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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3366-3380
Functional Characterization of the Human Platelet Glycoprotein V Gene
Promoter: A Specific Marker of Late Megakaryocytic Differentiation
By
Adeline Lepage,
Georges Uzan,
Nadège Touche,
Martine Morales,
Jean-Pierre Cazenave,
François Lanza, and
Corinne de
la Salle
From INSERM U. 311, Etablissement de Transfusion Sanguine de
Strasbourg, Strasbourg, France; and INSERM U. 506, Hôpital Paul
Brousse, Villejuif, France.
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ABSTRACT |
Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor
for von Willebrand factor and thrombin, is specifically found in
platelets and mature megakaryocytes. Studies of the GPV gene can
therefore provide insight into the mechanisms governing megakaryocyte
differentiation. The human GPV promoter was isolated, and elements
important for its tissue specific transcriptional activity were
localized using systematic DNase I protection and reporter deletion
assays. A  1413/+25 fragment inserted into a luciferase reporter
construct displayed promoter activity in Dami and HEL but not in K562,
HL60, or HeLa cells. Progressive 5' to 3' deletion showed a
putative enhancer region in the 1413/903 segment that contained
closely spaced GATA and Ets sites protected from DNase I digestion in
Dami extracts. Regions similar to a GPIIb gene repressor were found at
816 and 610, with the first exhibiting repressor activity in Dami
and HEL cells and the second protected from DNAse I. Deletions from
362 to 103, an area containing protected sites for Sp1, STAT, and
GATA, induced a progressive decrease in activity. The 103/+1
fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem
footprint, displayed 75% activity relative to the full-length promoter
and retained cell specificity. In summary, this work defines several
regions of the GPV gene promoter important for its activity. It
contains megakaryocyte-specific signals, including
erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like
repressor domains, and binding sites for ubiquitous factors such as
Sp1, ETF, and STAT.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
A CHALLENGING QUESTION in hematopoiesis
is how lineage-specific patterns of gene expression can arise from a
common precursor cell. The problem appears to be even more complex in the case of megakaryocyte differentiation, because megakaryocytes and
erythrocytes exhibit common features at different stages along the
differentiation path. Thus, several transcription factors, such as
GATA-1, Tal-1, Ets-1, and NF-E2, are detected in both lineages.1-4 In this context, how can a similar subset of
transcription factors differentially influence cell maturation into
megakaryocytes or erythrocytes?
Strong evidence has been obtained for the implication of GATA-1 in
regulation of the transcription of many megakaryocytic genes.5-9 GATA-1 is also important in vivo and in vitro to
direct the differentiation of definitive hematopoietic progenitors
along either the erythroid or the megakaryocytic pathway.
Overexpression of GATA-1 led to megakaryocytic differentiation of the
primitive myeloid cell line 416B, whereas no erythroid or mast cell
differentiation was observed. Levels of GATA-2 and GATA-3 transcripts
in these cells remained undetectable, which would suggest that GATA-2
and GATA-3 lie upstream of GATA-1. Moreover, enforced expression of GATA-2 or GATA-3 in 416B cells also induced megakaryocytic
differentiation, suggesting that GATA-1 is a dominant regulator of
maturation to this lineage.10
The original knock-out experiments did not show any direct link between
GATA-1 and megakaryocytic differentiation.11,12 GATA-1 / mice present severe anemia with a
total arrest of erythroid differentiation at the proerythroblastic
stage, and these precursors later die by apoptosis.13,14
Megakaryocyte-specific GATA-1 knock-out mice have been obtained more
recently. These mice have reduced platelet numbers, in association with
a deregulated proliferation of megakaryocytes and a block in their
terminal cytoplasmic maturation.15 In addition, mice with
disruption of GATA-1 express normal amounts of GPIIb and MPL (the
thrombopoietin receptor) together with about 50-fold higher levels of
GATA-2, which would suggest that GATA-2 is compensating for the loss of
GATA-1 and that GATA-1 negatively regulates GATA-2 during normal
erythroid maturation.16
Because the GATA-1 gene is located on the X chromosome, which is
randomly inactivated in every cell, heterozygous females can bear
either a wild-type or a mutant GATA-1 allele and consequently display
variable anemia or thrombocytopenia. Such heterozygous mutant mice have
a phenotype analogous to that of human myelodysplastic syndrome. There
is marked splenomegaly, anemia, and thrombocytopenia; proerythroblasts
and megakaryocytes accumulate massively in the spleen; and the animals
begin to die after 5 months. These findings suggest that the
hematopoietic progenitor cells start to proliferate but are unable to
terminally differentiate, which leads to progenitor proliferation in
the spleen and eventual death.17
Although binding sites for the transcription factor NF-E2 are present
in the promoters of many erythroid genes,18,19 inactivation of the p45 NF-E2 gene has only a mild effect on erythrocytic
differentiation. Conversely, this totally inhibits the terminal
differentiation of megakaryocytes by blocking proplatelet
formation20,21 and polyploidization,22
resulting in severe thrombocytopenia. PU.1 and Fli-1, two transcription
factor members of the Ets family, also play a role in erythroid and
megakaryocytic differentiation. PU.1 has been identified in mature
megakaryocytes and binding sites for this factor found in
megakaryocytic genes like those of glycoprotein IIb
(GPIIb)23 and  thromboglobulin
(TG).24 Overexpression of PU.1 in mice
induced erythroleukemia through blockage of erythroid
differentiation,25 and the overexpression of PU.1 on these
murine erythroleukemia cells reduced their NF-E2 expression and the
DNA-binding activity of GATA-1.26 Similarly, overexpression
of Fli-1 in K562 cells induced a megakaryocytic phenotype characterized
by markers such as GPIIb.27 Fli-1 has further been shown to
transactivate the GPIb and GPIX genes.28
In summary, GATA-1 blocks erythroid differentiation in erythroblasts
and induces the hyperproliferation of megakaryocytic progenitors. Other
factors, such as NF-E2, PU.1, and Fli-1, also have distinct effects on
erythrocyte and megakaryocyte precursors, suggesting that the same
factor could play different roles depending on its cellular context.
