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Blood, Vol. 92 No. 4 (August 15), 1998:
pp. 1247-1258
Oct-1 Is Involved in the Transcriptional Repression of the von
Willebrand Factor Gene Promoter
By
Jean-Luc Schwachtgen,
Jacques E. Remacle,
Nathalie Janel,
Reginald Brys,
Danny Huylebroeck,
Dominique Meyer, and
Danièle Kerbiriou-Nabias
From INSERM U143, Unité de Recherches sur l'Hémostase et
la Thrombose, Hôpital de Bicêtre, Bicêtre, France;
and the Laboratory of Molecular Biology (Celgen), University of Leuven
and Department of Cell Growth, Differentiation and Development,
Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium.
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ABSTRACT |
The negative regulation of transcription of the human von Willebrand
factor (vWF) gene was investigated in human umbilical vein endothelial
cells (HUVECs) and HeLa cells. A fragment spanning  89 to +244
nucleotides (nt), containing the first exon, is active in HUVECs only
but not in HeLa cells. The activity of this promoter is sharply reduced
by mutagenesis of the GATA binding site at +221. Extension of the
upstream sequences from nt 89 to 142 and to 496 results in
progressive reduction of the activity of the 89 to +244 promoter
identifying a negative regulatory element between nt 142 and 89.
A factor present in nuclear extracts from endothelial and
nonendothelial cells binds to an AT-rich sequence located between nt
133 and 125. Mutagenesis of the AT-rich sequence interferes with
nuclear protein binding and restores the activity of the 142 to
+244 fragment to the level of the 89 to +244 promoter. Binding
of the nuclear protein to the vWF AT-rich sequence in mobility shift
assays is inhibited by competition with a consensus Oct-1 binding site
and with a silencer octamer-like sequence from the vascular cell
adhesion molecule-1 (VCAM-1) promoter. Subsequent supershift
experiments identified Oct-1 as the transcription factor that binds to
vWF and VCAM-1 silencer elements. These results indicate that Oct-1
acts as a transcriptional repressor of promoters of genes expressed in
endothelial cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
VON WILLEBRAND FACTOR (vWF) is a
multimeric adhesive glycoprotein that binds platelets to the
subendothelium at sites of vascular damage at high shear
rates.1,2 vWF is also the carrier protein for the
coagulation factor VIII, stabilizing its activity in plasma. The
cellular distribution of vWF in vivo is restricted to endothelial cells, megakaryocytes, and platelets.3-5 However, its
expression varies among different subpopulations of endothelial cells.
In adult mammals, endothelial cells exhibit differential expression of
vWF based on the location of the cell in the vascular system, whereas
in mouse embryos, the vWF gene is expressed early in vascular development in a limited subset of endothelial cells.6 mRNA studies have shown that the heterogeneity of vWF expression is regulated at the transcriptional level.7,8
The human vWF gene sequence upstream of the transcription initiation
site has been cloned9-12 and several studies have addressed the transcriptional regulation of the human promoter in bovine endothelial cells.13-15 We have shown previously that an
ubiquitous core promoter, spanning nucleotides (nt) 89 to +19,
was repressed in all cell types by an upstream negative regulatory
region.13 A region spanning 487 nt of the 5 -flanking
region and 247 nt of the first nontranslated exon of the human vWF gene
was identified by another group as a cell-type specific promoter in
bovine aorta endothelial cells (BAECs).14,15 This region
includes the core promoter, a negative regulatory region spanning nt
312 to 487, and a cell-specific positive regulatory
region in the first exon. The positive regulatory region contains a
GATA transcription factor binding site that is required for expression.
The inhibitory effect of the negative regulatory region was
subsequently attributed to an NF1-like cis-acting element spanning nt
440 to 470.15 In transgenic mouse embryos,
the 487 to +246 promoter fragment was active in the yolk sac
vasculature, but in adult animals only brain endothelial cells
expressed the transgene.16
The aim of this study was to analyze the transcriptional activity of
human vWF gene promoter fragments in human umbilical vein endothelial
cells (HUVECs). We report that a promoter fragment spanning nt
496 to +244 is not active in HUVECs and identify a novel
negative regulatory element (NRE) that represses basal promoter
activity in human endothelial and HeLa cells. The core sequence of this
NRE is located directly 5 of a cell-specific promoter fragment
spanning nt 89 to +244 and contains an AT-rich octamer-like
binding sequence. The vWF promoter AT-rich NRE is characterized here as
an octamer-like binding site related to the previously described
vascular cell adhesion molecule-1 (VCAM-1) promoter silencer
sequences.17 The transcription factor binding in vitro to
the vWF and VCAM-1 NREs is identified as the ubiquitously expressed POU
family protein Oct-1.
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MATERIALS AND METHODS |
Plasmid constructions.
The pGvWCAT plasmids, containing 147, 420, and 1286 nt upstream of the
transcription initiation site and 19 nt of the first exon of the vWF
gene, have been described previously.13 The plasmids
containing inserts 89 to +19 and 500 to +19 have been constructed following the same methodology. To construct the plasmid pGvW(496/+244), a 496/+244 fragment was synthesized by
polymerase chain reaction (PCR) from the parental plasmid
pBvWE610 with the forward primer (Xba I site
underlined): 5-GAT CTA GAA TTC AAG ACC TTC ATC TTT AGC
CGA T-3 and the reverse primer (Sac I site underlined):
5-TAG AGC TCG AGC TGC AAA TGA GGG CTG CGG CT-3.
