©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cis-Acting Elements and Transcription Factors Involved in the Promoter Activity of the Human Factor VIII Gene (*)

Mauro S. Figueiredo (§) , George G. Brownlee

From the (1) Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Factor VIII is a glycoprotein that is essential for blood coagulation. Although factor VIII mRNA has been detected in a variety of human tissues, hepatocytes are considered to be the major source of plasma factor VIII. In this report we demonstrate that the 5`-flanking region of the factor VIII gene is able to transcribe a luciferase reporter gene in three human liver-derived cell lines: PLC/PRF/5, Chang, and HepG2. DNase I footprinting showed the presence of 19 protein binding sites (labeled A to S, proximal to distal) distributed along the region from nucleotide -1175 to -9 of the factor VIII promoter (+1 refers to the translation initiation codon, ATG). Functional analysis of 5` and 3` deletion mutants of the promoter region in PLC/PRF/5 cells revealed that the region from -279 to -64, including sites B to D, contains all the necessary elements for maximal promoter activity. By using electrophoretic mobility shift assays with nuclear extracts and purified transcription factors, and antibody supershift assays we were able to characterize four liver-enriched factors and one ubiquitous transcription factor interacting with the proximal promoter binding sites (sites A to E): hepatocyte nuclear factor (HNF) 1 (site A), NFB (site B), C/EBP and C/EBP (proximal and distal regions of site C, and site D), and HNF4 (site E). Additionally, mutation of the putative TATA box GATAAA (positions -201 to -196) to GACCGA resulted in less than 2-fold decrease in promoter activity, suggesting that the putative TATA box is not essential for factor VIII promoter activity. These results significantly contribute to the understanding of the control of the hepatic transcription of the factor VIII gene.


INTRODUCTION

Activated factor VIII (factor VIIIa) is as an essential cofactor for the phospholipid and calcium-dependent activation of factor X by factor IXa (1) . Deficiency or functional abnormality of factor VIII results in hemophilia A, an X-linked bleeding disorder that affects about 1 in 5,000 males worldwide (2) . As expected, the cloning of the factor VIII gene has allowed enormous progress in the understanding of this complex protein and the molecular characterization of the genetic defects causing hemophilia A (3, 4, 5, 6) . Even with the difficulties imposed by the large size of the factor VIII gene (186 kb() long and 26 exons encoding a 9-kb mRNA), it is now possible to detect mutations in virtually all affected patients (for a review of factor VIII gene mutations see Ref. 7).

Despite this progress, little is known about the transcriptional regulation of the factor VIII gene. Gitchier et al.(3) reported the DNA sequence of the 5`-flanking region of the factor VIII gene up to the nucleotide at position -1175 (+1 refers to the translation initiation site, ATG). By employing RNase protection mapping using RNA purified from human liver, these authors identified the transcription initiation site at position -170, implying that the factor VIII mRNA has a long 5` untranslated region. Using cDNA probes, it was shown that factor VIII mRNA detected in liver tissue is restricted to the hepatocyte fraction (8) . Factor VIII mRNA has also been found in various other tissues, such as spleen, kidney, lymph nodes, placenta, pancreas, and muscle, but not in endothelial cells, thymus, bone marrow, and peripheral lymphocytes (6, 8, 9) . Therefore, although showing a tissue-restricted pattern, the expression of the factor VIII gene is not liver-specific, in contrast to most other clotting factors (10) . Despite this, it is believed that hepatocytes constitute the major source of plasma factor VIII. This is supported by reports of correction of plasma factor VIII levels, resulting in cure of hemophilia A in patients submitted to liver transplantation (11, 12) .

In this report we show that the 5`-flanking region of the factor VIII gene is able to efficiently transcribe a luciferase reporter gene in different human liver-derived cell lines in tissue culture. We have identified a promoter region of about 200 bp that contains the necessary elements for adequate expression of the factor VIII-luciferase construct and furthermore we have characterized ubiquitous and liver-enriched transcription factors that interact with this region. Finally, by site-directed mutagenesis, we have investigated the importance of the putative TATA box of the promoter.


EXPERIMENTAL PROCEDURES

Construction of Luciferase Vectors

A plasmid containing a 1.2-kb SacI fragment of the factor VIII gene (positions -1175 to +15; +1 corresponds to the first base of the ATG translation initiation codon) was used to generate a series of 5` and 3` deletion mutants of the factor VIII promoter. Thirteen promoter segments were amplified by polymerase chain reaction (PCR) using a high fidelity DNA polymerase (Pfu DNA polymerase, Stratagene) and a pair of primers that were engineered to contain a SacI (forward primer) or an XhoI site (reverse primer) at their 5` ends: forward primers 5`-GCGCGAGCTCACCATGGCTACATTCTG-3` (-1175 to -1153), 5`-GCGCGAGCTCTAAATGTGGTGTTCCATATT-3` (-976 to -957), 5`-GCGCGAGCTCGATGAGAC-TACAGAGGTCAG-3` (-776 to -757), 5`-GCGCGAGCTCTAGTTGCCTAACCTCATGTT-3` (-586 to -567), 5`-GCGCGAGCTCCTAACAGGTTGCTGGTTACT-3` (-486 to -467), 5`- GCGCGAGCTCAGTAGGC-TAGGAATAGGAGC-3` (-386 to -367), 5`-GCGCGAGCTCCTTAAAGGTTCTGATTAAAG-3` (-317 to -296), 5`-GCGCGAGCTCTGCTCTCAGAAGTGAATGGG-3` (-279 to -259), 5`-GCGCGAGCTCGCTTCCC-ACTGATAAAAAGG-3` (-211 to -192), and 5`-GCGCGAGCTCATTAAATCAGAAATTTTA-3` (-105 to -86); reverse primers 5`-GCGCCTCGAGCTACAAATGTTCAACTGGAG -3` (-9 to -29), 5`-GCGCCTCG-AGCTCCCAGGAGGGGAAAAAAG-3` (-64 to -85), 5`-GCGCCTCGAGTACCCACTGGATGTGCTCAG-3` (-144 to -163), and 5`-GCGCCTCGAGTGGGAAGCAGCCACAGGAAG-3` (-202 to -223). PCR products were double-digested with SacI and XhoI, gel-purified, and cloned into SacI-XhoI-digested pGL2-Basic (Promega), a promoterless luciferase reporter gene vector. The factor IX-luciferase construct was generated by PCR amplification of factor IX sequences from -220 to +45 (13) and cloning of the amplified segment into pGL2-Basic vector as described above. The luciferase vector driven by the SV40 early promoter was obtained from Promega (plasmid pGL2-promoter). Both strands of each individual clone were sequenced to verify the correctness of the amplified promoter segment and its appropriate insertion into pGL2-Basic vector. All plasmids were purified twice by cesium chloride banding (14) , and for most constructs two different clones of the same construct were prepared.