A traditional approach to identifying the mechanisms involved in
megakaryocytopoiesis is to examine the transcriptional control of
megakaryocyte-specific genes in various differentiated cell types. At
the functional level, the promoters most studied to date are those of
GPIIb7,29-31 and platelet factor 4 (PF4).32,33 The MPL34 and
TG24,35 promoters have also been analyzed in functional studies.
The GPIb-V-IX complex is a receptor necessary for the von Willebrand
factor-dependent adhesion of platelets to the subendothelium of damaged
blood vessels.36,37 Absence or defective expression of this
complex leads to a severe hemorrhagic disorder known as Bernard-Soulier
syndrome. GPIb-V-IX is composed of 4 subunits: GPIb (145 kD) is
disulfide-linked to GPIb (28 kD) to form GPIb, which is, in turn,
noncovalently bound to GPIX (22 kD) and GPV (82 kD) in, respectively,
1:1 and 2:1 stoichiometry.37,38 All 4 proteins are members
of the leucine-rich glycoprotein (LRG) family of adhesive
receptors.39 GPIb contains binding sites for von
Willebrand factor and thrombin.40-42 The exact functions of
GPIb and GPIX are still unknown, whereas GPV contains a cleavage site for thrombin, but its role in platelet physiology is likewise unclear.
The GPIb,5 GPIX,8,43 and GPV genes are
restricted in their expression to the platelet lineage and appear late
in megakaryocytopoiesis.44 Cloning and characterization of
the GPV gene45 have shown it to comprise a short intron in
the 5' untranslated region and a coding sequence contained
entirely in the second exon. The 5'-flanking region contains
several putative cis-acting regulatory elements, including
consensus binding sites for GATA and Ets transcription factors, as in
megakaryocyte-specific genes,29,46-48 and for ubiquitous factors such as Sp1. The rat and mouse genes are identical in structure
and have conserved many of the putative human regulatory sequences.49
In the present study, we chose to undertake a functional
characterization of the human GPV gene promoter for the following reasons. First, GPV associates in 1:2 stoichiometry with GPIb-IX and is
absent or found at low levels in most Bernard-Soulier patients, suggesting a possible coordinated control of transcription. Second, GPV
appears late in megakaryocyte differentiation (Lepage et al, manuscript
submitted), unlike, for instance, the early GPIIb and MPL
genes, which are present in CD34+CD38+
hematopoietic progenitors.50,51 GPV is also absent from a majority of megakaryocytic leukemic cell lines, unlike other
platelet-specific proteins such as PF4, GPIIb, or even GPIb-IX. This
suggests that GPV could participate in later stages of megakaryocyte
maturation and respond to late appearing growth factors or
transcription factors. Characterization of the GPV promoter could thus
help to identify transcriptional elements involved in the coordinated expression of GPIb-V-IX and in modulating the timing of megakaryocyte maturation.
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MATERIALS AND METHODS |
Cell lines.
Cell lines (American Type Culture Collection, Rockville, MD)
were as follows: HeLa (epithelioid carcinoma), Raji (Burkitt's lymphoma), K562 (chronic myelogenous leukemia), HL60 (promyelocytic leukemia), HEL (erythroleukemia), and Dami. The Dami cell line originally described as a megakaryoblastic leukemia52 has
now been recognized as an HEL derivative.53 All cell lines
were grown in RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with
10% fetal calf serum (Boehringer, Mannheim, Germany), 2 mmol/L glutamine, and 100 U/mL penicillin and streptomycin (GIBCO BRL).
