(Received for publication, September 6, 1995; and in revised form, November 11, 1995)
From the
Factor VII is a vitamin K-dependent coagulation protein essential for proper hemostasis. The human Factor VII gene spans 13 kilobase pairs and is located on chromosome 13 just 2.8 kilobase pairs 5` to the Factor X gene. In this report, we show that Factor VII transcripts are restricted to the liver and that steady state levels of mRNA are much lower than those of Factor X. The major transcription start site is mapped at -51 by RNase protection assay and primer extension experiments. The first 185 base pairs 5` of the translation start site are sufficient to confer maximal promoter activity in HepG2 cells. Protein binding sites are identified at nucleotides -51 to -32, -63 to -58, -108 to -84, and -233 to -215 by DNase I footprint analysis and gel mobility shift assays. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Sp1, are shown to bind within the first 108 base pairs of the promoter region at nucleotide sequences ACTTTG and CCCCTCCCCC, respectively. The importance of these binding sites in promoter activity is demonstrated through independent functional mutagenesis experiments, which show dramatically reduced promoter activity. Transactivation studies with an HNF-4 expression plasmid in HeLa cells also demonstrate the importance of HNF-4 in promoting transcription in nonhepatocyte derived cells. Additionally, the sequence of a naturally occurring allele containing a previously described decanucleotide insert polymorphism at -323 is shown to reduce promoter activity by 33% compared with the more common allelic sequence.
Factor VII, a vitamin K-dependent clotting protein, plays a critical role in coagulation. Vessel wall injury exposes the membrane-bound glycoprotein tissue factor to circulating Factor VII, allowing the two proteins to form a complex. This facilitates cleavage of the Factor VII zymogen to its active form, Factor VIIa, which in concert with tissue factor further activates the coagulation cascade (1) .
Six proteins of the coagulation system undergo a
unique posttranslational modification, -carboxylation of glutamic
acid residues in the amino terminus, which requires the presence of
vitamin K(2, 3) . These vitamin K-dependent clotting
proteins (prothrombin; protein C; protein S; and Factors VII, IX, and
X) share many structural and functional similarities. More
specifically, Factors VII, IX, and X and protein C have 8 exons, each
of which corresponds to conserved functional domains of the
proteins(4) . Unlike the promoter regions of most eukaryotic
promoters, the promoter regions of the vitamin K-dependent clotting
proteins lack TATA
boxes(5, 6, 7, 8) . Additionally,
the sequence element ACTTTG is found in similar locations in the
proximal promoters of Factors VII, IX, and X. In Factor IX, this
element binds to the liver-enriched transcription factor hepatocyte
nuclear factor-4 (HNF-4)(
)(9) .
Among the coagulation proteins, Factor VII has the shortest half-life, approximately 2-3 h(10) . Factor VII also circulates at extremely low concentrations (0.5 µg/ml)(11) , less than one-tenth of the concentrations of the other vitamin K-dependent coagulation proteins(12) . However, the molecular mechanisms controlling these low Factor VII plasma concentrations have not been defined.
Clinical studies have shown correlations between Factor VII levels and a large number of variables, most notably cardiovascular disease. In 1986, the Northwick Park Heart Study, a prospective study of adverse cardiovascular events in 1511 middle-aged males, showed that higher concentrations of Factor VII coagulant activity were associated with an increased risk of ischemic heart disease(13) . Factor VII levels have also been correlated with age(14, 15) , cholesterol, and triglyceride levels(16) , dietary saturated fat intake(17) , menopause(15, 18) , oral contraceptive pills(19) , body mass index(20) , testosterone concentrations in men(21) , and a polymorphism within exon 8 encoding amino acid residue 353(22) . The presence of a common polymorphic decanucleotide insert (allele frequency 0.2) in the proximal human Factor VII promoter region (23) was recently shown to correlate with both a lower Factor VII antigen and coagulant activity(24) .