As a result of our sequencing experiments, we detected two differences from the reported sequence of the 5`-flanking region of the human factor VIII gene (3) : an extra C inserted at position -156 (GCACA instead of GCAA) and the absence of a G at position -890 (TGGAA instead of TGGGAA). The first difference had been detected previously by others (15) . In both cases we confirmed the results by carrying out both manual and automated sequencing. Additionally, we sequenced the 1.2-kb region of the factor VIII promoter from DNA samples from 5 unrelated individuals and obtained the same results.

Cell Cultures, DNA Transfections, and Luciferase and -Galactosidase Assays

The human liver-derived cell lines PLC/PRF/5, HepG2, and Chang were maintained in minimum essential medium supplemented with 10% fetal calf serum and 1% nonessential amino acids. The cells were plated in 60-mm dishes and transfected by the calcium phosphate precipitation method (16) with 10 µg of factor VIII promoter-luciferase constructs and 1.5 µg of pCH110 -galactosidase expression vector (Pharmacia Biotech Inc.), which was used as an internal control for transfection efficiency. Luciferase assays were performed using the Luciferase Assay System (Promega) as recommended by the manufacturer. The luciferase activity associated with each construct was corrected for differences in transfection efficiency based on the results of the -galactosidase assay (17) . Typically, the transfection efficiency varied 2-4-fold within a given transfection experiment (14-20 dishes). At least three independent transfection experiments in duplicate were performed for each construct. The coefficient of variation between duplicates was always below 15%. The final results for luciferase activity are expressed as mean ± S.D. The C/EBP expression vectors MSV-C/EBP, MSV-C/EBP, and MSV-C/EBP used in cotransfection experiments were kindly provided by S. McKnight (Tularik, Inc., San Francisco, CA). For these experiments PLC/PRF/5 cells were transfected with 5 µg of the luciferase vector containing the -279/-9 segment the factor VIII promoter, 5 µg of C/EBP expression vector, and 1.5 µg of -galactosidase expression vector. To keep the total amount of DNA constant, 5 µg of an irrelevant pUC vector was used in the transfection mixture of those cells that were not transfected with C/EBP expression vectors.

Preparation of Nuclear Extracts and DNase I Footprinting Assay

Rat liver and PLC/PRF/5 cell nuclear extracts were prepared as described by Maire et al.(18) and Dignam et al.(19) , respectively. Final protein concentrations were determined by the Bradford assay (16) . DNase I footprinting was performed as described by Chambaz et al.(20) with minor modifications. Footprinting assays were performed for both strands of the factor VIII promoter (except for sites R and S), with nuclear extracts from both rat liver and PLC/PRF/5 cells.

Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay

The following synthetic double-stranded oligonucleotides were used for EMSA and supershift assays (only one strand is indicated): factor VIII site A, 5`-TATTTTAGAGAAGAATTAACCTTTTGCT-3 (-58 to - 31); factor VIII site B-I, 5`-TTCATTAAATCAGAAATTTTACTTTTTTCC-3` (-106 to -77); factor VIII site B-II, 5`-TGCAAAGAAATTGGGACTTTTCATTAAA-3` (-125 to -98); factor VIII site C-I, 5`-TGCTTAGTGCTGAGCACATCCAGTGGGTA-3` (-172 to -144); factor VIII site C-II, 5`-CCCACTGATAAAAAGGAAGCAATCCTATCG-3` (-207 to -178); factor VIII site D, 5`-GCTCTCAGAAGTGAATGGGTTAAGTTTAGC-3` (-277 to -248); factor VIII site E, 5`-GTTCTGATTAAAGCAGACTTATGCCCCTAC-3` (-308 to -279); mutant a of the factor VIII site C-II, same as site C-II except for a G T mutation at position -201; mutant b of the factor VIII site C-II, same as site C-II except for a TAA CCG mutation between -199 and -197; human 1-antitrypsin HNF1 site, 5`-CCTTGGTTAATATTCACCTTCC-3`; NFB consensus oligonucleotide, 5`-AGTTGAGGGGACTTTCCCAGGC-3` (Promega); Oct1 consensus oligonucleotide, 5`-TGTCGAATGCAAATCACTAGAA-3` (Promega); Sp1 consensus oligonucleotide, 5`-ATTCGATCGGGGCGGGGCGAGC-3` (Promega); NF1 consensus oligonucleotide, 5`-CCTTTGGCATGCTGCCAATATG-3` (Promega); fibronectin cAMP response element, 5`-AGTCCCCCGTGACGTCACCCGGGAGCC-3`; rat albumin NF-Y site, 5`-GGGTAGGAACCAATGAAATGAAAGGTTAGT-3`; rat fibrinogen chain CP2 site, 5`-CAGTTCCAGCCACTCTTAGTCCCGCCCAGA-3`; rat transthyretin HNF3 site, 5`-TTGACTAAGTCAATAATC-AGAATCAG-3`; human collagenase AP1 site, 5`-ATAAAGCATGAGTCAGACACCTC-3`; human factor IX C/EBP site, 5`-CTTACCACTTTCACAATCTGCTAG-3`; rat albumin D (DBP, C/EBP) site, 5`-GATGGTATGATTTTGTAATGGGGTAGG-3`; human endothelin-1 GATA site, 5`-GGCCTGGCCTTATCTCCGGCTGC-3`; adenovirus TATA site, 5`-TCCTGAAGGGGGGCTATAAAAGGGGGTGGGGGCGCG-3`; and 1-antitrypsin HNF4 site, 5`-GACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTT-3`.

