 transactivation or, in analogy to the apoB gene
regulation, this function could also be fulfilled by C/EBP, or a
hypothetic C/EBP-like factor, binding to the putative site at position
-690 to -682. None of the additional binding sites for
liver-enriched transcription factors like HNF-3 or HNF-1, which have
been shown to be involved in, for example, apolipoprotein or
prothrombin gene regulation, have a counterpart in the factor VII
promoter. Although coordinated liver-specific gene expression certainly
involves a complex regulatory network, HNF-4 may play a key role. It is
known that HNF-4 is a positive regulator of the liver-specific
transcription factor HNF-1, which is involved in expression of genes
like prothrombin,
1-antitrypsin, and
transthyretin(20, 21, 59, 60, 61) ,
and HNF-1 in turn down-regulates expression from its own promoter and
other HNF-4-regulated genes like apoCIII and apoAI(62) . The
genes apoB, CIII, AI, and AII can be down-regulated by some or all of
the steroid receptor superfamily transcription factors ARP-1, EAR-2,
and EAR3/COUP-TF which bind with different affinity to HNF-4-binding
sites (51, 56, 57, 58) , but, as
Mietus-Snyder and colleagues concluded, the amount of HNF-4 is
significantly higher than the level of ARP-1 and EAR-3 in liver and
intestine, and thus the transcriptional activity of the apoCIII gene is
at least in part dependent upon the intracellular balance of HNF-4,
ARP-1, and EAR-3(57) .
We demonstrate in this paper that, like factor IX and factor X, expression of factor VII is probably regulated by HNF-4. These genes are part of a complex regulatory network that lead finally to coordinated liver-specific expression or repression. In analogy to the regulatory mechanism described for the apolipoprotein genes, additional orphan steroid receptors may also be involved which we are currently investigating.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U14580[GenBank].