In the present study, we characterize
important cis- and trans-acting functional elements in the human Factor
VII promoter, including sequence analysis of 3958 bp of 5`-flanking
DNA, delineation of transcription start sites, analysis of promoter
activity using reporter gene assays, and determination of protein
binding regions by DNase I footprint analysis and gel mobility shift
assays. We also compare promoter activity between the two alleles with
and without the decanucleotide insert 323 bp 5` of the translation
start site(23) . We show by Northern blot analysis that
synthesis of Factor VII mRNA is restricted to the liver. Using
mutagenesis of protein-binding sites along with reporter gene assays,
we demonstrate the critical role played by cis elements at -63
-58 and -108
-84, and their cognate
binding proteins, HNF-4 and Sp1.
Figure 1:
Northern analysis of
mRNA from multiple human organs demonstrates liver-specific expression
of human Factor VII mRNA. A nylon membrane containing approximately 2
µg/lane of poly(A) RNA was hybridized to a
radiolabeled probe containing a 1.5-kb HindIII-EcoR I
cDNA fragment of the human Factor VII gene. The 2.7-kb band as
indicated by an arrow corresponds to the Factor VII
transcript. A
-actin control on the same blot permits
normalization. Heart and skeletal muscle contain a muscle-specific
-actin transcript at 1.8 kb in addition to the 2.0-kb transcript
present in all other tissues shown. Abbreviations are as follows: H, heart; B, brain; Pl, placenta; Lu, lung; Li, liver; SM, skeletal muscle; K, kidney; Pa, pancreas; Sp, spleen; Th, thymus, Pr, prostate; Te, testis; O, ovary; SI, small intestine; C, colon; PB, peripheral blood lymphocytes.
Figure 2: Nucleotide sequence of the proximal promoter of the human Factor VII gene. The full 3958-base pair 5`-flanking DNA sequence can be found in GenBank (accession number U40852) The proximal 455 bp of sequence are derived from an allele, which does not contain the previously described decanucleotide insert at -323 bp. The sequence from -3958 to -456 was obtained from an allele that contains the decanucleotide insert. The major transcription start site is marked with an asterisk at -51. The 5`-flanking region is shown in lower case. The first 42 bp in exon 1 are shown in capital letters with the corresponding single-letter amino acid code underneath. Protein-binding sites identified by DNase I footprint analysis and gel mobility shift assays are shaded. Putative binding sites identified within the first 400 bp are as follows: AP-1, GATA factors, Sp1, AP-4, TFEB, XPF-1 (exocrine pancreas factor-1), HRE (hormone response element), Sp1, GAGA factor, H4TF-1 (Histone H4 transcription factor), GR (glucocorticoid receptor), and HNF-4.
The TRANSFAC data base was used to search
for putative transcription factor binding sites. Potential binding
sites were seen for the liver-enriched transcription factors HNF-4 at
-67 to -56 and LF-A1 (Liver Factor-A1) (43) at
-2178 to -2162. Putative binding sites were also seen for
the following transcription factors at the indicated positions (using
the numbering system of O'Hara et al.(6) where
-1 represents the first nucleotide 5` to the translation start
site): Sp1 at -286 to -276, -204 to -195, and
-101 to -92; XPF-1 (exocrine pancreas factor-1) at
-221 to -207; AP-1 at -391 to - 382; AP-2 at
-1684 to -1675; AP-4 at -3487 to -3478 and
-234 to -225; GR (glucocorticoid receptor) at -91 to
-82; H4TF-1 (histone H4 transcription factor-1) at -100 to
-92; PTF-1 (pancreatic acinar cell-specific factor) at
-1403 to -1392; GATA transcription factors at -363 to
-353, TFEB at -224 to -213; a hormone response
element at -226 to -212; GAGA factor at -143 to
-130, and ISGF-1 (interferon-stimulated gene factor-1) at
-3552 to -3543. An Alu repeat is present at -956 to
-634. Additionally, two nucleotide differences were noted between
an individual containing a decanucleotide sequence insert
(5`-CCTATATCCT-3`) at -323 and the sequence of an individual
lacking the insert; these changes were at -122 (T to C) and
-401 (G to T). The sequence data beyond -456 is based on
the sequence present in the cosmid clone, which was constructed from an
individual in whom the previously described decanucleotide insert was
present.