For the DNA binding reactions, 2-7 µg of nuclear extracts from rat liver or PLC/PRF/5 cells were mixed with 1 10 cpm of end-labeled double-stranded oligonucleotide in a total volume of 10-20 µl containing 20 mM Hepes, pH 7.6, 50 mM KCl, 1 mM MgCl, 0.5 mM dithiothreitol, 4% Ficoll, and 0.1 mg/ml double-stranded poly(dI-dC) in the presence or absence of unlabeled competitor oligonucleotide. The probe was added to the reaction mixture after preincubation of the other components for 15 min on ice, and the reaction mixture was incubated for further 15 min at room temperature and loaded onto a 4% nondenaturing polyacrylamide gel in 0.5 Tris borate-EDTA. Electrophoresis was carried out for 90-120 min at a constant voltage of 200 V. The gel was dried onto Whatman DE81 paper and exposed to x-ray film. Purified recombinant human TATA-binding protein and purified human p50 subunit of NFB factor were purchased from Promega. A truncated form of the transcription factor HNF1, that includes the DNA binding domain (21) was generously provided by R. Cortese (Istituto di Richerche di Biologia Moleculare, Rome, Italy). The conditions for DNA binding reactions with purified proteins are described in the respective figure legends. Antibodies to C/EBP, C/EBP, and C/EBP (22) (kindly provided by S. McKnight) and HNF4 (23) (kindly provided by F. M. Sladek, Rockefeller University, New York) were employed in supershift assays. 1 µl of antiserum at various dilutions was added to the initial reaction mixtures, the samples were preincubated for 30 min on ice, and the reactions were continued as above.

Mutagenesis

Promoter segments containing the mutants ``a'' and ``b'' of site C-II were generated by oligonucleotide-directed mutagenesis as described (24) . The oligonucleotides used for the mutagenesis were 30 bp long with the altered bases positioned centrally. The flanking primers were the same as described for the construction of the deletion mutants (see above). The final mutant promoter segments were digested with SacI and XhoI and cloned into SacI-XhoI-digested pGL2-Basic vector. All constructs were verified by DNA sequencing.

Primer Extension Analysis

Primer extension (16) was used to map the 5` terminus of factor VIII promoter-luciferase transcripts produced by transfected PLC/PRF/5 cells. mRNA was isolated by oligo(dT)-cellulose chromatography (14) from cells transfected with factor VIII promoter-luciferase constructs (two constructs were used, one containing the promoter segment from -279 to -9 and the other from -1175 to -9). An end-labeled oligonucleotide complementary to the luciferase cDNA sequence (5`-CTTTATGTTTTTGGCGTCTTCCA-3`) was annealed to the mRNA preparations and extended using avian myeloblastosis virus reverse transcriptase. The extension products were analyzed on an urea-acrylamide sequencing gel.

RESULTS

Factor VIII Promoter Activity in Human Liver-derived Cell Lines

To test for presumptive promoter activity of the 5`-flanking region of the human factor VIII gene, we used a luciferase reporter gene assay in transient expression experiments. The factor VIII segment from -1175 to -9 (+1 refers to the translation initiation codon, ATG) was cloned into the promoterless luciferase expression vector pGL2-Basic, and the factor VIII-luciferase construct was transfected into three human liver-derived cell lines: HepG2, PLC/PRF/5 (PLC), and Chang. Promoter activity (expressed relative to the activity observed for the promoterless construct in the same cell line, arbitrarily defined as 1) was 10.1 ± 3.7 in HepG2 cells, 61.2 ± 11.5 in PLC cells, and 24.5 ± 6.1 in Chang cells (mean ± S.D., n 3). These results show that the -1175/-9 segment of the factor VIII gene has promoter activity significantly above background in these liver-derived cell lines and indicate that the levels detected in PLC cells were substantially higher than those obtained in either HepG2 or Chang cells. For that reason, all subsequent functional experiments were performed exclusively in PLC cells. To evaluate the relative strength of the factor VIII promoter, we compared it with two other well characterized promoters in PLC cells: the factor IX promoter and the SV40 early promoter. Promoter activity (expressed as percentage of the activity observed for the factor VIII-luciferase construct in the same cell line) was 63 ± 8% for the factor IX-luciferase construct, and 495 ± 139% for the SV40-luciferase construct (mean ± S.D., n 6).

Deletion Analysis of the Factor VIII Promoter

To identify the regions of the factor VIII promoter that are important for its activity, 5` and 3` deletion mutants of the promoter segment from -1175 to -9 were cloned into the pGL2-Basic luciferase reporter gene vector for transient transfection experiments in PLC cells (see ``Experimental Procedures''). The promoter segments tested in this study and their respective activities are shown in Fig. 1. Initially, we performed 5` to 3` deletions fixing the 3` end at position -9. Deletion of positions between -1175 and -976 had no significant effect upon promoter activity. Further deletion from -976 to -776 caused a small (1.3-fold) reduction of activity and additional deletions between -776 and -386 produced no further significant change. Deletion from -386 to -317 produced a 1.5-fold increase of the promoter activity. Although small, this effect was significant and may suggest the presence of weak negative elements between -386 and -317. Deletion of positions from -317 to -279 produced no significant alteration, whereas further deletion from -279 to -211 caused a large, 8-fold reduction of promoter activity, suggesting that the region -279 to -211 contains important positive promoter elements. Finally, deletion from -211 to -105 virtually abolished all activity. Based on these results, we performed additional 3` to 5` deletions in the -279/-9 segment, which showed the highest promoter activity. No significant difference was observed between the constructs -279/-9 and -279/-64. However, deletion from -64 to -144 significantly reduced the activity (2.6-fold) and an additional deletion from -144 to -202 caused an additional 8.4-fold reduction. Taken together, these results suggest that the most important elements of the factor VIII promoter, as assessed in PLC cells, are located between positions -279 and -64.