Flow cytometry and antibodies.
Cells were washed in phosphate-buffered saline containing 1% bovine
serum albumin (PBS-BSA) and resuspended in PBS-BSA at a final
concentration of 5 × 105/mL. Aliquots of cells (5 × 104 per tube) were incubated for 20 minutes at
4°C with a primary antibody (20 µg/mL), washed in PBS-BSA,
resuspended in the same buffer, and labeled for 20 minutes at 4°C
in the dark with fluorescein isothiocyanate-conjugated goat
F(ab')2 antimouse IgG (FITC-GAM; 15 µg/mL; Jackson
ImmunoResearch Laboratories Inc, West Grove, PA). After further washing
in PBS-BSA, the samples resuspended in the same buffer were analyzed on
a Becton Dickinson FACSort flow cytometer with CellQuest software
(Becton Dickinson, Mountain View, CA).
The monoclonal antibody (MoAb) 5B12, which recognizes the
IIb3 integrin (GPIIb-IIIa), was obtained
from DAKO (Glostrup, Denmark). Murine MoAbs against GPIb (ALMA.12),
GPIX (ALMA.16), and GPV (V.1) were produced in our
laboratory,54 whereas the mouse MoAb Gi 27 recognizing
GPIb was provided by S. Santoso (Justus-Liebig University, Giessen, Germany).
GPV mRNA analysis.
Total RNA was extracted from cell lines by the thiocyanate-guanidium
method.55 Cells (107) were lysed in 1 mL Tri
Pure (Boehringer) and extracted into 0.2 mL chloroform, and the
extracts were centrifuged at 12,000g for 15 minutes at 4°C.
RNA was precipitated from the aqueous phase with isopropanol, and the
pellet was washed in 70% (vol/vol) cold ethanol.
Reverse transcription was performed on 100 ng total RNA using
a specific P1 primer in the noncoding orientation (5'-TAT CAG GTC
ACT GAA GGT GCC GGG GGC AA-3' from nt 2715 to nt 2687; GPV numbering throughout the manuscript according to Lanza et
al,45 EMBL/Genbank: HSGPV-Z23091). Oligonucleotides were
synthesized in an Oligo 1000 synthesizer (Beckmann, Fullerton, CA). The
reaction was performed in 30 µL polymerase chain reaction (PCR)
buffer, pH 8.3, containing 1 U reverse transcriptase (Moloney murine
leukemia virus [MMLV]-RT; GIBCO BRL), 0.1 µmol/L P1
primer, and 1.25 µmol/L of each dNTP for 30 minutes at 37°C. PCR
amplification was performed in the same tube by adding 0.4 µmol/L
each of P1 and a coding P2 primer (P2: 5'-AGT TAC TTT GGA GTG CAG
AAC CAT TTC-3' from nt 1433 to nt 1459) and 1 U Taq polymerase
(Perkin Elmer Cetus, Norwalk, CT) and cycling in a DNA Thermal Cycler
(Perkin Elmer Cetus). After denaturation for 2 minutes at 94°C, 30 cycles of 1 minute at 94°C, 2 minutes at 55°C, and 2 minutes at
72°C were followed by a final extension for 7 minutes at 72°C.
The 324-bp PCR product was separated by electrophoresis on a 2%
agarose gel and visualized by ethidium bromide staining and UV
illumination. Contamination through the PCR amplification of genomic
DNA would give a 1,282-bp fragment due to the presence of the 958 nt intron.
Nuclear extracts and DNase I protection assays.
Nuclear extracts were prepared from HeLa, Raji, K562, HL60, HEL, and
Dami cells by the modified method of Dignam et al.56 All
steps were performed at 4°C. The cells (0.5 to 1 × 107) were rinsed twice in PBS, pelleted by centrifugation
at 1,850g for 10 minutes, resuspended in 5 times the pellet
volume of hypotonic buffer (10 mmol/L HEPES, pH 7.9, 1.5 mmol/L
MgCl2, 10 mmol/L KCl, 0.2 mmol/L phenylmethylsulfonyl
fluoride [PMSF], and 0.5 mmol/L dithiothreitol [DTT])
and allowed to stand for 10 minutes. The resulting sediment was
resuspended in twice the volume of hypotonic buffer and centrifuged at
1,850g. After discarding the supernatant, the nuclei pellet was
resuspended in half the volume of cold low salt buffer (20 mmol/L
HEPES, pH 7.9, 1.5 mmol/L MgCl2, 20 mmol/L KCl, 0.2 mmol/L
EDTA, 25% [vol/vol] glycerol, 0.2 mmol/L PMSF, and 0.5 mmol/L DTT),
and the same volume of cold high salt buffer (low salt buffer
containing 1.2 mol/L KCl) was added slowly. This extract was gently
homogenized for 30 minutes and then clarified for 30 minutes at
25,000g. After dialysis against 500 mL storage buffer (20 mmol/L HEPES, pH 7.9, 20% [vol/vol] glycerol, 100 mmol/L KCl, 0.2 mmol/L EDTA, 0.2 mmol/L PMSF, and 0.5 mmol/L DTT) for 5 hours, the
dialysate was centrifuged at 25,000g for 20 minutes and the
precipitate was discarded. The supernatant containing the nuclear
extract was frozen in aliquots and stored at 80°C.
