©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two Mutations in the Promoter Region of the Human Protein C Gene Both Cause Type I Protein C Deficiency by Disruption of Two HNF-3 Binding Sites (*)

(Received for publication, May 31, 1995; and in revised form, July 20, 1995)

C. Arnold Spek (1)(§) Judith S. Greengard (2) John H. Griffin (2) Rogier M. Bertina (1) Pieter H. Reitsma (1)

From the  (1)Hemostasis and Thrombosis Research Center, Department of Hematology, University Hospital, 2300 CR Leiden, The Netherlands and (2)The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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-1alpha (16) and -1beta(17) ; HNF-3alpha, -3beta, and -3(18) ; HNF-4(19) ; C/EBPalpha (20) and -beta(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 (alpha, beta, or ) and that HNF-3 (alpha, beta, 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.


MATERIALS AND METHODS

Amplification of Genomic DNA

Fragments of the human protein C promoter region, containing 386 bp of 5`-flanking sequence, the complete first nontranslated exon (53 bp) and 54 bp of the first intron, were amplified from genomic DNA of different individuals. DNA from an individual not deficient in protein C was used to obtain the wild-type PCR fragment. DNA from the proband of the PC-43-010 pedigree (31) was used to obtain a PCR fragment with the T A mutation, while a patient from the PC-1-La Jolla IV pedigree(13, 30) was used to obtain a PCR fragment with the A G mutation. To perform the amplifications we used the following oligonucleotides: 5`-CAGCGTCCCCGGGCTTGTATGGTGGCACATAAATACATGT-3` (-396 to -357; all nucleotide numbering is relative to the transcription start site(12) ) and 5`-CTCTTCTCTTCTCCCGGGGGCAGCCCTCCCTCCACACCCCTCATA-3` (+122 to +78). The underlined nucleotides in the oligonucleotide sequence represent modified nucleotides that are not present in the protein C promoter region. These modified nucleotides introduce a SmaI site in the PCR fragment at position -386. Amplifications were performed in a 50-µl reaction mixture containing 10 mM Tris-HCl, pH 8.0, 1 mM MgCl(2), 50 mM KCl, 350 ng of primers, 100 ng of genomic DNA, 250 µM dNTPs, 60 µg/ml bovine serum albumin, and 0.3 unit of Taq polymerase. After an initial incubation at 91 °C for 4 min, 32 cycles were carried out at 91 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min.

Plasmid Constructions

The pCAT00 vector (25) was used to make reporter constructs containing wild-type and mutated protein C promoter sequences driving chloramphenicol acetyltransferase (CAT) gene expression. PCR-amplified DNA fragments of 518 bp, spanning nucleotides -396 to +122 of the human protein C gene, from different individuals were digested with SmaI. The resulting 493-bp fragments, spanning nucleotides -386 to +107, were cloned into the SmaI site of pCAT00. The reporter construct, containing the wild-type protein C sequence, was named pCwtCAT. The reporter constructs, containing the protein C sequence with the T A or A G mutation, were named pC-27CAT and pC-32CAT, respectively. The integrity of all constructs was verified by sequencing.

Transient Transfection

The differentiated human hepatoma cell line HepG2 (ATCC HB8065) and human epithelial HeLa cells (ATCC CCL2) were cultured in minimal essential medium containing Earle's salts and nonessential amino acids supplemented with 15% heat-inactivated fetal calf serum. Cells were seeded at a density of approximately 1 times 10^5 cells/60-mm tissue culture dish. After 24 h a DNA mixture, containing 6 µg of protein C CAT reporter construct, 2 µg of beta-galactosidase expression vector (pCH110; (32) ), and 1.5 µg of nonspecific plasmid pUC13, was transfected into the cells by the calcium phosphate coprecipitation method(33) . For cotransfection experiments, 0.5 µg of pUC13 was replaced by 0.5 µg of HNF-3 expression vector. Forty-eight hours after transfection, cells were harvested and beta-galactosidase activity was measured(34) . The CAT activity of each construct was determined essentially as described by Seed and Sheen (35) and normalized to beta-galactosidase activity. All transfections were repeated two to four times in duplicate, with at least two different plasmid preparations, and data from representative experiments are shown.

In Vitro Transcription/Translation

HNF-3 was in vitro transcribed and translated using the TnT-coupled reticulocyte lysate system (Promega) according to the manufacturer's protocol, in a final volume of 50 µl. The crude reticulocyte lysate containing translated proteins was used directly in electrophoretic mobility shift assays (EMSAs).

Oligonucleotides Used in EMSAs

The following nucleotides for human protein C gene were used.

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) .