Figure 1: Deletion analysis of the human factor VIII gene promoter region. The left part of the figure shows the organization of the factor VIII gene promoter. The arrow indicates the transcription initiation site (position -170). The hatched boxes indicate protein binding sites identified by DNase I footprinting. The numbers indicate the 5` and 3` ends of each promoter insert (see ``Experimental Procedures''), numbered in relation to the translation initiation site (+1). The right part shows the relative luciferase activity ± S.D. (relative to the activity of the -1175/-9 construct, arbitrarily set at 100%) in PLC cells. n, number of independent transfection experiments.



Primer extension analysis of mRNA isolated from transiently transfected PLC cells (see ``Experimental Procedures'') mapped the transcription initiation site of factor VIII promoter-luciferase constructs at position -170 (adopting the numbering for the factor VIII gene). Therefore, the 5` end observed for our factor VIII-luciferase transcripts is the same as that determined previously for the factor VIII mRNA expressed in human tissues (3, 8) .

Identification of Nuclear Factor Binding Sites

We investigated the nuclear factor binding sites present in the region from -1175 to -9 of the factor VIII promoter by DNase I footprinting. The assays were performed using nuclear extracts from rat liver and PLC cells and both DNA strands were analyzed (see ``Experimental Procedures''). Experiments using rat liver nuclear extracts allowed identification of 19 binding sites: site A, from -59 to -31 (sense strand); site B, -125 to -78; site C, -205 to -148; site D, -267 to -249; site E, -311 to -277; site F, -349 to -331; site G, -411 to -393; site H, -453 to -424; site I, -506 to -492; site J, -637 to -518; site K, -684 to -662; site L, -766 to -740; site M, -845 to -827; site N, -896 to -863; site O, -991 to -952; site P, -1016 to -1007; site Q, -1105 to -1024; site R, -1121 to -1113; and site S, -1170 to -1133 ( Fig. 2and 3). Footprinting experiments using nuclear extracts heated for 10 min at 75 °C showed partial or complete protection of sites C (proximal and distal regions), D, G, J (central region), O (distal region), Q (proximal region), and S (central region), indicating that these regions interact with heat-stable or relatively heat-stable nuclear factors (Fig. 2). Analysis of protection of site A with heated nuclear extracts produced inconclusive results. Footprinting assays using PLC cell extracts gave essentially the same results, except for small differences in the extent of protection observed for some sites and a complete absence of protection in the region corresponding to site H (data not shown).


Figure 2: DNase I footprinting analysis of the human factor VIII promoter. The experiments illustrated in this figure were carried out using rat liver nuclear extracts as described under ``Experimental Procedures''. In all panels: lane G+A, Maxam and Gilbert chemical sequencing reactions; lanes 1 and 2, control reactions with no nuclear extract and increasing concentrations of DNase I; lane 3, 3 µg of nuclear extract; lane 4, 6 µg of nuclear extract; lane 5, 12 µg of nuclear extract; lane 6, 24 µg of nuclear extract; lane 7, 12 µg of nuclear extract heated for 10 min at 75 °C. The boxes and the numbers on the right side of each autoradiogram indicate the boundaries of the binding sites, labeled A-S. The hatched boxes indicate regions partially or completely protected by heat-stable nuclear factors. The results for only one DNA strand are shown, except for binding site O.



The characterization of the transcription factors that interact with the binding sites of the factor VIII promoter was carried out using EMSA and supershift assays (see ``Experimental Procedures''). Because of the multiplicity of sites identified by DNase I footprinting and the results of the deletion analysis (see above), we decided to restrict this characterization to sites A to E, and only these sites are considered further in this paper.

Members of the C/EBP Family of Transcription Factors Bind to Site D and Proximal and Distal Regions of Site C

Sites C and D contain homologous sequences to the consensus binding sequence for the heat-stable liver-enriched C/EBP family of transcription factors (TKNNGYAAK, where K = G or T, and Y = C or T) (25) : site D, TGGGtTAAG (-262 to -254, mismatch in lower case); site C, gGAAGCAAT (-193 to -185) and cTCAGCAcT (-167 to -159). Two oligonucleotides were employed for the EMSA of site C: site C-I (-172 to -144) and site C-II (-207 to -178). Site C-I formed a set of incompletely resolved complexes with rat liver nuclear extracts (Fig. 4A), and competition experiments showed that all complexes were completely abolished when the albumin D site (a high affinity C/EBP site) (26) was used as competitor. In addition, the pattern of complexes was similar to that produced by the albumin D site probe. Accordingly, cross-competition experiments (Fig. 4B) showed that the C-I site was able to abolish the complexes formed by the albumin D site probe, although with less efficiency than that of the homologous albumin D site competitor. Site C-II also produced complexes that migrate similarly to those formed by the albumin D site, in addition to a strong complex that comigrated with the lower C/EBP complexes (Fig. 4A). The albumin D site abolished the C/EBP-like complexes but not the major lower complex (Fig. 4A). Site C-II was also able to compete for the formation of complexes by the albumin D site probe with a slightly lower efficiency than that of site C-I (Fig. 4B). Site D formed a pattern of complexes that were not competed out by the albumin D site (Fig. 4A). The identity of the nuclear factors that form the major complexes with the factor VIII D site is unknown. Oligonucleotides containing binding sites for the transcription factors AP1, Oct1, NF-Y, CP2, NF1, Sp1, NFB, cAMP response element, HNF1, HNF3, and HNF4 were also unable to compete for the formation of site D complexes (data not shown). However, when the D site was used as competitor in high concentrations, it was able to prevent the formation of complexes by the albumin D site (Fig. 4B) or factor IX C/EBP site (not shown).


