From the
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), NF
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(
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.
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.
For the DNA
binding reactions, 2-7 µg of nuclear extracts from rat liver
or PLC/PRF/5 cells were mixed with 1
To
explore further the functional importance of these proximal C/EBP sites
we cotransfected PLC cells with various C/EBP expression vectors
(
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).
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).
Site D contains a low
affinity binding site for C/EBP
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.
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
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
B (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.
)
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).
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.
Cell Cultures, DNA Transfections, and Luciferase and
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 Assays
-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`; NF
B 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`.
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 NF
B 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.
,
, 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 NF
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 Binding Site
B. 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 NF
B
competitor (Fig. 6A). When the NF
B 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 NF
B/rel family proteins (GGGAMTNYCC, where M
= A or C, and Y = C or T)
(29) . To confirm a
possible interaction of NF
B-like factors with site B-II, we used
recombinant human p50 subunit homodimers in EMSA
(Fig. 6B). The NF
B and site B-II probes formed
complexes with p50 that were indistinguishable from one another,
confirming the presence of a NF
B-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 NF
B, factor VIII site
B-II, or albumin C/EBP site oligonucleotides and purified human p50
subunit of NF
B 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, NF
B-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.
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) .
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.
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 NF
B
(41) . Therefore, the presence of
multiple C/EBP sites and a NF
B 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.
Table: Luciferase activity of TATA box mutant
constructs
-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.
/EMBL Data Bank with accession number(s) U24224.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.