(Received for publication, May 31, 1995; and in revised form, July 20, 1995)
From the
Protein C is a vitamin K-dependent zymogen of a serine protease
that inhibits blood coagulation by the proteolytic inactivation of
factors Va and VIIIa. Individuals affected with protein C deficiency
are at risk for thrombosis. Genetic analyses of affected individuals,
to determine the cause of the protein C deficiency, revealed a large
variety of mutations in the protein C gene, including several in the
promoter region of this gene. Comparison of the region around two of
these mutations, A
G and T
A, with transcription factor consensus sequences suggested
the presence of two overlapping and inversely oriented HNF-3 binding
sites. Direct evidence for the presence of the two HNF-3 binding sites
in the protein C promoter was obtained using electrophoretic mobility
shift assays and UV cross-linking experiments. These experiments
revealed that HNF-3 can bind specifically to both putative HNF-3 sites
in the wild-type protein C promoter. Due to the T
A mutation, one binding site is completely lost, while the
other site still binds HNF-3, but with strongly reduced affinity. As a
consequence of the A
G mutation, the protein
C promoter loses all its HNF-3 binding capacity. Transient transfection
experiments demonstrated that the binding of HNF-3 to the protein C
promoter is of physiological significance. This followed from
experiments in which the introduction of the A
G or T
A mutation resulted in a
4-5-fold reduced promoter activity in HepG2 cells. Furthermore,
transactivation of the wild-type protein C promoter construct with
HNF-3 showed a 4-5-fold increased promoter activity in HepG2
cells. In HeLa cells, significant wild-type promoter activity was only
observed after transactivation with HNF-3. When a promoter construct
containing the T
A mutation at position -27 was used, the
transactivation potential of HNF-3 was 2-fold reduced in HepG2 cells,
whereas in HeLa cells no transactivation was observed. With the
promoter construct containing the A
G
mutation, no transactivation by HNF-3 was found either in HepG2 or in
HeLa cells.
Protein C, which is synthesized in the liver as a vitamin K-dependent zymogen of a serine protease, plays an important role in the regulation of the hemostatic system. After activation by the thrombin-thrombomodulin complex, activated protein C inhibits blood coagulation in the presence of protein S(1) , phospholipids, and calcium ions through the proteolytic inactivation of factors Va and VIIIa(2, 3) . Furthermore, activated protein C stimulates fibrinolysis through the neutralization of plasminogen activator inhibitor-1(4) .
The physiological significance of the protein C anticoagulant activity is clearly shown in individuals homozygous or compound heterozygous for protein C deficiency. These individuals suffer from massive disseminated intravascular coagulation or neonatal purpura fulminans (5, 6, 7) . Individuals affected by heterozygous protein C deficiency, although more mildly affected, are at risk of thrombophlebitis, deep vein thrombosis, or pulmonary embolism(8, 9) .
The human
protein C gene, located on chromosome 2q13-q14(10) , contains
nine exons spanning 11 kilobase pairs of genomic
DNA(11, 12) . Of these nine exons the first and part
of the second exon (21 bp) ()consist of non protein coding
sequences. Genetic analyses of individuals with hereditary protein C
deficiency revealed a large variety of mutations in the protein C gene,
including several in the promoter region of the gene(13) .
Transcription of eukaryotic genes by RNA polymerase II involves DNA
elements located within the promoter region and transcription factors
that associate with these DNA elements(14) . Certain
transcription factors, such as the TATA box-binding protein (TBP),
specify the transcription initiation site(15) , whereas others
regulate the efficiency of transcription initiation. This latter group
of transcription factors comprises both ubiquitous and tissue-specific
factors. Because tissue-specific regulation in the liver has been
studied extensively, a significant number of liver-specific or
liver-enriched transcription factors have been cloned and
characterized, including HNF-1 (16) and
-1
(17) ; HNF-3
, -3
, and -3
(18) ;
HNF-4(19) ; C/EBP
(20) and -
(21) ;
DBP(22) ; and LAP(23) .