Figure 4: Sites C and D contain low affinity binding sites for C/EBP and C/EBP. Panel A, EMSA using rat liver nuclear extracts and the labeled factor VIII sites C-I, C-II, and D, and the albumin C/EBP site oligonucleotides, in the presence or absence of 150-fold molar excess of competitor oligonucleotides, as indicated. The reactions were performed with 7 µg of nuclear extracts, except for the reaction with the albumin C/EBP site, which contained 3 µg. Bracket, location of the C/EBP complexes; arrow, location of the major complex formed by site C-II probe, which comigrates with the lower C/EBP complexes; NS, nonspecific complex; free, free DNA probe. Panel B, competition experiments using labeled albumin C/EBP site, 3 µg of rat liver nuclear extracts, and increasing amounts of competitor oligonucleotides (50-, 150-, and 400-fold molar excess), as indicated. Panel C, supershift experiments using labeled factor VIII sites C-I, C-II, and D oligonucleotides, 7 µg of rat liver nuclear extracts, and no antiserum, C/EBP-specific antiserum (1 µl of 1:10 dilution), or C/EBP-specific antiserum (1 µl of 1:5 dilution), as indicated. The supershift band formed by site C-II and C/EBP antiserum was very faint in the original and did not reproduce well in the photograph.



To investigate further the possible interaction of C/EBP factors with factor VIII sites and to identify which C/EBP isoforms are involved, we employed supershift assays using antisera containing specific antibodies to C/EBP or C/EBP (22) . As shown in Fig. 4C, the addition of antibody to C/EBP resulted in a decrease of C/EBP-specific complexes and the formation of faint supershift bands with the C-I and C-II probes. C/EBP-specific supershift bands with the site D probe could only be seen when higher amounts of nuclear extracts were used (result not shown). C/EBP-specific antiserum shifted the lower C/EBP complexes of the C-I site and produced a clear supershift band with the C-I, C-II, and D sites (Fig. 4C). Taken together, these results suggest that factor VIII sites C-I, C-II, and D contain low affinity binding sequences for C/EBP factors (the order of affinity from the highest to the lowest was: C-I site C-II > site D). Additionally, DNase I footprinting experiments in the presence of competitor oligonucleotides showed that factor IX or albumin C/EBP binding sites were able to prevent the protection conferred by heat-treated rat liver nuclear extracts in at least four other regions of factor VIII promoter: sites S, Q, O, and J (data not shown). This suggests the presence of multiple C/EBP binding sites distributed along the factor VIII promoter.

To explore further the functional importance of these proximal C/EBP sites we cotransfected PLC cells with various C/EBP expression vectors (, , and ) and a luciferase reporter gene vector containing the -279/-9 segment of the factor VIII promoter (see ``Experimental Procedures''). Cotransfection with C/EBP expression vector caused a 4.8 ± 1.2-fold increase in the promoter activity (mean ± S.D., relative to the activity of the -279/-9 luciferase construct cotransfected with an irrelevant pUC plasmid), whereas cotransfection with C/EBP had no effect (1.1 ± 0.1 relative activity). Cotransfection with C/EBP, a factor that is not expressed in unstimulated liver or liver-derived cell lines (27, 28) , produced a 6.7 ± 0.8-fold enhancement of promoter activity.

The Transcription Factor HNF4 Binds to Site E

The E site probe formed a major DNA-protein complex with rat liver and PLC cells nuclear extracts (Fig. 5A). Competition experiments showed that the site E-specific complex was completely abolished by 1-antitrypsin (AT) HNF4 site (23) competitor (Fig. 5A). The region from -296 to -284 within the E site contains the sequence GCAGACTTATGCC, which is partially homologous to the HNF4 binding site of the AT gene (GTGGACTTAGCCC) (23) . Cross-competition experiments showed that site E oligonucleotides blocked the formation of the AT HNF4 site-protein complex, although it competed much less efficiently than the homologous competitor (Fig. 5B). Finally, antibodies raised to rat HNF4 protein (23) shifted the site E-specific complex formed with rat liver and PLC cell extracts (Fig. 5C). These results strongly suggest that the E site contains a low affinity sequence for HNF4.


Figure 5: The transcription factor HNF4 binds to site E of factor VIII promoter. Panel A, EMSA using nuclear extracts from rat liver (4.5 µg) or PLC cells (6 µg), labeled factor VIII site E oligonucleotide, in the presence or absence of competitor oligonucleotides, as follows: factor VIII site E and 1-antitrypsin HNF4 site, 100 and 400-fold molar excess in experiments with rat liver nuclear extracts and 400-fold molar excess in experiments with PLC cell extracts; CP1, 400-fold molar excess. Arrow, HNF4-specific complex; free, free DNA probe. Panel B, competition experiments using labeled 1-antitrypsin HNF4 site (ATHNF4), 3 µg of rat liver nuclear extracts, and increasing amounts of competitor oligonucleotides (100-, 200-, and 500-fold molar excess of AT HNF4 and factor VIII site E oligonucleotides, and 200- and 500-fold molar excess of CP2 oligonucleotide), as indicated. Panel C, supershift experiments using labeled factor VIII site E, 3 µg of rat liver or PLC cell nuclear extracts, and no antiserum, 1 µl of 1:10 dilution of antiserum containing antibodies to HNF4, or 1 µl of 1:10 dilution of antibody to influenza A virus nucleoprotein (NP), as indicated. ss, supershift band.