Figure 3:
A, primer extension analysis of the human
Factor VII gene. Primer extension analysis was performed as described
under ``Experimental Procedures'' using an oligonucleotide
spanning from +21 to +42 in exon 1 as a primer. Lane 1 shows P-labeled FX174 HaeIII-digested DNA to
indicate the total length of the reverse transcripts. Lanes
2-5 show the DNA sequence (lanes G, A, T, and C) of the 5`-flanking region using the
+21 to +42 oligonucleotide as a sequencing primer. In lane 6, bands are seen at 172, 159, and 147 (bracket); at 113,
111, and 110 (upper triple arrow); at 93, 92, and 90 (lower triple arrow); and at 74 (single arrow). These
map respectively to positions -130, -117, -105;
-71, -69, -68; -51, -50, and -48,
and -32 within the Factor VII 5`-flanking sequence. Lane 7 is a control in which the probe is hybridized with tRNA. B, RNase protection assay of the human Factor VII gene
proximal promoter region. A 550-bp fragment, spanning the sequence from
-508 5` of the translation start site to +42 of exon 1 was
used as probe. RNA-RNA hybrids were digested with RNase T1 alone or
with RNase T1 + RNase A. Lane 3 shows RNA markers of 100,
200, and 300 bp in length. Lanes 1 and 2 show a
positive control
-actin probe (249 bp, arrow on left) digested
with RNase T1 alone (lane 2) or RNase T1 + and RNase A (lane 1). Lanes 4 and 5 show RNase
protection of the Factor VII probe after digestion with RNase T1 alone (lane 4, 95 bp band, upper arrow on right)
or with RNase T1 + and RNase A (lane 5, 93 and 92 bp
bands, lower arrows on right). Lane 6 shows
the Factor VII probe incubated with tRNA and digested with RNase
T1.
RNase protection was performed using total human liver RNA
hybridized to a riboprobe (Fig. 3B). Results were
obtained using digestion with RNase T1, which cleaves 3` to guanine
residues, and with RNase T1 + RNase A, which together cleave 3` to
guanine and pyrimidine residues. The product incubated with RNase T1
alone revealed a prominent band of 95 bp, which maps to a transcription
start point at -53 (lane 4, upper arrow on right). This was two to three base pairs longer than the most
prominent RNase T1 + RNase A digested products, which yielded two
adjacent bands corresponding approximately to positions -51 and
-50 (lane 5, lower arrows on right).
The positive control, a 249-bp -actin riboprobe, was seen at the
appropriate size (arrow on left) with the RNase
T1-digested product (lane 2) but was again several base pairs
longer than the RNase T1 + RNase A product (lane 1). No
bands were seen in the negative control lane (lane 6) using
tRNA as a source for hybridization with the Factor VII probe.
Taken together, the primer extension and RNase protection data suggest a major start site at approximately -51. The primer extension data also suggest the presence of start sites between -80 and -30, although these were not confirmed by RNase protection.
Optimization
experiments were first performed to determine the quantity of DNA and
incubation times to use in transient CaPO transfections.
For HepG2 cells, 3 µg of test plasmid DNA (1 pmol) in 12-h
incubations were used; samples were taken 48 h later. Identical
conditions were used for transfections of HeLa cells; however,
transfections were performed with 15 µg of test plasmid DNA (5
pmol). To correct for variations in transfection efficiency in both
HepG2 and HeLa cell experiments, a luciferase internal control plasmid
(2.5 µg) was cotransfected along with the growth hormone reporter
gene constructs. All results were normalized in relation to luciferase
expression (Fig. 4A). Results in HepG2 cells showed
that the first 75 bp alone were insufficient to increase reporter gene
activity above base line. However, the addition of the adjacent 5` 34
bp in a 109-bp construct dramatically increased promoter activity to
approximately 20-fold above base line. Promoter activity increased
slightly upon including the next 53 bp (162-bp construct) and again
with adding the next 24 bp (186-bp construct). The 186-bp construct,
-185 to +1, conferred maximal activity. A statistically
significant decrease (p < 0.01) to 59% of the 186-bp
construct was seen after including the adjacent 105 bp. Levels
increased again at 501 bp and rose to a maximum at 1.746 kb. However,
the increase above the promoter activity seen with the 186-bp construct
was not significant. A decrease was seen between 1.7 and 3.9 kb.