EMSA

EMSAs were performed with 4 µl of in vitro transcribed/translated HNF-3 in a 10-µl reaction mixture containing 10 mM HEPES (pH 7.9), 100 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl(2), 0.05 mM EDTA, 0.1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 0.03 mg/ml bovine serum allbumin, and 1 ng of P-end-labeled oligonucleotide. In competition experiments different amounts of unlabeled oligonucleotides were included. After an incubation of 25 min at room temperature, free DNA and DNA-protein complexes were separated by electrophoresis on an 8% polyacrylamide gel with 0.33 times TBE buffer at 4 °C. Subsequently, the gel was subjected to autoradiography at -80 °C for 16 h.

Antisera produced in rabbits against HNF-3alpha and -3beta (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.

Oligonucleotides Used in UV Cross-linking Experiments

The following nucleotides for human protein C gene were used.

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).

UV Cross-linking

A fusion protein between glutathione S-transferase (GST) and the HNF-3beta DNA binding domain (a generous gift from D. G. Overdier) was affinity purified using RediPack GST purification modules (Pharmacia Biotech Inc.) according to the manufacturer's protocol. UV cross-linking experiments were performed with 1 µg of the fusion protein (GST-HNF-3beta) in a final volume of 10 µl. The reaction mixture contained 10 mM HEPES (pH 7.9), 100 mM KCl, 1 mM dithiothreitol, 2.5 mM MgCl(2), 0.05 mM EDTA, 0.1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 1 µg of dI-dC, and 1 ng of P-end-labeled oligonucleotides. After an incubation of 25 min at room temperature, the reaction mixture was irradiated for 5 min with UV (255 nm) to irreversibly cross-link DNA and protein. Subsequently, the molecular mass of the cross-linked complexes was determined by SDS-PAGE on a 10% polyacrylamide gel and autoradiography at -80 °C for 16 h. As molecular weight markers, the prestained markers of Sigma were used (M(r) 6,500-205,000).


RESULTS

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-3alpha, -3beta, and -3 each formed two distinctive complexes with the pCwt oligonucleotide. In the case of HNF-3alpha and -3beta, 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-3beta).


Figure 2: EMSAs of in vitro translated HNF-3alpha, -3beta, 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-3alpha (lane2), -3beta (lane3), or -3 (lane4).




Figure 3: Specific binding of HNF-3beta to the human protein C promoter. EMSAs were done with labeled oligonucleotide pCwt and in vitro translated HNF-3beta 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-3alpha, -3beta, and -3. As a consequence of the addition of anti-HNF-3alpha and anti-HNF-3beta 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-3alpha, -3beta, 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-3alpha incubated with anti-HNF-3alpha antibodies (lane2), anti-NF-1 antibodies (lane3), or without antibodies (lane1). B, HNF-3beta incubated with anti-HNF-3beta 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-3alpha, -3beta, 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-3alpha, -3beta, nor -3 formed a complex with the oligonucleotide containing the -32 mutation (Fig. 5B).


Figure 5: EMSAs of in vitro translated HNF-3alpha, -3beta, 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-3alpha (lane2), -3beta (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-3alpha, -3beta, 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-3alpha (lane2), -3beta (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-3beta 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-3beta 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-3beta 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-3alpha, -3beta, or -3. As shown in Fig. 8A, cotransfection of HepG2 cells with HNF-3alpha and -3 increased transcription from the wild-type protein C promoter about 4-fold, whereas HNF-3beta transactivated the promoter activity about 5-fold. In contrast when the pC-27CAT construct was used, the transactivation potential of HNF-3alpha, -3beta, and -3 was reduced to about a factor of 1.5, 3, and 1.3, respectively (Fig. 8A). Essentially no transactivation with HNF-3alpha and -3 was observed using the pC-32CAT construct, while HNF-3beta transactivated this construct approximately 2-fold. In Fig. 8B it can be seen that HNF-3alpha, -3beta, and -3 all transactivated the wild-type protein C promoter in HeLa cells. As shown in HepG2 cells, HNF-3beta 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-3alpha, -3beta, and -3. Note the difference in scale between panelsA and B.




DISCUSSION

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-3alpha and HNF-3 are more abundant than HNF-3beta, whereas in adult liver cells HNF-3beta and HNF-3 are most abundant(47) . In vitro HNF-3alpha, -3beta, 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-3alpha (95% decrease) and HNF-3beta (20% decrease) expression. Furthermore, they showed that this reduced HNF-3alpha and -3beta 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-3alpha and -3beta. 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.


FOOTNOTES

*
This work was supported by Grant 92.004 from the Trombosestichting Nederland and in part by National Institutes of Health Grant HL-52246. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Hemostasis and Thrombosis Research Center, Dept. of Hematology, Bldg. 1, C2-R, University Hospital, P. O. Box 9600, 2300 CR Leiden, The Netherlands. Tel: 31-71-261889; Fax: 31-71-225555; reitsma{at}rulgca.leidenuniv.nl.

(^1)
The abbreviations used are: bp, base pair(s); TBP, TATA box-binding protein; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.


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