Fragments of GPV promoter for DNase I protection assays were obtained
by PCR. PCR amplification was performed in an Expand High Fidelity PCR
System (Boehringer) in a 100 µL reaction volume with 200 µmol/L of
each dNTP, 0.4 µmol/L of the reverse and forward primers, 1.5 mmol/L
MgCl2, 2 µL cDNA, and 2.6 U of a mix containing Taq and
Pwo DNA polymerases. PCR conditions were as described above, except for
the annealing temperature, which ranged from 52°C to 59°C,
depending on the primer pair. The primer pairs were as follows:
fragment I, FP1S (5'-GGA ACT GAA AGA TCT CCC GCG ATA C-3',
nt 6-30) and FP1AS (5'-TGA TGA ACT CGA GCA CCT TTT CAC
AT-3', nt 408-383); fragment II, FP2S (5'-ATA TCT TCT GAG
ATC TAG CCT TTG TCA-3', nt 163-189) and FP2AS (5'-GCC GCT
CAC ATC TCG AGT TTA ACT GGG G-3', nt 600-573); fragment III, FP3S
(5'-TTT TGC CTA TAG ATC TAT GGG CAA AAG-3', nt 314-340) and
FP3AS (5'-TCA AGC AAT TCT CGA GCC TCG GCC TC-3', nt
800-775); fragment IV, FP4S (5'-AGA AGC ACA GGA GAT CTC ACA AAT
GGC A-3', nt 498-524) and FP4AS (5'-TCT CAA ATC CTC GAG TTT
TTT CTT CC-3', nt 992-967); fragment V, FP5S (5'-GGT AAA
ACC CCA GAT CTA CTA AAA TAC AA-3', nt 698-726) and FP5AS
(5'-GTG ATA CAG CTC GAG CAC TGC AGT AG-3', nt 1174-1149);
fragment VI, FP6S (5'-CAA GTT CAA AGA TCT TCT TAG CCT
TA-3', nt 929-954) and FP6AS (5'-AGG TCC CTA TCT CGA GCC
CTT GTT CT-3', nt 1332-1306); and fragment VII, FP7S
(5'-ATG AAA AGG AAG ATC TAG GGG AAG TG-3', nt 1203-1128)
and FP7AS (5'-GTT CTG CAC TCG AGA GTA ACT GAA A-3', nt
1453-1429). The fragments were cloned in the pCR2.1 vector using a TA
Cloning Kit (InVitrogen, Leek, The Netherlands). After digestion with
EcoRI, the fragments were dephosphorylated, purified, labeled
with [ 32P] ATP and T4 polynucleotide kinase
(Boehringer), digested on the 3' side, and repurified.
Footprinting studies were performed essentially as described by Ohlsson
and Edlung.57 Briefly, nuclear extracts (50 µg) were
incubated for 20 minutes at room temperature with 2 µg
poly(dIdC).poly(dIdC) in a final volume of 50 µL HEPES buffer (25 mmol/L HEPES, pH 7.8, 50 mmol/L KCl, 0.05 mmol/L EDTA, 0.5 mmol/L DTT,
0.5 mmol/L PMSF, and 5% [vol/vol] glycerol). A 5'-end-labeled
DNA fragment (2 ng, 30,000 cpm) was added, and the mixture was
incubated for 40 minutes at room temperature and then digested for 1 minute on ice with 1 to 3 U of DNase I (RQ1; Promega, Madison, WI) in
the presence of 5 mmol/L MgCl2. The enzyme was inactived by
incubation for 20 minutes at 37°C with 100 µL Tris buffer (100 mmol/L Tris-HCl, pH 7.8, 100 mmol/L NaCl, 1% sodium dodecyl sulfate
[SDS], and 10 mmol/L EDTA) containing 100 µg/mL proteinase K and 25 µg/mL tRNA. After phenol/chloroform extraction and ethanol
precipitation, the one-end-labeled DNA fragments were separated on
denaturing 8% polyacrylamide gels. The gels were dried and
autoradiographed under an intensifying screen.
Plasmid constructs, cell transfection, and gene reporter assays.