The PCR fragment was cut by Xba I and Sac I,
gel-purified, and inserted between the Xba I and Sac I
sites of the multiple cloning site of the vector pGCAT-A.18
To create the plasmid pGvW(89/+244), the construct
pGvW(496/+244) was cut with HindIII at 89 inside
the vWF gene 5-flanking sequence and the restriction site was
treated with Klenow DNA polymerase. After cutting with Sac I,
the resulting vWF promoter fragment 89/+244 was cloned between
the Sma I and Sac I sites of the multiple cloning site of pGCAT-A. To construct pGvW(142/+19) and
pGvW(142/+244), two fragments 142/+19 and 142/+244
were generated by PCR from the parental plasmids pGvW(500/+19)
and pGvW(496/+244), respectively. The forward primer
5-CAA GGC AGT TAA TTA AGG CAG C-3 was complementary to
the vWF promoter, whereas the reverse primer 5-GAG CAA ATG ACT
GAA ATG CCT-3 was complementary to the CAT gene. The amplified hybrid vWF-CAT fragment was cloned into pCRScript(Amp) (Stratagene Inc,
La Jolla, CA). The vWF gene fragment was isolated by BamHI and
Kpn I digestion and inserted into the multiple cloning site of
pGCAT-A. Plasmids pGvW(142m/+19) and pGvW(142m/+244) were constructed in the same manner with the upstream primer 5-CAA GGC AGT TAC CTA AGG CAG C-3 containing point mutations
at nt 130 and 129 (mutations of the wild-type sequence
are underlined). To generate plasmid pGvW(89/+244)GATAm, a
mutation of the GATA cis-element at +221 in the first exon of
the vWF promoter was obtained with the transformer mutagenesis kit
(Clontech, Palo Alto, CA) using the reverse mutagenesis primer
5-GGG CTG CGG CTG TTT CCA AGG TCC C-3
(mutations of the wild-type sequence are underlined). The control
plasmid pSVGCAT-A has been described.18
Fragments of the 5-flanking sequence of the vWF gene were
inserted in front of the Herpes simplex virus thymidine kinase (TK)
promoter of plasmid pBLCAT2.19 The fragments spanning nt 147 to 89 and nt 420 to 89 were isolated
from the pGvW(147/+19) and pGvW(420/+19) constructs by
digesting 5 in the multiple cloning site of pGCAT-A with
BamHI and 3 with HindIII at nt 89 inside
the vWF gene 5-flanking sequence. The cohesive ends were filled
in with Klenow DNA polymerase and ligated into the blunt-ended BamHI site of pBLCAT2.
The orientation and boundaries of all constructs and complete sequences
of fragments obtained by PCR were confirmed by sequencing with the T7
polymerase sequencing kit from Pharmacia LKB Biotechnology Inc
(Uppsala, Sweden).
Cell cultures and transfections.
HeLa, COS-7, and megakaryoblastic Dami cells were from the American
Type Culture Collection (ATCC; Rockville, MD). The cells were cultured
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum (FCS), penicillin (100 U/mL), and streptomycin (100 µg/mL). Calf pulmonary artery endothelial (CPAE) cells (ATCC) and
BAECs were cultured in OptiMEM (Life Technologies, Cergy-Pontoise, France) supplemented with 10% FCS, penicillin (100 U/mL),
and streptomycin (100 µg/mL). HUVECs were isolated from human
umbilical cord veins as described.5 The cells were cultured
in growth medium consisting of MCDB 107, containing 20 U/mL penicillin, 20 µg/mL streptomycin, 30 µg/mL endothelial cell growth supplement (Collaborative Biomedical Products, Becton Dickinson, Bedford, MA), 90 µg/mL heparin (Sigma, St Quentin, France), and 10% FCS (Hyclone
Europe, Aalst, Belgium). MCDB 10720 is prepared by adding
15.1 mg/L glycine and 149 mg/L KCl to medium MCDB 105 (Sigma).
Transfections of BAECs, CPAE cells, and HeLa cells were performed by
the calcium phosphate-DNA coprecipitation method.21 The
precipitate, obtained with 15 µg of the various CAT reporter plasmids
and 0.5 µg of the control plasmid pCMV (Clontech), coding for
-galactosidase, was incubated with the cells for 24 hours. The
medium was changed and cells were cultured for another 24 hours.
HUVECs were transfected by electroporation as described
previously.22 Briefly, cells at passage 2 were synchronized
in the G2/M phase, harvested, and resuspended in electroporation buffer (MCDB 107 without any addition but 5% FCS) at 4 × 106 cells/mL. The cell suspension (0.75 mL) and the plasmid
solution were added to 4-mm electrode gap cuvettes (Bio-Rad, Ivry,
France; or Eurogentec, Liège, Belgium) containing 20 or 30 µg
of reporter plasmid, 0.5 µg of pCMV as internal control plasmid,
and 30 µg of plasmid pBS (Strategene, La Jolla, CA) as carrier
DNA. Electroporation was performed at room temperature at a voltage of
360 V and a capacitance of 1,500 µF with a Cellject electroporator
(Eurogentec, Liège, Belgium). After electroporation, the cells
were immediately transferred into 4 mL of complete growth medium in
60-mm diameter culture dishes coated with gelatin (1% solution in
phosphate-buffered saline [PBS]).
