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(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
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 ( 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 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 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-
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
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
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 protein
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
[
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-
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
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].
Volume 270,
Number 39,
Issue of September 29, pp. 22988-22996, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
)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) . 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) .
Isolation and Sequencing of a Recombinant Cosmid
Clone
Sequence from the promoter previously published extends
only 522 bp upstream of the ATG(4) . A 654-bp fragment was
amplified from human genomic DNA (Clontech) by PCR and sequenced. This
showed some differences with the published sequence which included a
10-bp insertion (CCTATATCCT) located at position -324 with
respect to the ATG that has been previously described as a polymorphism
by Marchetti and co-workers(13) . To obtain additional
5`-flanking sequences, six aliquots of 1 
10
clones
each of a human placenta genomic DNA cosmid library (Clontech) were
screened by PCR using the same primers. One positive aliquot was
subjected to screening by colony hybridization using the PCR fragment
as probe, and one positive cosmid clone was obtained. Based on
restriction digests and Southern blotting the clone was estimated to
contain at least 5 kb of 5`-flanking sequences. A 3.5-kb EcoRI
fragment and a 0.8-kb SmaI fragment overlapping each other in
the already known sequence were subcloned into vector pBIIKS+
(Stratagene). To recombine both fragments, the 3.5-kb EcoRI
fragment was cloned into the EcoRI sites in the vector
containing the 0.8-kb SmaI fragment (Fig. 1).
Additional 5`-flanking sequences were subcloned as a 2.5-kb ApaI fragment that overlaps the EcoRI fragment.
Enzymatic sequencing of both strands was performed by both
ExoIII/S1-generated nested deletion templates (Erase a base kit,
Promega) or primer walking techniques.
Construction of Plasmids
The luciferase reporter
gene plasmids of the pGL2 series were obtained from Promega, and
reporter plasmids were constructed in several steps. First the factor
VII open reading frame was deleted by subcloning the proximal sequence
as a 280-bp EcoRI/HindII fragment into EcoRI/SmaI of pBIIKS+. In the next step the
3.5-kb EcoRI fragment was inserted in order to obtain a long
consecutive promoter fragment.
Cell Culture and Transfections
Human hepatoma
cells HepG2 (14) and HeLa cells were obtained from the
American Type Culture Collection. COS-7 cells and L132 cells were taken
from our in-house collection. HepG2 cells were cultured in
Dulbecco's modified Eagle's medium/F12 nutrient mix 1:1
(Life Technologies Inc.) supplemented with 10% fetal calf serum
(Boehringer Mannheim). HeLa cells, L132 cells, and COS-7 cells were
grown in minimal essential medium with 5% fetal calf serum. Both media
were supplemented with gentamicin and cells were grown in a 10%
CO
atmosphere.
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. , 1 µM dexamethasone, 100 nM phorbol 12-myristate 13-acetate,
and 10 µM forskolin.Luciferase and
Luciferase assays were carried out in microtiter plates
using the Luciferase assay system (Promega) as described in the
manufacturer's protocol. -Galactosidase
Assays-Galactosidase assays were
performed in microtiter plates as follows: 100 µl of prewarmed
assay buffer (40 mM Tris-HCl, pH 7.5, 2 mg/ml O-nitrophenyl -D-galactopyranoside, 84 mM -mercaptoethanol) were added to 20 µl of cell extract and