An approximately 1.5-kb length of the GPV gene flanking region was
amplified by PCR with the primers C1 (5'-GAA GAT
CTT CCC GCG ATA CCT GGC AGA GGC AGT GGC-3', nt 20-48) and C2
(5'-GAA GAT CTT CTG CAC TCC AAA GTA ACT GAA AGA
CT-3', nt 1451-1425), which carry two Bgl II sites
(underlined). This segment was subcloned into the Bgl II site
of the pGL3-basic plasmid (Promega), which contains the firefly
luciferase gene and the polyadenylation signal from SV40, to generate
the plasmid pGL3/1413. A series of pGL3/1413-deleted constructs was obtained by unidirectional 5' to 3'
progressive digestion of pGL3/1413 with exonuclease III/
mungbean (Promega) and religation with T4 DNA ligase (Promega).
Activity of the 103/+25 region was studied further by mutating
the GATA-71/Ets-66 domain. PCR amplification using the pGL3/1413 plasmid as a template was used to generate 4 constructs with mutation of GATA-71 (GATA-71 mut), deletion of GATA-71 (GATA-71  ), mutation of Ets-66 (Ets-66 mut), and double mutation of GATA-71 and Ets-66 (tandem mut). The different constructs were obtained using antisense primer M1 (5'-GCT TAC TTA GAT CGC AGA TCT AAT GGT TCT
GC-3', nt 1457-1447 of GPV promoter attached to nt 36-56 of
pGL3-basic) and 1 of the following sense primers. The
"TTATCC" GATA-71 site was mutated to
"CCGTCC" using primer M2 (5'-GGG GTA CCT ACT CTG GTA AAG TCT CCG TCC TCA GGA TGC AAG G-3', nt 1339-1376). The first 3 bases of the "TTATCC" GATA-71 site were
deleted using primer M3 (5'-GGG GTA CCT ACT CTG GTA AAG
TCT TCC TCA GGA TGC AAG G-3', nt 1339-1376). The
"CAGGATGC" Ets-66 site was mutated to
"CAAAGTGC" using primer M4 (5'-GGG GTA
CCA AAG TCT TTA TCC TCA AAG TGC AAG G-3', nt 1351-1376). The
"TTATCCTCAGGATGC" GATA/Ets tandem was mutated to
"CCGTCCTCAAAGTGC" using primer M5
(5'-GGG GTA CCA AAG TCT TTA TCC TCA AAG TGC AAG G-3', nt 1351-1376). Primers M2-M5 and primer M1 carried
Kpn I and Bgl II sites, respectively (underlined), to
allow cloning in the pGL3-basic vector creating a 94/+25 or
82/+25 construct ahead of the luciferase gene.
All constructs were sequenced to check the exact boundaries and ensure
that no errors had occurred during the procedure. The pRL-TK plasmid
(Promega) expressing sea pansy luciferase under the control of the
thymidine kinase promoter was used as an internal standard, and the
pGL3/SV40 plasmid containing the firefly luciferase gene driven by the
SV40 promoter was used as a control of transcription efficiency.
Cells were transfected by electroporation using a CellJect apparatus
(Eurogentec, Seraing, Belgium) at 340 V,   , and 1,500 µF.
Assays were performed with 20 µg GPV promoter construct, 5 µg
pRL-TK, and 5 µg herring sperm DNA per 8 × 106
cells in a total volume of 800 µL. The cells were harvested 48 hours
after transfection and cell extracts were obtained by addition of
LucLite from the FireLite kit (Packard, Meriden, CT). Firefly luciferase activity was measured in a BCL Book luminometer (Promega) and initially expressed in arbitrary units. Sea pansy luciferase activity was then measured in the same luminometer after the addition of RenLite substrate. The firefly luciferase activity was normalized against the sea pansy luciferase activity to correct for variable transfection efficiencies among different samples.
Primer extension.
Total RNA was extracted from Dami cells and platelets as described for
mRNA analyses and poly(A)+ mRNA was purified with a
commercial kit (Pharmacia, Uppsala, Sweden). The PE1 oligonucleotide
(5'-AGC ACC GCG CAC AGT AGA GTC-3', nt 2453-2433) was
labeled with [32P] using a 5'-end-labeling kit
(Amersham, Uppsala, Sweden). This 5'-end-labeled synthetic
oligonucleotide was annealed to total or poly(A)+ RNA and
extended with MMLV reverse transcriptase according to the
manufacturer's instructions (Superscript II plus kit; InVitrogen). The
reaction products were analyzed by electrophoresis on denaturing 6%
polyacrylamide gels.
Reverse transcriptase-PCR (RT-PCR) mapping.