Site B Contains a NFB Binding Site

Site B was investigated using two oligonucleotides: site B-I (-106 to -77) and B-II (-125 to -98). Site B-II produced two specific complexes, a and b, with rat liver nuclear extracts (Fig. 6A). The major, a complex, was efficiently competed out by an oligonucleotide containing the consensus binding sequence for the ubiquitous transcription factor NFB. The identity of the lower complex, b, is unknown. PLC extracts also formed two complexes, c and d, and both were completely abolished by the NFB competitor (Fig. 6A). When the NFB oligonucleotide was used as probe, two specific complexes were also observed and both were competed out by the site B-II oligonucleotide (data not shown). The sequence of the region from -113 to -104 of site B-II (GGGACTTTTC) constitutes an almost perfect match for the consensus binding sequence for NFB/rel family proteins (GGGAMTNYCC, where M = A or C, and Y = C or T) (29) . To confirm a possible interaction of NFB-like factors with site B-II, we used recombinant human p50 subunit homodimers in EMSA (Fig. 6B). The NFB and site B-II probes formed complexes with p50 that were indistinguishable from one another, confirming the presence of a NFB-like binding sequence within site B-II.


Figure 6: Site B contains a binding site for the ubiquitous factor NFB. Panel A, EMSA using 6 µg of rat liver or PLC cell nuclear extracts, labeled factor VIII site B-II oligonucleotide, in the presence or absence of 400-fold molar excess of competing oligonucleotides, as indicated. a and b, site B-II-specific complexes with rat liver nuclear extracts; c and d, site B-II-specific complexes with PLC cell nuclear extracts; NS, nonspecific complex; free, free DNA probe. Panel B, EMSA using labeled NFB, factor VIII site B-II, or albumin C/EBP site oligonucleotides and purified human p50 subunit of NFB factor, in the presence or absence of 100- and 400-fold molar excess of competing oligonucleotides, as indicated. Binding reactions were performed in a total volume of 15 µl containing 10 mM Hepes, pH 7.6, 50 mM KCl, 0.2 mM EDTA, 2.5 mM dithiothreitol, 4% Ficoll, 0.05% (v/v) Nonidet P-40, 0.1 mg/ml poly(dI-dC), 1 10 cpm of labeled oligonucleotide, and 0.3 µl of purified protein. C, labeled albumin C/EBP oligonucleotide and no competitor; arrow, NFB-DNA complex; free, free DNA probe.



Site B-I produced a faint single complex of unknown identity with rat liver nuclear extracts (not shown). Competition experiments using oligonucleotides containing binding sites for several ubiquitous and liver-enriched transcription factors (see ``Experimental Procedures'') did not reveal the factor or factors that possibly interact with this region.

Site A Contains a HNF1 Binding Site

The region from -38 to -52 within site A presents a significant homology to the consensus binding sequence for the liver-enriched transcription factor HNF1 (29, 30) . As illustrated in Fig. 7A, the DNA-protein complexes produced by site A probe and PLC cell nuclear extracts were competed out by the 1-AT HNF1 site (21) competitor. Accordingly, cross-competition experiments showed that the A site was as efficient as the homologous competitor in preventing the formation of complexes by AT HNF1 probe (Fig. 7B). Finally, site A was able to interact with a truncated form of HNF1 protein (21) , producing an even stronger complex than that observed with the AT HNF1 site (Fig. 7C). These results suggest that HNF1 protein binds to the A site of the factor VIII promoter with an affinity that is at least comparable to that of the AT HNF1 site.


Figure 7: Site A contains a binding site for the liver-enriched transcription factor HNF1. Panel A, EMSA using labeled factor VIII site A oligonucleotide and 6 µg of nuclear extracts from PLC cells, in the presence or absence of 150-fold molar excess of competitor oligonucleotides, as indicated. Bracket, factor VIII site A-specific complex; NS, nonspecific complex; free, free DNA probe. Panel B, EMSA using labeled 1-antitrypsin HNF1 oligonucleotide, and 6 µg of PLC cell nuclear extract, in the absence or presence of 200- and 500-fold molar excess of competitor oligonucleotides, as indicated. C, 500-fold molar excess of albumin C/EBP site oligonucleotide as competitor; bracket, HNF1 complex. Panel C, EMSA using labeled 1-antitrypsin HNF1, factor VIII site A, or factor VIII site C control oligonucleotides and purified truncated HNF1 protein (21). Binding reactions were performed as described under ``Experimental Procedures,'' except that 0.5 mg/ml albumin was added to the reaction mixture. - and + indicate absence or presence of 1 µl of purified HNF1 protein in the reaction mixture; arrow, complex.



Mutagenesis of the Putative TATA Box of the Factor VIII Promoter

A TATA-like sequence (GATAAA) is present between positions -201 and -196 of the factor VIII gene, 31 bp upstream from the transcription initiation site (3) . In order to assess the functional importance of this region, we constructed two mutants: a G T mutation at position -201 (mutant a), which converts the wild type sequence to the canonical TATA box sequence (TATAAA); and a TAA CCG mutation at positions -199 to -197 (mutant b), which theoretically disrupts the TATA-like sequence. The factor VIII promoter region from -279 to -9 containing the wild type, or mutant a, or mutant b sequences were cloned into the luciferase reporter gene vector and functionally evaluated in transiently transfected PLC cells. Luciferase activity was calculated relative to the -1175/-9 promoter construct, which was set at 100% (). The activities of the wild type (126.3 ± 16.4%, n = 7) and the mutant a (154.5 ± 29.9%, n = 5) promoter constructs were comparable, suggesting that the introduction of a canonical TATA sequence had no substantial effect on the promoter activity of the -279/-9 segment. On the other hand, disruption of the TATA-like sequence (mutant b construct) caused a decrease in promoter activity (76.2 ± 13.8%, n = 6; p < 0.01). Although reduced, the promoter activity of the mutant b construct was still 60% of that of the wild type construct, suggesting that the proposed TATA box of the factor VIII gene may not be essential for its promoter activity.