Analyses of promoter
mutations causing inherited disease is very useful in identifying
transcription factors involved in the transcriptional control of a gene
of
interest(24, 25, 26, 27, 28, 29) .
In this study we examined the promoter region of the human protein C
gene around two mutations, A
G (13, 30) and T
A(31) , which are known to cause reduced protein C antigen and
activity levels in the blood (type I protein C deficiency). We show
that the wild-type promoter contains two binding sites for HNF-3
(
,
, or
) and that HNF-3 (
,
, or
)
transactivates the wild-type protein C promoter in both cells of
hepatic (HepG2) and epithelial (HeLa) origin in transient transfection
assays. Furthermore, we show that both mutations influence the binding
of HNF-3 to the protein C promoter and significantly reduce
(T
A) or completely abolish (A
G) transactivation of this promoter by HNF-3.
The following nucleotides for human transthyretin gene were used.
Nucleotide changes compared to the protein C and transthyretin wild-type sequence, respectively, are indicated in boldface type. In the TTR-2x oligonucleotide, we changed seven nucleotides compared to the TTR-1x oligonucleotide to introduce a second HNF-3 consensus sequence(36) .
Antisera produced in
rabbits against HNF-3 and -3
(a gift from R.H. Costa) were
used for ``supershift'' assays, while antiserum against
HNF-3
(a gift from E. Lai) was used for competition assays. As a
negative control we used antiserum against NF-1 (a gift from P. J.
Rosenfeld). In each case, 1 µl of 5-fold diluted antiserum was
added to the reaction mixture prior to the 25-min incubation at room
temperature.
TCCCGGTTCGTUTATAAACACCAATACCT
TCCCGGTTCGTTTATAAACACCAATACCT
TCCCGGTTCGTUTATAAACACCAATACCT
TCCCGGTTCGTTTATAAACACCAATACCT
Indicated in boldface type are 5`-bromouracil nucleotides, which are incorporated instead of thymidine nucleotides present in the wild-type protein C promoter region at position -27 (sense strand) or position -32 (antisense strand).
To investigate whether the T
A and
A
G mutations influenced the protein C
promoter activity, we analyzed the transcriptional activity of the
wild-type and mutated promoters in transient transfection assays.
Therefore we made constructs containing 493-bp protein C promoter
fragments cloned 5` to the CAT reporter gene. This protein C fragment
contains three polymorphic sites, which are associated with variation
in plasma protein C levels(37) . Sequencing of both the
pC-27CAT and pC-32CAT constructs revealed that the pC-27CAT construct
contains the haplotype associated with low protein C plasma levels
(CGT), whereas the pC-32CAT construct contains the haplotype associated
with high protein C plasma levels (TAA). However, the association is
not the consequence of differences in promoter activity. This followed
from three independent transient transfection experiments with two
different DNA isolations in which no difference in promoter activity
was observed between the two haplotypes in the wild-type context (data
not shown). Therefore we used the promoter constructs irrespective of
the haplotype. After introduction into HepG2 cells, the pC-27CAT
construct had an approximately 4-fold reduced promoter activity
compared to the wild-type promoter (Table 1). The use of pC-32CAT
reduced the promoter activity to about 20% of wild-type activity. When
we repeated the transfection experiments with HeLa cells, no CAT
activity could be detected with any of the three constructs.
To identify the transcription factor(s) binding to the promoter sequence around the -32/-27 region, we compared this sequence to a number of transcription factor consensus sequences. This suggested that the human protein C promoter region contains two putative HNF-3 binding sites, which are overlapping and in reverse orientation (Fig. 1). The first binding site (bs1), located from nucleotide -26 to -37, is 100% identical to the HNF-3 consensus sequence(36) , while the second binding site (bs2), located from nucleotide -33 to -24, showed an identity of 83% with the consensus sequence. The mutations at -32 or -27 each occur at nucleotide positions that are invariant among numerous HNF-3 binding sites. Furthermore, they decrease the identity of the promoter sequence with the HNF-3 consensus sequence to 92% and 67% for bs1 and bs2, respectively.