After 48 hours of incubation, transfected cells were lysed by four
freeze-thaw cycles in TE buffer (100 mmol/L Tris-HCl, pH 7.8, 5 mmol/L
EDTA). -Galactosidase activity was assayed as
described.22 CAT activity was measured by a dual-phase
diffusion assay23 in the presence of 1.4 µmol/L of
[3H] acetyl-coenzyme A (200 mCi/mmol/L; Amersham, Little
Chalfont, Bucks, UK), and 1 mmol/L chloramphenicol. The
[3H] acetylchloramphenicol partitioned into the organic
phase and was measured over a period of several hours in a TopCount
counter (Canberra-Packard, Aalst, Belgium) and the rate of
CAT enzyme activity during the linear phase was expressed as counts per
minute (cpm). CAT activity was normalized by the
-galactosidase activity and expressed as relative CAT activity.
Mobility shift assays and DNase I footprints.
To perform mobility shift assays and footprints, nuclear extracts from
HUVECs, CPAE cells, Dami cells, and HeLa cells were prepared by the
method of Dignam et al.24 Mobility shift assays were
performed with the indicated wild-type or mutated vWF promoter fragment
spanning nt 142 to 89, which were labeled with Klenow DNA
polymerase and [ -32P] dCTP (3,000 Ci/mmol/L; Amersham)
or double-stranded oligonucleotides end-labeled with
[ -32P]ATP or [-33P]ATP (3,000 Ci/mmol/L; Amersham) using polynucleotide kinase (Boehringer, Mannheim,
Germany), annealed, and purified by polyacrylamide gel electrophoresis.
Two micrograms of HeLa or 4 µg of HUVEC nuclear extracts was
preincubated for 5 minutes at 4°C in the absence or in the presence
of competitors in binding buffer consisting of 2 µg of poly(dI-dC), 1 mmol/L dithiothreitol (DTT), 10 mmol/L HEPES, pH 7.8, 50 mmol/L KCl, 1 mmol/L MgCl2, 0.1 mmol/L EDTA, 0.1% NP-40, 1 mmol/L spermidine, and 10% glycerol. The probes were added and the
reaction mixture was then incubated for another 15 minutes at 24°C.
The samples were loaded on a nondenaturing 5% polyacrylamide gel run
in TGE (25 mmol/L Tris, pH 8.3, 250 mmol/L glycine, 2 mmol/L
EDTA).25 Double-stranded oligonucleotides used as
competitors were as follows: ROR: 5-TCG ACT CGT TAT AAC TAG GTC
AAG CGC TG-3, which contains an AT-rich binding site for the
orphan nuclear receptor ROR26,27; MLP: 5-CTA CAC
CTA TAA ACC AAT CAC CTG T-3, containing the CCAAT sequence of
the Adenovirus major late promoter28; Oct: 5-TGT CGA
ATG CAA ATC ACT AGA A-3 (Promega, Madison, WI), the
binding site for Oct-1; and VCAM: 5-TAG TGA ATT TAC ATG ATG ATG
A-3, the octamer-like silencer sequence of the VCAM-1 promoter.17
In supershift assays, the antibodies antihuman Oct-1/2 and anti-Oct-1
(Upstate Biotechnologies, Lake Placid, NY) or Pbx 1/2/3 (Santa Cruz
Biotechnology, Santa Cruz, CA) were incubated (1/20 dilution) with the
nuclear extract for 20 minutes at room temperature before the addition
of the probes.
For DNase I footprinting, a fragment spanning nt 178 to +48 was
synthesized by PCR and cloned into the Sma I site of plasmid pBS. The plasmid was cut with EcoRI and labeled with the
Klenow DNA polymerase and [-32P] dCTP, and the DNA
probe was liberated by restriction enzyme digestion with Xba I. Nuclear extract (90 µg) was preincubated for 15 minutes at 24°C
in a binding buffer consisting of 2.5 µg of poly(dI-dC), 25 mmol/L
HEPES, pH 7.9, 50 mmol/L KCl, 0,1 mmol/L EDTA, 1 mmol/L DTT, 10%
glycerol. The labeled probe was added and the reaction mixture was
incubated for 30 minutes. Samples were digested for 1 minute with 1 to
4 U of RQ1 DNase I (Promega) in the presence of 5 mmol/L
MgCl2 and 0.5 mmol/L CaCl2. DNase I was
inactivated by the addition of 12.5 mmol/L EDTA and repeated extraction
with phenol-chloroform. The samples were loaded on a 6% sequencing gel
to visualize the DNase I digestion pattern. The gel was dried and
exposed to x-ray film.
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RESULTS |
Identification of positive and negative regulatory regions in the vWF
gene promoter.
To identify important regions for the basal transcription of the human
vWF gene in HUVECs, pGvWCAT constructs containing deletions of the
5-flanking sequence were transfected into HUVECs, together with
a plasmid expressing the lacZ gene as an internal control for
transfection efficiency. To normalize between experiments, the CAT
activity of the constructs is calculated as a percentage of the mean
CAT expression obtained with plasmid pGvW(89/+19). Figure 1A shows that the fragment spanning
nt 89 to +19 represents a functional promoter in HUVECs. The
activity of this promoter is 7.6-fold lower in HUVECs than the SV40
promoter construct pSVGCAT-A. Extending the promoter region to nt
142 in the construct pGvW(142/+19) resulted in a 60%
reduction of transcriptional activity compared with plasmid
pGvW(89/+19). This result suggests the presence of an NRE
located between nt 89 and 142.
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| Fig 1.