To investigate the interaction of this region with basal and liver-enriched factors, we used two additional oligonucleotides of site C-II, which encompass the GATAAA sequence, incorporating mutants a or b sequences. EMSA using site C-II probes and purified human TATA-binding protein (TBP) showed that the wild type site C-II is able to bind TBP, although with significantly less affinity than adenovirus TATA site oligonucleotide (Fig. 8C, lanes 1 and 2). As expected, the mutant a-TBP complex was much stronger than the complex formed by wild type probe, while still significantly weaker than the adenovirus TATA site-TBP complex (Fig. 8C, lanes 1-3). Mutant b produced no complex (Fig. 8C, lane 4). Competition experiments were in agreement with these results (Fig. 8D). When the site C-II oligonucleotides were mixed with rat liver nuclear extracts, we observed that mutants a and b were unable to form the major lower complex produced by the wild type probe, whereas both mutant probes formed the complexes shown above to contain C/EBP factors (Fig. 8B, lanes 1-3). Accordingly, competition experiments demonstrated that mutants a and b abolished the C/EBP-containing complexes formed by the site C-II wild type probe, but failed to interfere with the major lower complex (Fig. 8B, lanes 4-8).


Figure 8: In vitro analysis of mutants of the factor VIII putative TATA box. Panel A, sequences of the TATA box region of factor VIII site C-II wild type, mutant a, and mutant b oligonucleotides (mutated bases are underlined) aligned with the consensus binding sequence for GATA factors and the TATA canonical sequence. W, A or T; R, A or G. Panel B, EMSA using 6 µg of rat liver nuclear extracts, the indicated probe, and the indicated competitor oligonucleotides used at 200-fold molar excess (lanes 5-8). wt, site C-II wild type; mt a, site C-II mutant a; mt b, site C-II mutant b; bracket, location of C/EBP complexes; arrow, major site C-II-specific complex. Panel C, EMSA using purified recombinant human TBP and the following labeled oligonucleotides: adenovirus TATA site (TATA), site C-II wild type (wt), site C-II mutant a (mt a), and site C-II mutant b (mt b). Binding reactions were performed in a total volume of 10 µl containing 20 mM Tris-Cl, pH 7.9, 80 mM KCl, 10 mM MgCl, 2.0 mM dithiothreitol, 4% Ficoll, 6 10 cpm of labeled oligonucleotide, and 10 ng of purified protein. Arrow, TBP complex. The gel was overexposed to show the faint complex formed by wt probe. Panel D, competition experiments using labeled adenovirus TATA site oligonucleotide, purified human TBP, and the absence (-) or presence of competitor oligonucleotides: 100-, 250-, and 500-fold molar excess of adenovirus TATA site (ad. TATA), 250-fold molar excess of site C-II wild type (wt), site C-II mutant a (mt a), or site C-II mutant b (mt b). Arrow, TBP complex.



The above results led us to speculate that a GATA-like factor could be responsible for the appearance of the major complex detected with wild type probe, since the TATA-like sequence of factor VIII promoter represents a perfect match for the consensus binding sequence of GATA factors (29) (Fig. 8A). Moreover, the GATA factors would not be expected to interact with the sequences of mutants a and b (31) . To test the hypothesis that a GATA-like factor interacts with the factor VIII site C-II, we performed competition experiments using an oligonucleotide containing the GATA site of the human endothelin-1 gene (Et1 GATA site) (32) . As shown in Fig. 9 , the major complex formed by the C-II site probe and rat liver extracts could also be detected with PLC cells extracts (Fig. 9, lanes 1-4). The Et1 GATA site oligonucleotide added as competitor completely abolished this complex (Fig. 9, lane 5). Cross-competition experiments showed that the specific complex formed by the Et1 GATA site probe and rat liver nuclear extracts could be efficiently prevented by addition of factor VIII site C-II as competitor (Fig. 9, lanes 7-9). These results suggest that a GATA-like factor is present in liver tissue and may interact with the TATA-like sequence of the factor VIII promoter.


Figure 9: Major site C-II binding activity is competed out by a GATA oligonucleotide. Lanes 1 and 2, 4.5 µg of PLC cell nuclear extracts and site C-II wild type probe; lanes 3-6, 3 µg of rat liver nuclear extracts and site C-II wild type probe; lanes 7-10, 3 µg of rat liver nuclear extracts and human endothelin-1 GATA site probe. Competitors oligonucleotides (400-fold molar excess) were added as indicated. Bracket, location of C/EBP complexes; left arrow, major site C-II-specific complex with PLC cell extracts; right arrow, major site C-II and GATA site-specific complexes with rat liver extracts.



DISCUSSION

We demonstrate here that a segment of 1.2 kb that includes the 5`-flanking region and most of the 5`-untranslated region of the factor VIII gene is able to transcribe a luciferase reporter gene in different human liver-derived cell lines, confirming that promoter activity is associated with this region. PLC cells afforded the highest reporter gene expression, whereas in HepG2 cells this expression was less, in agreement with previous studies, which showed the presence of factor VIII mRNA in PLC cells (8) but not in HepG2 cells (3) .

Transcription of genes that are expressed in liver is typically controlled by a combination of ubiquitous and liver-enriched transcription factors (18, 33, 34, 35, 36, 37, 38) . The latter group includes HNF1, HNF3, HNF4, and the C/EBP family of transcription factors (39) . Of these, our results (summarized in Fig. 10 ) suggest that the factor VIII promoter contains binding sites for C/EBP factors (sites C and D, Fig. 4), HNF4 (site E, Fig. 5 ), and HNF1 (site A, Fig. 7).


Figure 10: Structure of the proximal region of the factor VIII promoter. Diagram of the factor VIII gene promoter showing the start site (arrow), the TATA-like sequence (GATAAA), and the cis-acting elements characterized in this study. The horizontal bars indicate the probable location of the binding sites. A question mark (?) indicates binding sites (dashed bars) of unknown factors.



Deletion analysis (Fig. 1) indicated that the promoter region from -279 to -64, which includes sites B, C, and D only, was the minimal segment which showed maximal promoter activity. Therefore, the physiological importance of the HNF1 and HNF4 binding sites (in sites A and E, respectively) remains uncertain.