RT-PCR assays were performed in a Titan RT-PCR System (Boehringer)
using total RNA from Dami cells and poly(A)+ mRNA from
platelets. Briefly, 100 ng RNA was incubated in 50 µL RT-PCR buffer
containing 0.2 mmol/L of each dNTP; 0.3 µmol/L of reverse primer PM4;
0.3 µmol/L of direct primer PM1, PM2, or PM3; 5 mmol/L DTT; 5 U RNase
inhibitor (Boehringer); 1.5 mmol/L MgCl2; 1 U avian
myeloblastosis virus (AMV)-reverse transcriptase; and 1 U Expand High
Fidelity enzyme blend (Taq and Pwo DNA polymerases) at 50°C for 30 minutes. The primers were PM1 (5'-AGT TAC TTT GGA GTG
CAG-3', nt 1433-1450), PM2 (5'-CAT GCA GAG CTC TAA
GTC-3', nt 1411-1428), PM3 (5'-GAT ACC ACC CTC TTC
CTG-3', nt 1376-1393), and PM4 (5'-GTG CGT GAG GTT GGT
GGG-3', nt 2583-2566). PCR amplification was performed in the
same vial on a DNA Thermal Cycler as described above, with annealing at
52°C for PM1+PM4 and PM2+PM4 or at 54°C for PM3+PM4.
| |
RESULTS |
Dami cells are GPV-positive megakaryocytic cells.
To study GPV promoter activity, cell lines with constitutive (Dami,
HEL, Meg-01, and CHRF-288) or inducible (K562) megakaryocytic features
or negative cells (HL60) were screened for GPV expression by FACS
analysis or for GPV mRNA expression by RT-PCR.
GPV was detected only on the surface of Dami cells
(Fig 1A), together with the GPIb-IX
subunits (data not shown). GPV was absent from the surface of HEL,
Meg-01, K562, and HL60 cells (data not shown), although the first 2 cell lines are positive for the IIb3
integrin.58,59 Similarly, GPV mRNA was absent from HEL, K562, and HL60 cells when these cells were analyzed by RT-PCR and was
only shown in Dami cells (Fig 1B). Dami was therefore used as the
reference megakaryocytic cell line expressing GPV in further studies of
GPV promoter function.
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| Fig 1.
Expression of GPV in Dami cells. (A) Dami cells were
analyzed by flow cytometry using MoAbs against GPV (V.1) or the
GPIIb-IIIa complex (ALMA.17) and FITC-conjugated goat antimouse IgG.
Histograms are representative of 5,000 cells. Neg corresponds
to isotype-matched negative control. (B) RT-PCR analyses were performed
on total RNA from HL60, K562, HEL, and Dami cells. The GPV PCR products
were identified by 2% agarose gel electrophoresis and ethidium bromide
staining and neg corresponds to RT-PCR without reverse
transcriptase.
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Identification of the GPV transcription start site.
To determine the exact 5'-end of GPV mRNA, we isolated
poly(A)+ RNA from platelets and Dami cells and performed
primer extension analyses (Fig 2A). In both
cell types, comparison with the reported genomic sequence led us to
assign a major transcription start site to nt 1433, located 29 nt
upstream of the intron donor splice site. Additional longer extension
products matching positions 1419 and 1407 were found in platelets,
consistent with the frequent observation of multiple start sites in
TATA-less genes.5,42
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| Fig 2.
Identification of the transcription start site of the
human GPV gene. (A) Primer extension mapping of the 5'-ends of
GPV transcripts from Dami cells and platelets (numbering according to
Lanza et al45). (Bold arrowhead) start site common to
platelet and Dami RNA; (open arrowheads) additional upstream sites in
platelet RNA. (B) Positions of the primers used for PCR mapping are
indicated by arrows: PM1 (nt 1433 to 1450), PM2 (nt 1411 to 1428), PM3
(nt 1376 to 1393), and PM4 (nt 2583 to 2566). The intron sequence is in
lowercase characters and the GPV intron donor and acceptor splice sites
are in bold characters. (C) RT-PCR mapping of the 5'-ends of GPV
transcripts. The primer pairs defined in (B) were tested on Dami total
RNA and platelet poly(A)+ RNA and neg corresponds
to RT-PCR without reverse transcriptase. PCR products were identified
by 2% agarose gel electrophoresis and ethidium bromide staining.
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The start site at position 1433 was confirmed by RT-PCR mapping of this
region (Fig 2B) using the direct primers PM1 (nt 1433-1450), PM2 (nt
1411-1428), and PM3 (nt 1376-1393) and the reverse primer PM4 (nt
2583-2566). PM1/PM4 amplified a 193-bp fragment in accordance with a
transcription start site at position 1433 (Fig 2C) from Dami cells and
platelets. PM2/PM4 amplified the expected 215-bp band with less
efficiency and specificity, whereas no band was detected in most
PM3/PM4 assays. These results strongly suggest that platelet GPV mRNA
has 31 nucleotides of 5'-untranslated sequence and that the first
exon is 29-bp long. The GPV promoter region was numbered accordingly
(Fig 3B).