incubated at 37 °C. After 2-45 min the reaction was stopped
by adding 75 µl of 1 M sodium carbonate and the A determined. Luciferase data were normalized
with respect to
-galactosidase values in order to correct for
differences in transfection efficiency.RNA Isolation and Northern Blotting
Single step
RNA isolation from cultured cells and Northern blotting were done as
described in (15) . Radioactive hybridization was performed
with [P]dCTP-labeled DNA fragments in 5
SSPE, 10 Denhardt's, 100 µg/ml single-stranded DNA,
50% formamide, and 2% SDS at 42 °C. After 16 h of incubation, the
filters were washed with 2 SSC, 0.5% SDS for 5 min at room
temperature and 2 times 30 min at 42 °C. For non-radioactive
hybridization DIG-labeled RNA probes were used according to the
manufacturer's recommendations (Boehringer Mannheim).DNase I Footprint Assays
Nuclear extracts from
HepG2, HeLa, L132, and COS-7 cells were prepared by the method of
Dignam et al.(16) with minor modifications as
described by Ausubel et al.(15) ., 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.Electrophoretic Mobility Shift Assay
Duplex
oligonucleotides were labeled at both ends by filling in with Klenow
fragment of DNA-polymerase I. Ten to 30 µg of crude nuclear
extracts were preincubated in presence of 2 µg of
poly(dI-dC)poly(dI-dC) in a total volume of 25 µl of DNase I
footprint buffer at room temperature. After 10 min, 0.25 ng of labeled
oligonucleotide (3 10 counts/min) were added, and
incubation was continued for an additional 10 min. ProteinDNA
complexes were fractionated on 5% polyacrylamide gels (29:1, 0.5
Tris-borode, 5% glycerol) with 0.4 TBE running buffer
at 4 °C.Antibody Supershift Assay
The electrophoretic
mobility shift assays were performed with HNF-4 antiserum kindly
provided by Dr. Frances Sladek (University of California, Riverside).
One µl of undiluted antiserum was added to 10 µg of crude
nuclear extract preincubated with labeled oligonucleotide.
5`-Flanking Sequence of the Human Factor VII
Gene
Based on the limited published sequence (4) we
cloned a PCR fragment of 523 bp immediately upstream of the factor VII
translational initiation codon from human genomic DNA (Clontech). This
clone identified one positive clone cosfVII on screening a human
placenta cosmid library. The cosmid DNA, which was shown to be colinear
with genomic DNA by Southern blotting using the PCR fragment as a
probe, contains at least 5 kb of new 5`-flanking sequence which was
sequenced on both strands. Compared to the published sequence two
additional nucleotides were found, an additional C at -140 and an
additional G at position -460 (Fig. 1), which were
probably not detected previously due to band compressions typically
found in GC-rich sequences.Tissue Distribution of Factor VII Transcription
In
order to determine the tissue distribution of factor VII expression, we
probed a human multiple tissue Northern blot from Clontech (catalog no.
7760-1) with a randomly labeled fragment encoding amino acids
-60 to +152 in factor VII. Factor VII expression is clearly
restricted to liver, where a strong signal corresponding to transcripts
of 2.4 kb of length was obtained while in all other tissues transcripts
can be detected only at very low levels (Fig. 2). Under the low
stringency washing conditions, another band of about 1.2 kb was
detected in pancreas, and two additional bands of 1.3 and 1 kb were
observed in skeletal muscle.
-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).
Transcriptional Control of the Factor VII Gene in HepG2
Cells
To investigate the transcriptional regulation of factor
VII, a promoterless luciferase reporter gene in the vector pGL2-basic
was fused to a variety of promoter fragments at position -34 as a HindII/SmaI fusion which is 3` of the transcriptional
start site (Fig. 3). The constructs were all transfected into
human hepatoma HepG2 cells, known to express factor VII(22) ,
as well as HeLa and COS-7 cells, and transient promoter activity was
monitored by measuring luciferase activity in cell extracts.