Figure 1:
Sequence
alignment of the -32/-27 region of the human protein C
promoter region with the HNF-3 consensus sequence
((A/C/G)A(A/T)TRTT(G/T)RYTY)(36) . Homologous nucleotides
between the protein C promoter and the HNF-3 consensus sequences are
indicated. Also indicated are the A
G and
the T
A
mutations.
To obtain evidence for the existence of the two
putative HNF-3 binding sites, EMSAs were performed. As shown in Fig. 2, HNF-3, -3
, and -3
each formed two
distinctive complexes with the pCwt oligonucleotide. In the case of
HNF-3
and -3
, the majority of HNF-3 binding was localized in
a low mobility complex. For HNF-3
, however, the formation of a
high mobility complex seemed slightly favored over the formation of the
low mobility complex. The binding activities were all specific since
both complexes could be effectively competed only by the corresponding
unlabeled oligonucleotides (shown in Fig. 3for HNF-3
).
Figure 2:
EMSAs of in vitro translated
HNF-3, -3
, and -3
binding to the wild-type human protein
C promoter. The labeled pCwt oligonucleotide was incubated with a
rabbit reticulocyte lysate serving as a negative control (lane1) or with in vitro translated HNF-3
(lane2), -3
(lane3), or
-3
(lane4).
Figure 3:
Specific binding of HNF-3 to the
human protein C promoter. EMSAs were done with labeled oligonucleotide
pCwt and in vitro translated HNF-3
in the absence (lane1) or presence of a 10-fold (lanes2 and 5), a 50-fold (lanes3 and 6), or a 100-fold (lanes4 and 7) molar excess of unlabeled oligonucleotide pCwt (lanes
2-4) or the nonspecific pCcomp oligonucleotide (lanes
5-7).
In order to ascertain whether the complex formation is due to HNF-3
binding, we tested specific antisera against HNF-3, -3
, and
-3
. As a consequence of the addition of anti-HNF-3
and
anti-HNF-3
to the reaction mixture, both the low and high mobility
complexes were ``supershifted'' (Fig. 4).
Anti-HNF-3
mainly competed for HNF-3 binding, while some fraction
of the complexes was also ``supershifted.'' The same
experiments with NF-1 antiserum showed no ``supershifted''
complex or competition.
Figure 4:
Specific binding of HNF-3, -3
,
and -3
to the wild-type human protein C promoter. EMSAs were done
with labeled oligonucleotide pCwt, in vitro translated HNF-3
and antisera. A, HNF-3
incubated with anti-HNF-3
antibodies (lane2), anti-NF-1 antibodies (lane3), or without antibodies (lane1). B, HNF-3
incubated with anti-HNF-3
antibodies (lane2), anti-NF-1 antibodies (lane3), or without antibodies (lane1). C, HNF-3
incubated with anti-HNF-3
antibodies (lane2), anti-NF-1 antibodies (lane3), or without antibodies (lane1).
Next, we determined the influence of the
mutations on the binding affinity of HNF-3 to the promoter. This showed
that as a consequence of the introduction of the -27 mutation,
only the high mobility complex could be formed by HNF-3, -3
,
and -3
(Fig. 5A). In addition, for all HNF-3
isoforms the amount of high mobility complex formed did not increase as
a consequence of the loss of formation of the low mobility complex,
indicating a significantly reduced binding affinity. Neither
HNF-3
, -3
, nor -3
formed a complex with the
oligonucleotide containing the -32 mutation (Fig. 5B).
Figure 5:
EMSAs of in vitro translated
HNF-3, -3
, and -3
binding to the mutated human protein C
promoter. The labeled oligonucleotides pCmt-27 (A) and pCmt-32 (B) were incubated with a rabbit reticulocyte lysate serving
as a negative control (lane1) or with in vitro translated HNF-3
(lane2), -3
(lane3), or -3
(lane4).