Site D contains a low affinity binding site for C/EBP and C/EBP (Fig. 3). Additionally, our DNA-binding assays indicated that one or more transcription factors of unknown identity are the major components of the site D-protein complexes. Segments of the factor VIII promoter containing mutants of site D, which impair its interaction with C/EBP factors and abolish the formation of site D-specific major complexes, show a very low promoter activity in PLC cells.() These observations, along with the results of the deletion analysis showing that construct -211/-9 (sites A to C) retains only residual promoter activity, indicate that site D contains important positive cis-acting elements. Our results cannot distinguish whether the importance of site D is due to the presence of the low affinity C/EBP site, to the interaction with the other unknown transcription factors, or to both.


Figure 3: Binding sites of the human factor VIII promoter. Sequence of the 5`-flanking region of the factor VIII gene showing the binding sites A-S as determined by DNase I footprinting of both DNA strands, except for the sites R and S, where the antisense strand (dashed line) was not analyzed. The sequence incorporates the differences that have been detected between our study and the published sequence (3) (see ``Experimental Procedures''). Nucleotides are numbered relative to the translation initiation site (+1). The transcription initiation site (arrow) and TATA-like sequence (horizontal bar) are also indicated.



Two other C/EBP binding sites were identified within site C (Fig. 10), and it is interesting to note that, as described for the factor IX promoter (35) , the factor VIII promoter contains a C/EBP site (site C-I) immediately downstream from the transcription initiation site. Our cotransfection experiments using C/EBP expression vectors strengthen the suggestion that C/EBP factors may play a significant role in the expression of the factor VIII gene. Both C/EBP and C/EBP transactivated the factor VIII promoter about 5-7-fold, whereas no enhancement of promoter activity was observed by overexpression of C/EBP. Unlike C/EBP, which is usually involved in base-line expression of liver-specific genes, C/EBP and C/EBP have been implicated in the regulation of acute phase response genes (27, 28, 40) . Another nuclear protein that has been shown to mediate enhanced expression of acute phase response genes in response to stimulating agents is the transcription factor NFB (41) . Therefore, the presence of multiple C/EBP sites and a NFB site (site B) in the factor VIII promoter suggests that expression of the factor VIII gene may be influenced by cellular signals, like inflammatory cytokines.

We also investigated the functional importance of the factor VIII putative TATA box. A TATA-like motif (GATAAA) is located around -30 bp upstream from the transcription initiation site (position -170) of the factor VIII gene (3) . We could demonstrate a weak in vitro interaction between TBP and a factor VIII oligonucleotide containing the GATAAA sequence (Fig. 8). Mutation of this region to a canonical TATA sequence (TATAAA) did not alter significantly the promoter activity, although it showed enhanced in vitro binding to TBP (Fig. 8). In addition, disruption of the TATA-like sequence caused a less than 2-fold decrease in the promoter activity. These results suggest that the TATA-like sequence is not essential for the transcription of the factor VIII gene, although it is clearly associated with higher promoter activity. A possible explanation for these results is that the GATAAA sequence, although showing a weak in vitro binding to TBP, functions as an adequate TATA box in the context of the factor VIII promoter. In our TATA-less (mutant b) promoter construct, protein-protein interactions would allow binding of TBP (42) , resulting in lower but still adequate transcription.

Using EMSA, we demonstrated that another nuclear factor of unknown identity present in rat liver and PLC cells extracts interacts with the GATAA motif of the factor VIII promoter. Interestingly, sequence analysis and competition experiments suggest that a GATA-like protein may be the nuclear factor that forms complex with the factor VIII GATAA sequence (Fig. 9). The relative importance of this unknown transcription factor and its potential interaction with basal transcription factors can only be investigated after its identification, which is beyond the scope of this study.

In conclusion, our results significantly contribute to the understanding of the regulation of the hepatic transcription of the factor VIII gene. Furthermore, the identification of critical cis-acting elements and the factors binding to them is a necessary basis for understanding the effect of promoter mutants in hemophilia A patients, should these be discovered in the future. Our results also provide a framework for future studies on the regulation of factor VIII expression, such as in the acute phase response, hormonal regulation, and the expression of factor VIII in different tissues.

  
Table: Luciferase activity of TATA box mutant constructs

Segments of factor VIII promoter containing wild type, mutant a, or mutant b sequences were cloned into a promoterless luciferase vector, and the constructs were transfected into PLC/PRF/5 cells. A -galactosidase expression vector was cotransfected and used as an internal control for correction of transfection efficiency. The results are expressed as luciferase activity ± S.D., relative to the -1175/-9 promoter construct, which was set at 100. n = number of independent transfection experiments.



FOOTNOTES

*
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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U24224.

§
Supported by Fellowship 92/2578-1 from Fundaão de Amparo Pesquisado Estado de São Paulo, Brazil. To whom correspondence should be addressed. Present address: Dept. of Clinical Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, 14048-900 Ribeirão Preto, Brazil.

The abbreviations used are: kb, kilobase pair(s); bp, base pair(s); PCR, polymerase chain reaction; TBP, TATA-binding protein; C/EBP, CCAAT/enhancer-binding protein; EMSA, electrophoretic mobility shift assay; AT, antitrypsin; NF, nuclear factor; HNF, hepatocyte nuclear factor.

M. S. Figueiredo and G. G. Brownlee, unpublished results.


ACKNOWLEDGEMENTS

We thank S. McKnight (Tularik, Inc., San Francisco, CA) for the generous gift of C/EBP expression vectors and antibodies, R. Cortese (Istituto di Richerche di Biologia Moleculare, Rome, Italy) for the gift of HNF1 protein, F. M. Sladek (Rockefeller University, New York) for the gift of HNF4 antibodies, and E. G. D. Tuddenham (Haemostasis Research Group, Middlesex, United Kingdom) for the gift of the plasmid containing the 1.2-kb fragment of the factor VIII gene.


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