Figure 4:
Functional analysis of the human Factor
VII promoter by transient transfections with reporter gene constructs
in HepG2 and HeLa cells. A, relative promoter activities of
deletion constructs are shown as percentages of the -185/+1
portion of the Factor VII 5`-flanking DNA sequence. A luciferase
internal control plasmid was used in all experiments to correct for
variations in transfection efficiency. The results represent an average
± S.D. of 5-16 transfections from six independent
experiments in HepG2 cells and three to six transfections from two
independent experiments in HeLa cells. For purposes of comparing
promoter activity in HepG2 and HeLa cells, the pOGH negative control in
HeLa cells was normalized to the HepG2 negative control. The standard
deviations in HeLa cell experiments ranged between 3 and 30% for all
constructs; error bars cannot be seen with HeLa transfections
in this graph due to the low values. B, reporter gene
constructs (109-bp constructs) containing mutations at -100 and
-94 in the G-rich region of the Sp1 motif (CCCCTCCCCC mutated to
CCACCTCCAC) were made as described under ``Experimental
Procedures.'' The normalized promoter activity of a wild-type
construct and three mutant constructs (109 bp MT94/100: C to A changes
at -94 and -100; 109 bp MT94: C to A change at -94
only; and 109 bp MT100: C to A change at -100 only) are shown as
a percentage of the 186-bp wild-type construct. The data represent the
mean ± S.D. based on two independent transfection experiments in
HepG2 cells performed in triplicate. Mutation of the ACTTTG HNF-4
binding motif (186 bp ACTTTG) between bases -63 and
-58 to GACAAT was generated within the 186-bp construct. The data
represent the mean ± S.D. based on four transient transfections
of HepG2 cells.
HeLa cells were used to compare promoter activity to that seen in HepG2 cells in order to study differences between a cell with hepatocyte-like characteristics and a nonhepatocyte derived cell. The 109-bp construct had maximal promoter activity but showed levels only 3 times the base-line value. For the remaining constructs, including 162 bp or more, activity was at or below base line. These results indicate that the Factor VII promoter is more active in HepG2 cells than in HeLa cells.
The promoter activity of two different polymorphic alleles was compared in HepG2 cells; one construct contained the FVII sequence from an individual carrying a decanucleotide insert at -323 (bases -324 to -333); the other did not contain this insert(23) . The construct containing the decanucleotide insert had a promoter activity that was only 67% that of the more common allele lacking the insert when assayed in four independent transfection experiments with duplicate preparations of each of the two constructs. This difference reached a statistical significance of p < 0.005 using the Student's t test to compare the results (67% ± 21% for the construct containing the decanucleotide insert and 100% ± 23% for the construct lacking the insert (n = 28)).
Figure 5: DNase I footprint analysis of the proximal 309 base pairs of the human Factor VII promoter reveals three protected regions at -51 to -32, -108 to -84, and -233 to -215, with both HepG2 and HeLa nuclear extracts. Lanes 1 and 2, Maxam and Gilbert sequencing ladder of the Factor VII promoter. Lane 3, DNA digested with 0.1 unit of DNase I in the absence of nuclear extracts. Lanes 4 and 5, DNA cleaved with 2.5 units of DNase I after incubating with 50 µg of HepG2 nuclear extract (lane 4) or 50 µg of HeLa nuclear extract (lane 5). The left and right panels show the same reactions. The right panel, electrophoresed for a longer period, more clearly demonstrates regions B and C. The nucleotide sequences of the coding strands of the three protected regions are shown below the photograph. A marks the sequence between -51 and -32; B marks the sequence between -108 and -84, and C marks the sequence between -233 and approximately -215.