To investigate whether the two complexes formed by HNF-3 with the pCwt oligonucleotide are the consequence of the binding stoichiometries of one and two HNF-3 protein monomers, respectively, in the high and low mobility bands, we performed EMSAs with HNF-3 and the TTR-1x oligonucleotide. The TTR-1x oligonucleotide contains the HNF-3 consensus sequence from the transthyretin promoter (38) and binds one HNF-3 monomer(39) . As shown in Fig. 6, only the high mobility complex was formed, indicating that the high mobility complex is formed due to binding of one HNF-3 protein. We next modified the TTR-1x oligonucleotide to create an oligonucleotide containing two HNF-3 consensus sequences in the same context as in the protein C promoter (TTR-2x). EMSAs with this TTR-2x oligonucleotide showed the appearance of high and low mobility complexes, just as observed with the pCwt oligonucleotide (Fig. 6).
Figure 6:
EMSAs of in vitro translated
HNF-3, -3
, and -3
binding to the human transthyretin
promoter or the human protein C promoter. The labeled oligonucleotides
TTR-1x (A), TTR-2x (B), and pCwt (C) were
incubated with a rabbit reticulocyte lysate serving as a negative
control (lane1) or with in vitro translated
HNF-3
(lane2), -3
(lane3), or -3
(lane4).
To explore further the nature of the low and high
mobility complexes, we performed UV cross-linking experiments. In these
experiments we used a GST-HNF-3 fusion protein and
oligonucleotides containing 5`-bromouracil nucleotides in both strands
(pCBrU2x), in one strand only (pCBrU1x-27 or pCBrU1x-32), or in neither
of the two strands (pCBrU0x). The 5`-bromouracil was incorporated in
place of the thymidines at the locations of the two mutations
associated with protein C deficiency, i.e. at position
-27 in the sense strand and/or position -32 in the
antisense strand, on the assumption that these thymidines play
important roles in HNF-3 binding. After UV irradiation the cross-linked
complexes were separated on a SDS-PAGE gel. As shown in Fig. 7(lane1), the oligonucleotide containing
two 5`-bromouracil nucleotides was cross-linked into complexes of
approximately 55 and 110 kDa. When only a single modified nucleotide
was present in either strand, only a 55-kDa complex was formed (lanes 2-4), whereas no complex was formed when the
oligonucleotide did not contain 5`-bromouracil nucleotides. Boiling of
the samples prior to SDS-PAGE resulted in diminishment of the 110-kDa
complex, whereas the intensity of the 55-kDa band was not affected (lanes 5-8).
Figure 7:
Molecular mass analysis of the complexes
formed between GST-HNF-3 and the wild-type protein C promoter.
Labeled oligonucleotides pCBrU2x (lanes1 and 5), pCBrU1x-32 (lanes2 and 6),
pCBrU1x-27 (lanes3 and 7), and pCBrU0x (lanes4 and 8) were incubated with
affinity-purified GST-HNF-3
fusion protein. The complexes formed
after UV cross-linking for 5 min were either directly loaded (lanes
1-4) or boiled for 5 min and then loaded (lanes
5-8) onto a 10% reducing SDS-PAGE gel. The complexes of
interest are indicated by arrows.
Finally, we investigated whether HNF-3
was able to transactivate the protein C promoter. HepG2 and HeLa cells
were cotransfected with the protein C reporter constructs pCwtCAT,
pC-27CAT, or pC-32CAT and expression vectors for HNF-3, -3
,
or -3
. As shown in Fig. 8A, cotransfection of
HepG2 cells with HNF-3
and -3
increased transcription from
the wild-type protein C promoter about 4-fold, whereas HNF-3
transactivated the promoter activity about 5-fold. In contrast when the
pC-27CAT construct was used, the transactivation potential of
HNF-3
, -3
, and -3
was reduced to about a factor of 1.5,
3, and 1.3, respectively (Fig. 8A). Essentially no
transactivation with HNF-3
and -3
was observed using the
pC-32CAT construct, while HNF-3
transactivated this construct
approximately 2-fold. In Fig. 8B it can be seen that
HNF-3
, -3
, and -3
all transactivated the wild-type
protein C promoter in HeLa cells. As shown in HepG2 cells, HNF-3
seems to be the most potent transactivater. Finally, in HeLa cells,
none of the HNF-3 family members could transactivate the promoter
constructs containing one of the mutations (Fig. 8B).