Expression of CAT reporter gene constructs containing
deletions of the 5-flanking region of the vWF gene and 19 nt or
244 nt of the first exon. (A) Depicted on the left side of the figures are the vWF promoter-CAT deletion constructs containing 19 nt or 244 nt
of the first exon (pGvW vectors) and pSVGCAT-A (containing the SV40
early promoter). Thirty micrograms of the CAT plasmids was transfected
into HUVECs in the presence of 0.5 µg of pCMV plasmid and 30 µg
of carrier plasmid pBS. Forty-eight hours later, extracts were
prepared and the level of CAT activity was measured, normalized for
-galactosidase activity, and expressed relative to the CAT activity
of plasmid pGvW(89/+19). Plasmid pGvW(+244/89) was
transfected into HUVECs as a reference for background CAT activity. The
results are the mean ± SD of 4 to 6 experiments. (B) HUVECs and HeLa
cells were transfected with the pGvW vectors indicated on the left side
as described in the Materials and Methods. The level of CAT activity
was measured, normalized for -galactosidase activity, and expressed
relative to the CAT activity of plasmid pGvW(89/+19). ( )
HUVECs; ( ) HeLa cells.
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To investigate whether the first exon could alleviate the repression
mediated by the NRE situated between nt 142 and 89, we
transfected HUVECs with plasmids containing the insert 89 to
+244, 142 to +244, or 496 to +244 (Fig 1A). The level of CAT activity of construct pGvW(89/+244) in HUVECs is comparable to the CAT activity of plasmid pGvW(89/+19), which contains the core promoter alone. The promoter activity of plasmid
pGvW(142/+244) is markedly reduced in HUVECs, whereas the
activity of pGvW(496/+244) is close to the background observed
with a control plasmid pGvW(+244/89). Therefore, the NRE,
identified between nt 89 and 142, represses the 89
to +244 promoter to the same extent as the 89 to +19 core
promoter in HUVECs. Sequences in the first exon are not sufficient to
relieve the inhibition mediated by this NRE. The results also suggest
that other NREs are located upstream of nt 142.
The 89 to +244 vWF gene promoter fragment that contains a GATA
sequence at position +221 exhibits transcriptional activity in HUVECs
(Fig 1A). The study of the human endothelin promoter showed the
importance of GATA binding sites for the control of endothelial
cell-specific expression.29,30 To examine further the role
of the GATA sequence in the first exon of the vWF gene, this element
was mutated to AACA to construct the plasmid
pGvW(89/+244)GATAm. As shown in Fig 1B, CAT activity of the
mutant construct in HUVECs is reduced to 20% compared with the
activity of plasmids pGvW(89/+19) and pGvW(89/+244). This
result demonstrates that the GATA element at +221 is essential for the
transcriptional activity of the 89 to +244 promoter in HUVECs.
The construct pGvW(89/+19) is also active in HeLa
cells13,14 (Fig 1B). However, the vectors
pGvW(89/+244) and pGvW(89/+244)GATAm are not active in
HeLa cells. These data suggest that the GATA binding site is not
functional in HeLa cells and that silencing sequences, which are
located in the first exon and which are distinct from the GATA binding
site, contribute to prevent expression of the vWF promoter.
In a study performed by another group in BAECs, the activity of a
90 to +20 core promoter linked to the growth hormone gene was
also repressed due to an upstream NRE located between nt 312 and
487.15 However, the activities of a 487 to
+247 promoter and of the core promoter were similar, indicating that
the repression mediated by the NRE was relieved when the entire first
exon was included in the constructs. We also analyzed an equivalent CAT reporter plasmid pGvW(496/+244) in BAECs, cultured between
passage 5 and 8, and in CPAE cells, which express vWF. In our
experiments (Fig 2), the construct
pGvW(496/+244) does not express CAT in either of those bovine
cells.
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| Fig 2.
Expression of CAT reporter gene constructs in bovine
endothelial cells. BAECs and CPAE cells were transfected with 10 µg of the CAT reporter vectors pGvW(89/+19) or
pGvW(496/+244) and 0.5 µg of pCMV plasmid. The level of CAT
expression, normalized for -galactosidase activity and expressed
relative to the CAT activity of plasmid pGvW(89/+19), is shown.
( ) BAECs; ( ) CPAE. The results are the mean ± SD of 3 experiments.
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The negative cis-regulatory regions of the vWF promoter inhibit the
transcriptional activity of a heterologous promoter.
To test whether the NREs identified between 496 and 89
could inhibit the transcriptional activity of a heterologous promoter, two fragments spanning nt 420 to 89 and 147 to
89, respectively, were subcloned upstream of the ubiquitous TK
promoter in plasmid pBLCAT2 to construct the plasmids
pBvW(420/89)TK and pBvW(147/89)TK. The CAT
activities of the constructs transfected into HUVECs and HeLa cells are
presented in Fig 3. When the longer
420 to 89 fragment is inserted into the construct, the
CAT activity of plasmid pBvW(420/89)TK decreases by more
than 70% in HUVECs and HeLa cells. The fragment 147 to
89 retains the capacity to reduce TK promoter activity by more
than 50% in both endothelial and nonendothelial cells. Furthermore,
the DNA fragment spanning nt 147 to 89 inhibits the TK
promoter when cloned in reverse orientation. These results confirm that
the NRE situated between nt 142 and 89 is sufficient to
inhibit the transcriptional activity of the heterologous TK promoter in
both orientations.
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| Fig 3.
Effect of negative regulatory elements in the vWF gene
promoter on the transcriptional activity of the TK promoter.
Blunt-ended fragments of the vWF gene 5-flanking sequence were
ligated in the sense or antisense direction into the BamHI site
of pBLCAT2 upstream of the TK promoter. Transfections were performed in
HUVECs and HeLa cells as described in the Materials and Methods.