Gel mobility shift assays confirmed the presence of proteins, which bound specifically to regions A, B, and C. Fig. 6A shows the gel mobility shift assay using an oligonucleotide spanning the sequence between -51 and -29 bp. In HepG2 and HeLa nuclear extracts, two faint bands are seen (lanes 1 and 3) in addition to a faster migrating complex. A slower migrating band is also seen (top arrow) more prominently in HeLa than in HepG2 nuclear extracts. The ability of cold competitor to eliminate the binding of the radiolabeled oligonucleotide indicates the specificity of this sequence for the nuclear proteins.
Figure 6: Gel mobility shift assays confirm binding of nuclear factors to the protected regions (A, -51 to -32; B, -108 to -84; and C, -233 to -215) of the human Factor VII promoter as determined by DNase I footprinting. Areas protected in the DNase I footprint analysis (Fig. 5) were analyzed for their ability to bind specifically to nuclear extracts in three different cell types: HepG2 cells, HeLa cells, and human liver cells. Increasing concentrations of unlabeled self competitors are used to differentiate specific from nonspecific binding. A, an oligonucleotide probe containing the sequence between -51 and -29 shows formation of four complexes (indicated by arrows) with nuclear extracts from HeLa cells (lanes 1 and 2) and HepG2 cells (lanes 3-6). Lane 2 contains 100-fold of cold self competitor. Lanes 4-6 contains self competitors at 10-fold, 100-fold, and 500-fold excess, respectively. B, an oligonucleotide spanning the sequence from -108 to -83 was incubated with nuclear extracts from HepG2 (lanes 1-4), HeLa (lanes 5-8), and human liver (lanes 9-12). Specific DNA-protein complexes are indicated by arrows. Indicated amounts of molar excess of cold self competitors were added. C, an oligonucleotide spanning the sequence from -236 to -201 was incubated with nuclear extracts from HepG2 cells (lanes 1-4), HeLa cells (lanes 5-8), and human liver (lanes 9-12). Two specific complexes are indicated by thick arrows. A faster migrating band as indicated by a thin arrow is not consistently seen and is therefore considered nonspecific. Molar excess of cold self competitors were added as indicated. Nucleotide sequence of the probe is shown at the bottom.
Fig. 6, B and C, shows gel mobility shift assays using HepG2 nuclear extracts (lanes 1-4), HeLa nuclear extracts (lanes 5-8), and human liver nuclear extracts (lanes 9-12) with probes designed to study regions B and C as defined by DNase I footprint analysis (Fig. 5). Two slowly migrating complexes are seen using an oligonucleotide spanning the sequence -108 to -83 (Fig. 6B). Both complexes are seen more prominently using the HeLa nuclear extracts (lanes 5-8) than using either HepG2 (lanes 1-4) or liver nuclear extracts (lanes 9-12), suggesting a higher abundance of proteins in these complexes in HeLa cells. Both also disappear in the presence of cold competitor, suggesting that binding is specific for this DNA sequence. A fast migrating complex is also present; this complex disappeared only in the presence of the highest concentrations of competitor (for HeLa and human liver nuclear extracts). In Fig. 6C, using a probe that extended from -236 to -201, two closely spaced complexes are seen that are reliably competed away by excess self competitor (lanes 2-4, 6-8, and 10-12). A third faster migrating band was variably present and not thought to represent specific binding to this sequence.