Figure 8:
Expression of protein C reporter
constructs in HepG2 (A) and HeLa (B) cells. Plasmid
constructs with the CAT gene under control of the wild-type (pCwtCAT),
-27 mutated (pC-27CAT), or -32 mutated (pC-32CAT) protein C
promoter were tested for CAT activity in the absence or presence of
HNF-3, -3
, and -3
. Note the difference in scale between panelsA and B.
The T
A and the A
G mutations in the protein C promoter region are both
associated with type I protein C
deficiency(13, 30, 31) . The aim of this
study was to determine the cause of this association. We show that both
mutations significantly reduce protein C promoter activity in hepatic
HepG2 cells, whereas in non-hepatic HeLa cells no constitutive promoter
activity is observed. This tissue-specific promoter activity indicates
the necessity of liver-enriched (or liver-specific) transcription
factors in the expression of the protein C gene.
In the protein C
promoter region, two putative HNF-3 binding sites were identified by
comparing the sequence of the promoter region around the -27 and
-32 mutations with transcription factor consensus sequences. Both
binding sites were largely overlapping and reversely orientated. The
presence of the T
A mutation, which
decreases the identity of the promoter sequence with both HNF-3
consensus sequences, abolishes one binding site, whereas the other
binding site is still capable of binding HNF-3, although with clearly
reduced affinity. Due to the presence of the A
G mutation, which also decreases the identity between
promoter region and HNF-3 consensus sequence, no HNF-3 binding was
observed. Therefore, we conclude that the adenosine at position
-32 in the protein C promoter is essential for HNF-3 binding. In vitro, with relative large amounts of HNF-3 present the
thymidine at position -27 seems very important but not essential
for HNF-3 binding.
Evidence for the simultaneous binding of two HNF-3 monomers to the wild-type protein C promoter was obtained from UV cross-linking experiments. In these experiments the naturally occurring nucleotides at position -32 and -27 were replaced by 5`-bromouracil nucleotides, which dramatically enhance the irreversible cross-linking of proteins to DNA after UV irradiation(40) . After separation on a SDS-PAGE gel, we observed complexes of approximately 110 kDa, corresponding to the oligonucleotide with two HNF-3 monomers and complexes of 55 kDa, corresponding to the oligonucleotide with one HNF-3 monomer. Boiling of the samples prior to loading resulted in the disappearance of the 110-kDa complex, whereas the 55-kDa complex was not affected. We interpret the loss of the 110-kDa complex as due to denaturing of the double-stranded oligonucleotides with as a consequence the formation of two complexes of approximately 55 kDa, consisting of one HNF-3 monomer linked to a single-stranded oligonucleotide.
With the use of x-ray crystallography, HNF-3 has been found to interact with the transthyretin promoter as a monomer over a linear distance of about 40 Å along the axis of the double helix(39) . As stated above, the wild-type protein C promoter binds two HNF-3 monomers at largely overlapping binding sites. Since these binding sites are separated 5 bp (or about half a turn of the double helix), we hypothesize that the HNF-3 monomers bind at opposite faces of the DNA helix (Fig. 9).
Figure 9:
Schematic representation of transcription
factors binding to the wild-type human protein C promoter. Indicated is
the DNA double helix in 10-bp turns, with the associated transcription
factors HNF-1 and HNF-3. Both the T
A and
A
G mutations are indicated on both strands
by asterisks (*), whereas the transcription start site is
indicated by an arrow.
The binding of two or more HNF-3 monomers close to each other has been reported previously(41, 42, 43) . Furthermore, it has been shown that HNF-3 transcription factor binding sites often overlap with other transcription factor binding sites(41, 43, 44, 45) . However, this report is novel in demonstrating the binding of two HNF-3 monomers to two largely overlapping HNF-3 binding sites. The functional role of this motif of overlapping binding sites remains unknown.