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| Fig 3.
Sequence of the GPV gene promoter and DNA fragments of
the 5'-flanking sequence used for DNase I protection assays. The
GPV promoter was numbered from an arbitrary start site common to Dami
cells and platelets, which conforms to the consensus start of platelet
TATA-less gene (Table 2). (A) Alignment of the fragments I to VII with
the 1432/+21 GPV 5'-flanking segment. The fragments were
checked by sequencing before 5'-end-labeling and use in DNase I
protection assays. Footprinting analyses of III, IV, VI, and VII are
reported in Figs 4 through 7. (B) Sequence of the GPV promoter. The
transcription start site is denoted +1, the intron sequence is in
lowercase characters, and the intron donor splice site is in bold
characters.
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This major transcription initiation region is 100% conserved between
human, mouse, and rat GPV49
(Table 1), matches the consensus sequence
for initiation found in the mouse terminal transferase gene, and is
highly homologous to sites present in other megakaryocyte genes, such
as the IIb29 and
260 integrin, human PF4 and
PF4alt,32 rat PF4,61
TG,35 and GPIX42 genes. These regions
correspond to the consensus initiator site for promoters lacking TATA
and CAAT boxes.62
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Table 1.
Comparison of the Transcription Start Sites of
Platelet-Specific Genes With the Human GPV Transcription Start Site
Common to Dami Cells and Platelets
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Identification of multiple binding sites for nuclear factors on a
1,500-bp promoter fragment by DNase I footprinting.
Seven overlapping fragments (I to VII; Fig 3A) covering the 1432
to +21 5'-flanking region were generated by PCR amplification and
blunt-end cloned in the pCR2.1 vector. The fragments were excised with
EcoRI, 5'-end labeled with [32P], and cut
with selected restriction enzymes to generate one-strand-labeled fragments. These DNA segments were analyzed on both strands for DNase I
protection using nuclear extracts from megakaryocytic (Dami and HEL)
and nonmegakaryocytic (K562, HeLa, Raji, and HL60) cell lines.
No protected region was detected on fragment I (1432 to
1025). Fragment II (1270 to 833) contained 3 protected sites (data not shown) that were confirmed on fragment III
(1119 to 633; Fig 4). One
15-bp footprint centered at 989 was specifically protected from
nucleolytic attack when tested with Dami cell extracts. This domain
contained 2 inverted sequences, named GATA-989 and GATA-984, that
resemble the consensus GATA box63: 5'-WGATAR-3' (W = A or T; R = A or G). A second 25-bp footprint, centered at 962, contained the 5'-CAGGAAGT-3' motif, whereas a
third 20-bp domain was centered at 938 and contained the
5'-AGAGGAAGC-3' motif. These 2 regions, named Ets-960 and
Ets-936, match the consensus sequence for an Ets box64:
5'-RSMGGAWRYY-3' (R = A or G; S = C or G; M = A or C; W = A
or T; Y = C or T) and were protected by Dami (Fig 4), Raji, HL60, K562,
and HEL extracts (data not shown), but not by HeLa extracts.
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| Fig 4.
DNase I footprint analysis of the GPV promoter fragment
III using Dami and HeLa cell extracts. The 5'-end-labeled
fragment III (nt 1119 to 633) was incubated with 50 µg HeLa or
Dami nuclear extract in the presence of 2 µg poly(dIdC).poly(dIdC)
and digested with 1, 2, or 3 U DNase I. Control corresponds to
digestion of III with 0.6 or 1.2 U DNase I in the absence of nuclear
extract. Digestion products were separated on an 8% acrylamide
sequencing gel in the presence of 8 mol/L urea, and bands were compared
with those of a DNA molecular weight marker (Hpa II digest of
pBR-322). The protected regions are indicated on the right and named
according to their homology with known transcription factor binding
sites ("GATA" and "Ets" for putative binding sites for GATA
and Ets transcription factors). The corresponding protected nucleotide
sequences are indicated in the lower part of the figure and the
consensus binding sites are underlined and in bold characters.
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Fragment IV (935 to 441) showed 2 footprinted areas
(Fig 5). The first, a 19-bp sequence
centered at 604, was protected only by HeLa extracts. It
contained the 5'-TGAGCC-3' motif, which has been named
RIIb-610 due to its resemblance to the
5'-TGAGTCC-3' motif in the 5' sequence of the GPIIb
gene (120), which is known to exhibit repressor
activity.31 A second 28-bp domain was centered at
560 and displayed no cell specificity, because it was protected by all of the nuclear extracts tested. This site, named ETF-566, contained a polypyrimidine 5'-CCCCTCCC-3' motif
matching the consensus binding site for ETF,65 a member of
the Sp1 family. Fragment V (735 to 259) contained 2 footprints already identified in fragment IV as RIIb-610
and ETF-566 (data not shown).