Figure 7: A, electrophoretic mobility shift assays of wild-type and mutant oligonucleotides reveal binding of Sp1 and another protein to the Sp1 motif, CCCCTCCCCC. The -108 to -84 bp region was incubated with HeLa nuclear extracts in lanes 1-3 (wild-type) and lanes 5-7 (mutants). The sequences of the oligonucleotides are shown at the bottom. Instead of nuclear extracts, recombinant Sp1 was added in lanes 4 and 8. The wild-type oligonucleotide formed two DNA-protein complexes in HeLa cells as shown in lane 1. Competition with 500-fold excess unlabeled wild-type oligonucleotide eliminates binding of the radiolabeled oligonucleotide to both proteins demonstrating that the binding is specific (lane 2). Competition with 500-fold cold consensus Sp1 oligonucleotide (lane 5) eliminates binding of the slower migrating complex. Incubation with 1 µl Sp1 antibody (lane 3) results in loss of this complex and appearance of a ``supershifted'' complex. Mutation of the cytosine residues at -100 and -94 to adenosines eliminates binding of both slowly migrating complexes from HeLa nuclear extracts (lane 6). Binding of purified Sp1 is seen with the wild-type oligonucleotide sequence (lane 4) but not with the mutated sequence (lane 8). B, mutation of cytosine to adenosine at position -94 but not at position -100 eliminates binding of Sp1 and the protein in complex B. Wild-type (lane 1) and mutant (lanes 2-4) oligonucleotides spanning -108 to -83 were incubated with HeLa nuclear extracts. Sequences of the probes are shown at the bottom. The specific DNA-protein complexes are indicated by arrows.
Two mutations were installed in the G-rich (lower strand) region between -103 and -92 base pairs at residues -100 and -94 by changing cytosine residues to adenosines. An oligonucleotide probe containing this -94/-100 mutation (MT94/100) fails to bind either the slower migrating protein or the protein in complex B (Fig. 7A, lane 6) and no longer binds to purified Sp1 protein (Fig. 7, lane 8). Independent mutations of the -94 and -100 bp sites revealed that the C to A change at -100 noticeably reduced binding on gel shift assay (Fig. 7B, lane 3) whereas mutation of the -94 bp site alone has a more profound effect, completely eliminating binding of both Sp1 and complex B on gel shift assays (Fig. 7B, lane 2). This result, the loss of two distinct complexes on gel shift assay with the mutation of a single nucleotide, suggests the existence of overlapping binding sites for Sp1 and another nuclear protein.
Figure 8:
A, gel mobility shift assays reveal
binding of a protein from liver and HepG2 nuclear extracts but not HeLa
nuclear extracts to an oligonucleotide sequence containing the ACTTTG
motif (bases -63 to -58) in the proximal Factor VII
promoter. A probe spanning the sequence from -76 to -47
forms a complex (arrow at left) with a protein
present in HepG2 nuclear extracts (lanes 1-3) and human
liver nuclear extracts (lanes 4-6). Addition of excess
unlabeled self competitor results in a gradual disappearance of the
complex at 10 (lanes 2 and 5) and 100
(lanes 3 and 6) concentrations. HeLa nuclear extracts
do not show binding of a protein in this region (lanes 7 and 8). Mutation of the ACTTTG to GACAAT between -63 and
-58, within the -76 to -47 sequence, eliminates
binding of human liver nuclear extracts to the mutated radiolabeled
probe (lane 9). B, anti-HNF-4 antibodies supershift
the binding of human liver nuclear extracts to a probe containing the
sequence between -76 to -47 bp. Lane 1 shows the
formation of a complex (left arrow) by the -76 to
-47 radiolabeled probe with human liver nuclear extract. Lane
2 shows the binding of the same components with the addition of 1
µl of antiserum raised against the carboxyl terminus of HNF-4. The
position of a slower moving immune complex is indicated (right
arrow).
To study the
functional importance of this ACTTTG element, a 6-bp mutation (
ACTTTG: ACTTTG
GACAAT) was installed in the 186-bp pOGH
construct and used in transfection experiments with HepG2 cells and in
transactivation studies with HeLa cells. This 6-bp mutation (
ACTTTG) was previously shown to dramatically reduce activity in the
Factor X promoter(7) . In HepG2 cells, mutation of the putative
HNF-4 binding site reduced promoter activity to 2% of the 186-bp
wild-type (Fig. 4B). Transactivation experiments were
performed to assess whether the presence of HNF-4 in trans could
increase expression of the human growth hormone reporter gene above
base-line levels. A 4-fold increase in promoter activity of the 186-bp
wild-type construct was seen when cotransfected with 10 µg of
pLEN4S, an expression plasmid containing the HNF-4 cDNA. This was not
seen with cotransfection of an equimolar amount of the pLEN expression
plasmid, lacking the HNF-4 cDNA insert (wild-type + pLEN4S: 392%
± 33.9% versus wild-type + pLEN: 100% ±
9.6%). When this experiment was repeated using a 186-bp reporter gene
construct containing a mutation at the putative HNF-4 binding site
(p186
ACTTTG), no transactivation occurred (p186
ACTTTG +
pLEN4S: 137% ± 15.9% versus p186
ACTTTG +
pLEN: 136% ± 20.9%). The levels of promoter activity were
comparable with levels of the wild-type construct cotransfected with
pLEN. These results suggest that the binding of HNF-4 to the ACTTTG
element in Factor VII is critical for promoter activity.