Forty-eight hours after transfection, cell extracts were prepared and
aliquots, normalized for transfection efficiency, were used for the
determination of CAT activity. The inserts are schematized on the left.
The CAT activity of the vector pBLCAT2 with the TK promoter alone is
set at 100%. The CAT activities associated with the other vectors are
expressed relative to the activity of pBLCAT2 and are given on the
right. () HUVECs; () HeLa cells. The results are the mean ± SD
of 4 to 6 experiments.
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HUVECs and HeLa nuclear proteins bind to the 142 to 89
NRE.
To determine whether the silencing activity of the 142 to
89 region correlates with the binding of nuclear factors from endothelial and nonendothelial cell nuclear extracts, DNase I footprintings were performed with a 32P-end-labeled
restriction fragment spanning the vWF gene from 178 to +48.
Analysis of the sense strand of the probe shows that a region between
nt 126 and 133 is protected from DNase I digestion in the
presence of endothelial (CPAE and HUVECs), megakaryoblastic (Dami), and
HeLa nuclear extracts (Fig 4). This region
includes the sequence TTAATTAA, an octamer-like AT-rich palindrome.
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| Fig 4.
DNase I footprint of the 142 to 89 NRE. A 178 to
+48 fragment was labeled at 178 on the antisense strand and
incubated for 30 minutes with 90 µg of nuclear extract from CPAE
cells, HeLa cells, Dami cells, or HUVECs or 90 µg of BSA and used in footprinting analysis as described in the Materials and Methods. The
126 to 133 protected sequence is shown on the right. G+A lane
corresponds to cleavage at bases G and A by Maxam-Gilbert chemical
sequencing of the probe.
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To confirm that the AT-rich sequence is part of a binding site for one
or several nuclear factors, mobility shift assays were performed with
labeled wild-type (WT) 142 to 89 or mutant (Mut) 142 to 89 fragment as probes. In the Mut probe, the
AT-rich sequence TTAATTAA was mutated to TTACCTAA
(Fig 5A). For competition studies,
reactions were performed with an excess of cold WT or Mut fragment.
After incubation with HUVEC nuclear extracts, the 142 to
89 probe forms two retarded DNA-protein complexes in the absence
of competitor (arrows 1 and 2 in Fig 5B, left panel). Complexes in
bands 1 and 2 are specific, because binding to the labeled WT probe is
almost abolished with 25-fold molar excess of unlabeled WT fragment and
binding is no longer observed with higher molar excess of competitor.
The mutant competitor does not interfere to the same extent as the WT
fragment with the formation of the major complex (band 1). Even at a
100-fold molar excess of Mut fragment, complex 1 can still be detected.
Band 2 is weak, but the relative intensities of band 1 and band 2 appear to be unchanged in the presence or the absence of Mut
competitor. Unrelated oligonucleotides were next used as competitors in
the mobility shift assays with HUVEC nuclear extract to confirm that
the 142 to 89 probe binds sequence-specific nuclear
proteins. The oligonucleotides used were ROR, which contains an AT-rich
binding site for the orphan nuclear receptor ROR, and MLP,
containing the CCAAT sequence of the Adenovirus major late promoter.
Complexes 1 and 2 are not affected by competition with a 500-fold molar
excess of these oligonucleotides, except for a slight decrease of the
intensity of band 1 in the presence of large quantities of ROR. The
labeled mutant probe does not bind significant amounts of HUVEC nuclear proteins (Fig 5B, last lane in left panel), confirming that binding of
the nuclear factors in complexes 1 and 2 is greatly reduced by mutation
of the AT-rich sequence.
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| Fig 5.
Detection of nuclear proteins interacting with the 142
to 89 NRE of the vWF gene by mobility shift assays. (A) 5-end
of the sequence of the 142 to 89 wild-type probe and of the
142 to 89 mutant probe. The mutated basepairs are underlined. (B) The restriction enzyme fragments were labeled and incubated with nuclear extracts from HUVECs or HeLa cells before electrophoresis as
described in the Materials and Methods. The competitors were wild-type
fragment (WT), mutant fragment (Mut), ROR, and MLP oligonucleotides at
the indicated molar excess. Arrows designate the specific retarded complexes as described in the text.
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With nuclear extracts from HeLa cells (Fig 5B, right panel), two bands
corresponding to complexes 1 and 2 are also detected. Again, a 25-fold
molar excess of WT fragment competes more efficiently for the formation
of the major complex 1 as compared with a 25-fold molar excess of Mut
fragment. An additional band, indicated by arrow 3, is also visible
without competitor or with a 10-fold excess of WT competitor. In the
presence of 100-fold excess of Mut competitor, the complex in band 3 is
completely abolished in contrast to the complex in band 1. Therefore,
the formation of this complex seems to be independent of the mutated
nucleotides in the AT-rich sequence.
To further investigate the binding of the specific nuclear factors to
the AT-rich sequence, a double-stranded oligonucleotide (NRE) covering
the same 142 to 89 region as the restriction enzyme
fragment (Fig 5) was synthesized and used as probe in a mobility shift
assay. Two shorter fragments encompassing nt 142 to 114
(NRE1) and nt 113 to 85 (NRE2), respectively, were used as competitors. The full-length 142 to 89 NRE was
end-labeled and incubated with HUVEC nuclear extracts in the absence
and in the presence of NRE, NRE1, or NRE2 as competitors.