Differences in transfection efficiencies were corrected for by
cotransfection of pCMV that carries the -galactosidase
reporter gene under the control of the CMV promoter. As negative
control the promoterless plasmid pGL2-basic was used while as a
positive control plasmid the luciferase gene was transcribed from the
SV40 early promoter-enhancer in pGL2-control which gave rise to very
high luciferase values in all three cell lines tested. In HepG2 cells,
expression from the factor VII promoter in plasmid pLUC-3.9/-34 was
significantly higher than the negative control pGL2-basic, but, even in
hepatocytes, the factor VII promoter is remarkably weak as compared to
the SV40 promoter, which gave about 50-fold higher luciferase values. Hepatocyte Specificity of Factor VII
Expression
Direct comparison of expression data from HepG2 and
HeLa cells was difficult because transfection efficiencies differed by
a factor greater than 20, as judged from -galactosidase values,
but expression from the factor VII promoter was detected in both cell
lines. Virtually no promoter activity was detectable in COS-7 cells as
none of the factor VII promoter constructs expressed luciferase better
than the negative control pGL2-basic (data not shown). In HeLa cells
sequential truncation of the promoter led to a steady reduction in
activity down to about 50% of the original values in pLUC-165/-34 and
to 10% in plasmid pLUC-46/-34 (Fig. 3) which does not identify
any specific promoter in the 5`-flanking sequence. Constructs
containing the heterologous SV40 enhancer were more informative as only
the constructs containing sequences upstream of position -1212
can be activated by a factor of three to four by the SV40 enhancer,
which suggests that the enhancer interacts with some promoter-like
structures located upstream from position -1212 (Fig. 3).Identification of Regulatory Sequences in the Factor VII
Promoter
A homology search for putative transcription
factor-binding sites, using the Findpatterns algorithm of the GCG
software package on the data base Transcription Factor Sites Release
6.5(23, 24) , revealed four putative Sp1 binding sites
at positions -2295, -1862, -1847, and -1785 and
consensus sequences for AP-1 binding at position -2488,
-1006, and -750 although no inducibility of factor VII
expression by phorbolesters was observed (data not shown). A putative
C/EBP-/NF-IL6-binding site at position -690 to -682
seems not to be relevant as adding IL6, IL1-, and combinations of
IL6 with IL1- or dexamethasone did not effect transient
transfection assays nor RNA levels measured by Northern blots. This is
consistent with a report by Hassan et al.(25) that
IL6 does not affect factor VII mRNA levels in Hep3B cells.HNF-4 Binds to the Factor VII Promoter
The
transcription factor HNF-4 (26) has been shown to be involved
in liver-specific expression of coagulation factors IX (17, 27) and X(3, 18) . So, to
investigate whether the presence of the putative HNF-4-binding site is
of any significance for factor VII transcriptional regulation,
electrophoretic mobility shift assays (EMSA) were performed with
double-stranded P-labeled oligonucleotides that correspond
to wt factor VII sequence from -77 to -43 and a mutated
oligonucleotide carrying a T
A transversion (Table 1).
EMSA analysis with nuclear extracts from HepG2 cells revealed a
concentration-dependent shift of protein
DNA complexes as compared
to free probe (Fig. 4A, lanes 1-4, C, and F). This shift could be specifically competed
for by preincubation with unlabeled wt oligonucleotide (lanes 5 and 6), but not by the T A transversion mutant
oligonucleotide (lane 7) which when present in the factor IX
promoter causes the Leyden phenotype of factor IX
deficiency(27) . The affinity of a DNA-binding protein to this
mutated oligonucleotide was strongly reduced in our experiments (lane 9) and could be competed for much more efficiently by
the wt sequence as compared to the mutated oligonucleotide itself (lanes 10 and 11) demonstrating that protein binding
is highly specific and dependent on the sequence of the binding site.
DNA 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
proteinDNA complex did not occur.
The Factor VII Promoter Is Regulated by Additional
Cis-acting Regulatory Sequences
To identify further cis-acting
regulatory sequences in the promoter, DNase I protection assays were
performed. Besides the footprint FPVII4 originating from HNF-4 on the
antisense strand at position -50 to -76, five more regions
protected against DNase I digestion were detected (Fig. 5). All
footprints were confirmed on the sense strand, on which HNF-4 protects
position -49 to -71 (data not shown), but none of
additional sites were similar to known cis-acting regulatory sequences.