A longstanding assumption in the field of coagulation has
been that the vitamin K-dependent coagulation proteins are synthesized
exclusively in the liver. Recent data indicate, however, that this
assumption is not true, at least at the mRNA level, where several
groups have documented the presence of transcripts in non-liver tissues
of protein S(45) , protein C(46) ,
prothrombin(47) , and Factor X. ()For human
coagulation Factor VII in contrast, within the sensitivity of the
assay, Northern analysis using poly(A)
RNA from a wide
variety of tissues demonstrates that expression is confined to the
liver (Fig. 1). Thus, at least for Factor VII, the older notion
in fact appears true.
Factor VII, along with Factor X, is located on the long arm of chromosome 13 (13q34) (48) only 2.8 kb 5` of the Factor X gene(8) . Densitometric analysis of autoradiograms demonstrated that steady state levels of Factor VII mRNA were only 6% those of Factor X mRNA levels, correlating well with the 10-20-fold difference in plasma levels. Thus, the difference in plasma concentrations of the two factors is due to a difference in rates of transcription or in RNA stability (or some combination of the two).
A qualitative comparison of our Factor VII promoter data with those previously published for Factor X (7, 8) shows similarities in that both promoters are TATA-less with the essential regulatory elements located within the first several hundred bp 5` to the translation start site. An HNF-4 binding site, including the same ACTTTG motif seen in the promoters of Factors IX (9) and X(7, 8) , is shown to be critical in the Factor VII promoter. Although a CAAT box, a conserved element present in approximately 30% of eukaryotic promoters(49) , is critical in the promoters of both factor IX (9) and Factor X(7) , no such element is apparent in the Factor VII promoter.
Identification of the transcription start site is an essential step
in the characterization of a promoter. Like the promoters of many other
clotting factor genes, the Factor VII promoter lacks a TATA box, a
sequence present in approximately 80% of RNA polymerase II eukaryotic
promoters(49) , which is usually located about 30 bp upstream
from the transcription start site. Many TATA-less promoters are thought
to depend on the presence of the transcription factor Sp1 upstream from
the start site(50, 51) . TATA-less promoters are also
frequently characterized by the presence of an initiator element
encompassing the transcription start site(52) . In this report,
a major transcription start site is identified at -51 by both
primer extension and RNase protection. This start site occurs in the
context TCAGTCCC, a site that is in 100% agreement
with both a mathematically calculated consensus sequence CANYYY (49) based on the cap signal of 502 eukaryotic promoters, and
an experimentally defined consensus PyPyA
NT/APyPy
determined using synthetically prepared promoter
sequences(53) . An Sp1 binding site, which we show to be
crucial in promoter activity of Factor VII, is located approximately 41
base pairs upstream of the identified start site. This position
corresponds well with the observed optimal distance between a GC-rich
Sp1 binding element and the transcription start
site(52, 54) . It should also be noted that, as is the
case for most other TATA-less promoters, multiple other start sites are
identified in the region surrounding the major transcription start
site.