Figure 6 shows that, in the absence of
competitor, a major band corresponding to complex 1 is again observed
with the NRE probe. However, additional bands are also present, among
them the minor complex 2. The wild-type NRE sequence competes for the
formation of complex 1. Complex 1 is completely abolished in the
presence of NRE1 spanning nt 142 to 114 and including the
AT-rich binding site. The downstream oligonucleotide NRE2 (nt
113 to 85) is not effective in preventing the formation
of the DNA-protein complexes, indicating that it does not contain
high-affinity nuclear protein binding sites. Results from Figs 5 and 6
are consistent with the hypothesis that a nuclear protein interacts
with the octamer-like AT-rich sequence spanning nt 133 to
126 to form the major complex 1.
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| Fig 6.
The high-affinity nuclear protein binding site in the vWF
gene promoter NRE is located inside the sequence NRE1 (142 to
114). The double-stranded oligonucleotide NRE (142 to 89) was
labeled, incubated with nuclear extracts from HUVECs, and analyzed as
described in the Materials and Methods. Unlabeled double-standed
competitors NRE, NRE1 (142 to 114) and NRE2 (113 to 85)
were included at 50-, 100-, and 200-fold molar excess. The major
complex 1 is indicated by an arrow.
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The AT-rich binding site is a negative cis-acting regulatory element.
The octamer-like AT-rich sequence, located between nt 133 and
126 of the negative regulatory region of the vWF gene promoter, is a binding site for nuclear protein(s) present in nuclear extracts from HUVECs and HeLa cells. To examine whether this AT-rich motif is
responsible for the silencing of the 142 to +19 and 142
to +244 promoter fragments, the same point mutations at position 130 and 129, which inhibit DNA-protein interaction (Fig
5A), were introduced into the constructs pGvW(142m/+19) and
pGvW(142m/+244). The reporter construct pGvW(142/+19)
exhibits reduced transcriptional activity in HUVECs and HeLa cells
compared with pGvW(89/+19) (Fig 7A).
Mutation of the AT-rich sequence in plasmid pGvW(142m/+19) restores the level of CAT activity to that obtained with the core (89/+19) promoter. As shown in Fig 7B, the double base mutation in the AT-rich sequence also increases the transcriptional activity of
construct pGvW(142m/+244) to 75% of the activity of the
89 to +244 promoter in HUVECs. These results suggest that the
nuclear protein(s) binding to the AT-rich sequence is involved in the negative regulation of the activity of the human vWF gene promoter.
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| Fig 7.
Functional analysis of mutations in the AT-rich sequence.
HUVECs and HeLa cells were transfected as described in the legend to
Fig 1. (A) The vectors pGvW(89/+19), pGvW(142/+19), and
pGvW(142m/+19) are depicted on the left side of the figures. The
construct pGvW(142m/+19) is mutated at nt 130 and 129 inside
the AT-rich sequence. The CAT activities associated with these vectors
are expressed relative to the activity of pGvW(89/+19), which is
set at 100%, and are given on the right. () HUVECs; () HeLa
cells. (B) The vectors pGvW(89/+244), pGvW(142/+244), and
pGvW(142m/+244) are shown on the left side of the panel. The CAT
activities in HUVECs are expressed relative to the activity of the
plasmid pGvW(89/+19). The results are the mean ± SD of 4 to 6 experiments.
|
|
Oct-1 binds to related NREs in the vWF and VCAM-1 promoters.
The AT-rich NRE of the vWF gene promoter conforms to the canonical
homeodomain binding sequence TAATTA and exhibits limited homology with
the octamer-1 (Oct-1) transcription factor binding site ATGCAAATAA.
Octamer-like sequences in the human VCAM-1 promoter were shown to act
as negative regulatory elements in endothelial and nonendothelial
cells.17 To investigate whether both the vWF AT-rich and
VCAM-1 octamer-like NREs are recognized by the same octamer binding
factors, we used as competitors in mobility shift assays the
oligonucleotides Oct, containing the cognate Oct-1 binding sequence,
and VCAM, consisting of the VCAM-1 silencer sequence. The probe NRE1
was incubated with HeLa nuclear extracts and different amount of these
competitors (Fig 8). At a 100-fold molar
excess, Oct and VCAM compete as effectively as NRE1 for the binding of
the nuclear factor in complex 1, suggesting that a transcription factor
of the Oct family binds both to the vWF and VCAM NREs. To identify the
protein involved in vWF and VCAM-1 transcriptional repression, a
monoclonal antibody that recognizes the Oct-1 and Oct-2
POU-homeodomains was added to the binding reactions. In the presence of
the antibody, complex 1 is abolished and partly supershifted (Fig 8B,
left panel). This result identifies the vWF repressor as Oct-1, because
Oct-2 is only expressed in lymphoid cells. A control antibody, directed
against the homeodomain of the Pbx family of transcription factors, had
no effect on the formation of the band. In Fig 8B (right panel), a
second antibody directed specifically against the Oct-1 POU-domain also
abolishes the formation of complex 1. Mobility shift assays with the
labeled Oct-1 consensus sequence (probe Oct) gives rise to a nuclear
protein-DNA complex migrating at the same level as complex 1 (Fig 8C).
The vWF NRE1 and the VCAM octamer-like sequence, but not NRE1m, are able to compete for the binding of this nuclear protein to its cognate
octamer binding site. This protein is also clearly supershifted by the
Oct-1/2 antibody. Finally, the formation of complex 1 with the VCAM-1
probe is abolished (Fig 8D) by competition with NRE1 and Oct. Complex 1 is again supershifted in the presence of the Oct-1/2 antibody.