Both the
T
A and A
G
mutations influence the binding of HNF-3 to the protein C promoter, and
the transactivation capacity of the promoter is also influenced. As
shown by transfection assays in HepG2 cells, the -27 mutated
promoter is transactivated to a lesser extent by HNF-3 than the
wild-type promoter. This indicates that the residual binding of HNF-3
to the -27 mutated promoter is still sufficient to transactivate
the protein C promoter. Transfection experiments in HeLa cells show
that only the wild-type promoter is transactivated by HNF-3. The
observed stimulation of protein C transcription by exogenous HNF-3 in
HepG2 cells, despite the presence of HNF-3 proteins in these cells,
could be attributed to the limiting amounts of HNF-3 in these cultured
cells(46, 47) .
Another liver-enriched transcription factor known to be involved in protein C gene expression is HNF-1(48) . This transcription factor is, according to Berg et al., necessary but not sufficient for protein C gene expression. However, our observations in HeLa cells, which do not express endogenous HNF-1, argue against the necessity of HNF-1. Importantly, HNF-1 is expressed in HepG2 cells as opposed to HeLa cells. This difference in HNF-1 expression might explain the different response of the protein C promoter containing the -27 mutation to HNF-3 in HepG2 and HeLa cells. In HepG2 cells complex formation between HNF-1 and HNF-3 can occur with transactivation of the promoter as a consequence. In HeLa cells such complex formation between HNF-1 and HNF-3 cannot occur, and consequently no transactivation takes place. Therefore, we hypothesize that in vivo a functional interplay occurs between HNF-1 and HNF-3 in the transcriptional regulation of the protein C promoter. This hypothesis is supported by the fact that the HNF-1 binding site in the protein C promoter is localized directly downstream of the HNF-3 binding sites. Cooperation between HNF-3 and other factors bound to closely situated DNA binding sites have been reported previously(43, 49, 50) .
HNF-3
consists of a family of liver-enriched transcription factors, called
HNF-3/forkhead. During liver differentiation the members of this family
show a different expression pattern. In fetal cells HNF-3 and
HNF-3
are more abundant than HNF-3
, whereas in adult liver
cells HNF-3
and HNF-3
are most abundant(47) . In
vitro HNF-3
, -3
, and -3
seem to bind with a similar
affinity to the protein C promoter. This excludes the different
expression pattern of the HNF-3 proteins as a possible regulatory
mechanism for protein C gene expression. More likely, the fact that all
HNF-3 proteins bind with the same affinity to the protein C promoter
ensures the presence of protein C independent of the age of the
organism.
Recently, Qian et al.(45) showed that
acute-phase livers exhibit a dramatic reduction of HNF-3 (95%
decrease) and HNF-3
(20% decrease) expression. Furthermore, they
showed that this reduced HNF-3
and -3
expression coincided
with reduced expression of one of their target genes, the transthyretin
gene. In this report we showed that the protein C gene is also a target
gene of HNF-3
and -3
. This indicates that the observed
reduction of protein C activity during septic
shock(51, 52, 53, 54) , severe
infection(53) , and after major surgery (55) might
well be caused by a decrease in protein C transcription.
Sequence comparison of the protein C promoter region around position -27/-32 with transcription factor consensus sequences revealed more than just the presence of putative HNF-3 sites. The sequence also contains a core sequence that resembles the TBP consensus sequence (TATA(A/T)A(A/T))(56) . This transcription factor, which is essential for basal transcription(57) , might therefore bind to the core sequence. However, due to the -27 mutation the identity with the TBP consensus is increased (from 71 to 86%), which argues against the hypothesis that this mutation interferes with TBP binding and thereby would be responsible for the protein C deficiency in the patients.
In conclusion, we have identified the liver-enriched transcription factor HNF-3 as a potential physiologic regulator of protein C gene expression. Furthermore, we propose a functional interplay between HNF-3 and HNF-1 to drive protein C gene expression.