Functional characterization of the human Factor VII promoter was performed in both HeLa cells, a non-hepatocyte-derived cell line, and HepG2 cells, a hepatoma-derived cell line previously shown to secrete Factor VII(55) . Analysis of promoter activity using varying lengths of 5`-flanking region adjacent to the translation start site revealed a dramatic increase in promoter activity from base line to maximal activity upon the addition of a G-rich (lower strand) region, which includes an Sp1 binding site. The importance of this binding site was demonstrated by a dramatically reduced promoter activity (2% of wild-type) in a construct containing a single base change (C to A), which eliminated binding of Sp1 by gel mobility shift assay. However, the possibility cannot be excluded that a second protein, with an overlapping binding site to that of Sp1, plays an equally important role.
In reporter gene assays, a reproducible and
statistically significant decrease of 41% was seen between constructs
containing 186 and 291 base pairs of Factor VII promoter sequence. This
region includes a site at -233 to -215, which was protected
on DNase I footprint analysis and bound nuclear proteins on gel
mobility shift assays. Interestingly, part of the protected sequence
(5`-PuGGTCANNTGACCPy-3`) bears great resemblance to the sequence of
several hormone response elements(56) . Experimental studies
testing the influence of spacing, orientation, and the exact sequence
of the motif and flanking sequence have documented the importance of
all these factors in determining receptor binding to a particular
combination of half-sites(26) . The sequence, which contains
two PuGGTCA half-sites in an inverted orientation separated by 2 bp,
does not exactly match any documented hormone responsive
elements. It should be noted that putative binding sites
for several other factors were also seen in this region.
Experiments revealed that an intact binding site for HNF-4, a known member of the steroid hormone receptor superfamily(35) , is critical for Factor VII promoter activity in vitro. First, a protein present in both human liver and HepG2 nuclear extracts but not in HeLa cells could be supershifted with the addition of HNF-4 antiserum. Mutation of the sequence ACTTTG (-63 to -58), which is well documented to bind HNF-4 in the Factor IX promoter(9) , eliminated binding in gel mobility shift assays. Furthermore, an HNF-4 expression plasmid was able to increase promoter activity 4-fold when cotransfected with a 186-bp Factor VII promoter construct in HeLa cells. Mutation of the ACTTTG not only dramatically reduced promoter activity in HepG2 cells (2% of wild-type) but also resulted in the loss of ability of the HNF-4 expression plasmid to transactivate the Factor VII construct in HeLa cells. These experiments demonstrate that the HNF-4 binding site is critically required for Factor VII promoter activity. Given these findings, it is perhaps surprising that protection of the HNF-4 binding site is not seen on footprint analysis, but in this case this appears to result from the resistance of this region of DNA to digestion by DNase I, since no ladder is seen in the ``no protein'' lane (Fig. 5, lane 3).
A recent report documented a reduced level of Factor VII antigen in individuals heterozygous for a decanucleotide insert at position -323 in the Factor VII promoter (81% antigen level in individuals heterozygous for the insert, 112% antigen level in individuals without the insert, p < 0.005)(24) . We investigated whether the presence of this decanucleotide in reporter gene constructs could influence promoter activity in HepG2 cells. Our findings showed that a 33% reduction in activity was associated with the naturally occurring allelic sequence containing the insert. This provides additional evidence that the presence of a decanucleotide insert may be directly related to the lower Factor VII levels seen in the population tested.
A schematic model of the promoter region of Factor VII based
on data presented in this report is shown in Fig. 9. Four cis
elements were shown to bind nuclear proteins. These elements are
located at -51 to -32, -63 to -58, -108
to -84, and -233 to -215. Binding sites for the
liver-enriched transcription factor, HNF-4, and the ubiquitous factor,
Sp1 were identified at -63 -58, and -101 to
-94, respectively. A major transcription start site was
identified at approximately -51, located between two protein
binding regions. This start site was favorably located approximately 40
bp downstream of a functionally important Sp1 binding site. A sequence
with homology to a hormone responsive element was seen at -227 to
-213 within a region protected by DNase I footprint analysis.
Additionally, transfection studies of the FVII promoter comparing
polymorphic alleles, one of which included a decanucleotide insert at
-323, suggested that this region may also influence promoter
activity.
Figure 9: Schematic diagram of regulatory sequences in the human coagulation Factor VII gene 5`-flanking region.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40852[GenBank].