The strong footprint FPVII6, at position -13 to +9,
interestingly covers the ATG translational initiation codon, and we
have tested the specificity of this footprint in EMSA experiments using
two complementary oligonucleotides spanning nucleotides -20 to
+12 on the sense strand and nucleotides +14 to -18 on
the antisense strand (Table 1). A proteinDNA complex
produced on incubation with HepG2 nuclear extracts (Fig. 6, lanes 1-3, C) was shown to be specific as
unlabeled double-stranded oligonucleotide competed successfully (lane 4), but the HNF-4 oligonucleotide did not (lane
5). In COS-7 cell nuclear extracts a distinct but different
binding activity was present (lane 6), leading to a complex
with different migration pattern.
-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).
Factor VII Expression Is Tissue-specific
Factor VII
promoter transient transfections showed cell line specificity of
luciferase expression to human hepatocyte cell line HepG2 (Fig. 2). In contrast, the factor VII promoter is not active at
all when compared with the promoterless vector pGL2-basic in non-human
COS-7 cells. Although HeLa cells did express luciferase from the factor
VII promoter, it seems likely that in HeLa cells the slight decrease of
expression upon successive truncations is the effect of a rather
unspecific fortuitous transcription initiation in HeLa cells, which is
further substantiated by the observation that the heterologous SV40
enhancer only increases expression in HeLa cells when present with 1.6
kb of promoter whereas it increases expression in HepG2 cells with a
promoter fragment of only 165 bp (Fig. 3). Thus it is unlikely
that identical sequences act as promoters in HepG2 and HeLa cells.Factor VII Expression Is Regulated by Several
Factors
Functional studies of promoter deletions in the HepG2
cell line showed that deletion of sequences from -1601 down to
-1212 increases expression about 2-fold which suggests a negative
element is present upstream of -1212 (Fig. 3). A 25-bp
sequence element at position -1230 to -1214 that has a
similarity of 76% to position -122 to -98 of the apoAIV
promoter element AIVC is a good candidate for a binding site of such a
negatively acting factor. The element AIVC was defined by a footprint
covering position -148 to -92 in the apoAIV promoter, and
proteins binding to the 5`-half of the motif at position -142 to
-128 have been identified but not to the 3`-half (41) . 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) .Transcription Factor HNF-4 Binds to the Factor VII
Promoter
One of the footprints which we detected corresponds to
the liver-enriched transcription factor HNF-4. HNF-4 is an orphan
member of the steroid hormone receptor superfamily which has been
highly conserved throughout evolution, suggesting that it might respond
to an as yet unidentified ligand. Initially, it was characterized as a
factor that activates apolipoprotein CIII and transthyretin (TTR) gene
expression (26, 52) and it also positively regulates
numerous genes (for a review, see (52) ). HNF-4 exists in two
different isoforms as a result of differential splicing from which the
largest is by far the most common in HepG2 cells(53) . Despite
the presence of binding activity specific for the HNF-4 site in all
cells investigated, HNF-4 is mainly restricted to hepatocytes. With
high levels in liver and moderate levels in kidney and intestine, the
expression pattern of HNF-4 reflects the tissue distribution of genes
regulated by HNF-4 (54) . HNF-4 has been demonstrated to confer
liver-specific expression on coagulation proteases factor IX (30, 33, 34) and factor
X(3, 18) . A number of point mutations in the factor
IX promoter between position -21 and +13 cause the Leyden
phenotype of the severe bleeding disorder hemophilia B (Table 1;
(26, 31, and references therein), and the mutations at position
-21 and -20 disrupt the HNF-4-binding site. As shown by our
DNase I protection experiments and EMSA assays, HNF-4 specifically
binds to position -71 to -49 on the factor VII promoter ( Fig. 4and Fig. 5).
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) .
), interleukin 1-; IL6, interleukin 6; wt,
wild-type; PCR, polymerase chain reaction; CMV, cytomegalovirus.
We are indebted to Dr. Frances Sladek for providing
the HNF-4 antibody used in this study. We thank Dr. Peter
Fürst for fruitful discussions throughout the
project, Dr. Fred Asselbergs for providing plasmid pCMV and the
mammalian cell lines, and Dr. Frank Buxton and Dr. Gino van Heeke for
carefully reading the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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