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| Fig 8.
Identification and characterization of Oct-1 binding to
the vWF AT-rich NRE, to the consensus Oct-1 binding site, and to the VCAM-1 promoter octamer-like sequence. Double-stranded oligonucleotides were labeled and incubated with nuclear extracts from HeLa cells before
electrophoresis as described in the Materials and Methods. (A)
Sequences of the probes used for MSA. These probes spanned the vWF
AT-rich NRE between nt 142 and 114 (NRE1), the binding site for
the bipartite POU domain of Oct-1 (Oct), and the octamer-like silencer
element in the VCAM-1 promoter (VCAM). The competitors NRE1, NRE1m (the
mutated base pairs in the AT-rich element are underlined), Oct, and
VCAM were used at the indicated molar excess. (B, C, and D) Mobility
shift assays with probes NRE, Oct, and VCAM. Arrows show the specific
retarded complex 1 and the supershifted complexes between Oct-1 and the
probes (S) as described in the text. Supershifts with probe NRE were
performed with two different antibodies, an anti-Oct-1/2 antibody (B,
left panel) and an antibody specific to Oct-1 (B, right panel). The
supershifted complex S is situated just underneath the level of the
wells of the polyacrylamide gel.
|
|
Taken together, these results establish that the protein that forms
complex 1 with the vWF AT-rich NRE and the VCAM-1 octamer-like silencer
is the ubiquitously expressed POU-domain transcription factor Oct-1
acting as a repressor.
| |
DISCUSSION |
To determine the molecular mechanisms contributing to the expression of
vWF in human endothelial cells, we have studied the transcriptional
regulation of the vWF gene. Using nested deletions of the
5-region linked to a CAT reporter gene, we identified a core
promoter, spanning nt 89 to +244, that is active in HUVECs but
not in HeLa cells. This DNA fragment contains the first nontranslated exon of the vWF gene and was previously shown to be a functional promoter in BAEC.14 The transcriptional activity of this
promoter region in HUVECs depends on the presence of an intact GATA
binding site at position +221. Indeed, the mutation of the GATA binding site abolishes the activity of the core promoter in HUVECs and does not
restore transcriptional activity in HeLa cells. These results show that
the 89 +244 vWF gene promoter contains a silencer sequence,
which is different from the GATA element. The silencer is offset by the
positive GATA element in HUVECs only. It may thus be hypothesized that
the GATA site contributes to the activity of the 89/+244
promoter fragment in endothelial cells by overcoming the effect of a
repressor localized in the first exon. However, endogenous GATA-binding
proteins (such as GATA-2) are expressed by HeLa cells.29
Therefore, the 89 to +244 fragment may not be an effective
promoter in HeLa cells, because the level of expression of the
functional GATA-binding protein is too low. Another possibility is that
a distinct (and perhaps endothelial cell-restricted) GATA-binding protein is responsible for cell-specific derepression in HUVECs.
vWF is expressed exclusively in two cell types, endothelial cells and
megakaryocytes. The basal transcription of other genes, which are
either expressed in endothelial cells (endothelin,29,30 platelet/endothelial cell adhesion molecule-1,31
endothelial nitric oxide synthase32) or which are expressed
in megakaryocytes (glycoprotein IIb33), requires the
presence of GATA cis-acting regulatory sequences. Several
megakaryocytic gene promoters contain GATA sequences that are active in
conjunction with Ets sequences to establish positive promoter
activity.33-38 Recently, we have identified functional Ets
binding sites in the core vWF promoter,39 and it could be
hypothesized that GATA and Ets family members contribute in a
coordinated fashion to the endothelial cell-restricted activity of the
vWF promoter.
In this study, we have identified an NRE between nt 142 and
89 of the vWF gene promoter that represses the transcriptional activity of the 89 to +244 promoter fragment in HUVECs. The NRE also inhibits the 89 to +19 promoter fragment and the TK
promoter in both HUVECs and HeLa cells. The core sequence of the NRE
has been mapped to an AT-rich palindromic octamer (TTAATTAA) between nt
133 and 126. Site-directed mutagenesis in this core
sequence sharply reduces protein-DNA interaction and abolishes the
NRE-mediated repression in transfection assays.
Our experimental results confirm that the vWF NRE is bound by nuclear
proteins related in their binding specificity to the Oct family of
transcription factors. Indeed, the protein binding to the AT-rich NRE
is recognized by antibodies directed against the POU domain of the
widely expressed transcription factor Oct-1. Therefore, we conclude
that the protein that binds to the NRE and that is involved in the
negative regulation of vWF gene promoter is Oct-1. The AT-rich NRE of
the vWF gene promoter conforms to the consensus homeodomain binding
sequence, TAATTA.40,41 The homeodomain, a structural DNA
binding module that contains the helix-turn-helix (HTH) motif, occurs
in a large family of proteins that regulate
transcription.42,43 Among transcription factors exhibiting
the homeodomain motif are Oct-1 and Oct-2 (only expressed in lymphoid
cells44), which contain the composite POU domain, a
conserved DNA binding region of approximately 160 amino
acids.45 The structure of the Oct-1-DNA complex has been
determined by nuclear magnetic resonance and by crystallography,
showing that the preferred binding site of Oct-1 is related to the vWF
AT-rich element.46,47 The ability of Oct-1 and other POU
domain factors to bind to a variety of degenerate octamer motifs is
well established48 and could represent an important feature
of their transcriptional regulatory function.
The transcriptional regulation of the VCAM-1 gene in endothelial cells
has been extensively studied.17,49,50 Octamer-like sequences act as silencers to prevent VCAM-1 expression in unstimulated HUVECs and in other cell types.17,49 Experimental evidence presented in this report clearly shows that the extensively
characterized VCAM-1 repressing element located at nt 1554 is
related to the vWF AT-rich NRE and to the Oct-1 binding site. We
demonstrate in this study for the first time that the VCAM-1
octamer-like silencer is bound by Oct-1. Therefore, Oct-1 may play an
important role in the well-characterized promoter repression mechanism
of the VCAM-1 promoter51 and in the negative regulation of
the vWF promoter both in HUVECs and in other nonendothelial cell types.
The role of Oct-1 as a transcriptional repressor is further
strengthened by recent studies that have implicated Oct-1 in the downregulation of the human pituitary-specific transcription factor pit1/ghf1 gene promoter52 and the repression of the
human immunodeficiency virus (HIV) long terminal
repeat.53 In addition, a detailed study54 of
the human thyrotropin subunit (hTSH ) gene promoter has
shown that Oct-1 mediates silencing of this promoter through AT-rich,
degenerate Oct-1 binding sites. As is the case for the vWF AT-rich NRE,
the Oct-1-mediated silencing was acting in all cell types, including
HeLa cells. Oct-1 was found to possess an intrinsic silencing activity
in the carboxy-terminal domain.54
In response to TNF, NF- B transcription factor family members bind
to the VCAM-1 promoter in HUVECs and activate transcription by
overcoming the negative effect of the octamer-like silencers. In
skeletal muscle cells, the inhibition of the VCAM-1 promoter can also
be lifted by a cell-type-specific and position-specific enhancer
located between the TATA box and the transcription initiation start
site.17 A similar mechanism is found in the hTSH
promoter, in which Oct-1-mediated silencing is overcome in thyrotrops
by an upstream enhancer in the 10,000 to 1,200
region.54 By analogy, we would hypothesize
that endothelial cell-specific enhancer sequences are located in
the vWF promoter upstream or downstream of the sequences analyzed in
this study. In this study, the extension of the upstream flanking
sequence to nt 496 does not lift the repression of the
496 to +244 vWF promoter but leads to even stronger inhibition
in HUVECs. The results from these deletion experiments therefore point
to the presence of other NREs between nt 496 and 142 and
suggest that putative enhancer sequences could be located in the
introns and/or in more upstream sequences.
Jahroudi and Lynch14 have reported that the
GATA site at position +221 was necessary and sufficient for
cell-type-specific transcriptional activity in BAE cells of a human
vWF promoter fragment spanning nt 487 to +247, which was active
at the same level as a 90/+20 promoter. In this report, we show
that an analogous 496 to +244 promoter region fused to the CAT
reporter gene lacks the specific sequences required for high promoter
activity in HUVECs, in CPAE cells, and in BAECs. These results suggest
that this promoter fragment is dependent on the cell line used or that the different reporter vectors used in these two studies (growth hormone v CAT) influence cell-type-specific activity. However, our previous study on the transcriptional regulation of the human vWF
promoter in CPAE cells13 and the results in human
endothelial cells presented in this report show a mechanism of promoter
repression involving Oct-1, which was not observed in the study in
BAECs. Furthermore, when the same 487 to +246 vWF promoter
fragment that shows transcriptional activity in BAECs was fused to the Escherichia coli lacZ gene and was used to generate
transgenic mice,16 only a limited subpopulation of
embryonic yolk sac endothelial cells and adult brain endothelial cells
expressed the LacZ construct. No expression was detected in
adult mouse endothelial cells outside the brain. Recently, Aird et
al55 demonstrated that a longer human vWF gene fragment,
spanning 2,182 bp of 5 flanking sequence, the first exon, and
first intron, is active in the endothelial lining of blood vessels in
the brain, heart, and skeletal muscle, contrasting again with the much
more widespread expression of the endogenous vWF gene. The expression
of the transgene was dependent on the tissue environment. Taken
together, the results of studies in bovine endothelial
cells,13-15 in HUVECs, and in transgenic mice suggest that
transcriptional repression of the human vWF gene promoter by POU domain
proteins and other trans-acting factors is overcome in a
tissue-specific manner through positive cis-acting elements
that may be different in endothelial cells of various origins and
depend on the tissue environment.
POU domain proteins play an important role in embryonic development and
the establishment of distinct cell lineages.56 As we have
shown in this study, Oct-1, a member of the POU domain transcription
factor family, may be involved in the transcriptional regulation of two
important genes expressed by endothelial cells.
| |
FOOTNOTES |
Submitted September 3, 1997;
accepted April 9, 1998.
J.-L.S. was supported by a grant from the Ministère de
l'Education Nationale, Luxembourg. N.J. was supported by the
Ministère de l'Enseignement Supérieur et de la Recherche,
France. J.E.R. and D.H. were supported by funds from the Research
Council of the University of Leuven (OT/94/22).
Address reprint requests to Jean-Luc Schwachtgen, PhD,
Vascular Diseases Unit, Glaxo Wellcome, Medecines Research Centre, Gunnels Wood Road, Stevenage, Herts., SG1 2NY, UK.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Lydie Barek and Annick Ganieux for excellent
technical assistance. We thank Dr Philippe Huber for stimulating discussions.
| |
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1997[Abstract/Free Full Text]
56.
Scholer HR:
Octamania: The POU factors in murine development.
Trends Genet
7:323,
1991[Medline]
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Abstract
Introduction
Abstract