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| Fig 5.
DNase I footprint analysis of the GPV promoter fragment
IV using HeLa, Dami, and Raji cell extracts. The 5'-end-labeled
fragment IV (nt 935 to 441) was analyzed as described in the
legend to Fig 4, using HeLa, Dami, and Raji nuclear extracts.
"RIIb" is a putative repressor binding site with
homology to a repressor site in the GPIIb promoter,31
whereas "ETF" is a putative binding site for ETF, a member of the
Sp1 family.65
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Fragment VI (504 to 101) displayed 4 protected regions
(Fig 6). The first 25-bp footprint, which
was centered at 290, was protected by all of our nuclear
extracts. It contained a 5'-GGGGTGTGGC-3' sequence
resembling an Sp1 binding site and was named Sp1-292. A second 20-bp
domain, which was centered at 216 and named STAT-213, was
protected by all nuclear extracts and contained a STAT-like 5'-TTATGGAAA-3' motif (STAT consensus motif66:
5'-TTNNNGNAA-3'). The third 25-bp region was centered at
177 and contained the 5'-AATCA-3' motif that
corresponds to an inverted GATA site. It was named GATA-175, presented
an apparent DNAse hypersensitive site shown in Fig 6, and was protected
only by Dami extracts (Fig 7). A fourth
10-bp domain, named GATA-147, also matched a GATA binding site but was
less cell specific, because it was protected by HeLa, Dami, Raji, and
HL60 extracts.
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| Fig 6.
DNase I footprint analysis of the GPV promoter fragment
VI using Dami and HeLa cell extracts. The 5'-end-labeled fragment
VI (nt 504 to 101) was analyzed as described in the legend to Fig
4, using HeLa and Dami nuclear extracts. "STAT" is a putative
binding site for a transcription factor of the STAT
family.66
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| Fig 7.
DNase I footprint analysis of the GPV promoter fragment
VII using Dami and HeLa cell extracts. The 5'-end-labeled
fragment VII (nt 362 to +28) was analyzed as described in the
legend to Fig 4, using HeLa and Dami nuclear extracts.
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The proximal fragment VII (362 to +28) also contained 4 protected areas (Fig 7), with the first corresponding to GATA-175. A
second 15-bp domain, which was centered at 111 and named
GATA-111, contained the 5'-AGATAG-3' motif and was
protected only by Dami extracts. The third region differed depending on
the strand analyzed. In the coding strand, a 15-bp sequence centered at
68 was protected by Dami and HeLa extracts, as shown by a strong
DNAse hypersensitive site in Dami and HeLa nuclear extracts, whereas
the 27-bp footprint centered at 68 in the noncoding strand was
protected by all of our nuclear extracts. This domain combined 2 motifs, an inverted GATA sequence (5'-TTATC-3') and an Ets
site (5'-CAGGATGC-3'), which were named GATA-71 and Ets-66,
respectively. The last 20-bp region, named Ets-42, was only detected on
the plus strand, where it was centered at 40, and was evident
when using the lowest (1 U) DNase I concentration. It contained the
5'-CTTCCTGT-3' motif, a putative Ets binding site.
In summary, this systematic DNase I scanning of the 1432 to +21
5'-region of the GPV promoter showed a total of 14 protected domains. Six of these corresponded to GATA and 4 corresponded to Ets
binding sites, and several sites displayed putative
megakaryocyte-restricted binding properties
(Table 2). A number of regions proved to be highly conserved between human, mouse, and rat GPV. Regions
RIIb-610 and ETF-566, present on the human Alu sequence,
were, however, absent from the rat and mouse genes due to lack of the
Alu repeat in these 2 species.
Localization of positive and negative regulatory regions using
reporter gene deletion constructs.
To determine its promoter strength, the 1413 to +25 region of
the human GPV gene was inserted into the pGL3-basic vector upstream of
the luciferase reporter gene. This construct (pGL3/1413; Fig 8A) was transfected into the
GPV-positive megakaryocytic Dami and the GPV-negative HeLa cell lines.
pGL3/1413 displayed significant promoter activity amounting to
81% ± 4% of the control SV40 activity in Dami cells but was
inactive in HeLa cells (Fig 8B). The same region inserted in the
inverse orientation gave a completely inactive construct
(pGL3/1413R).
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| Fig 8.
Analysis of GPV gene reporter/Exonuclease III deletion
constructs after transient transfection in Dami and HeLa cells. (A)
Schematic representation of the 1413 to +25 5'-flanking
region of the human GPV gene linked to the luciferase reporter gene
(luc) in the pGL3 vector. Binding sites for transcription factors,
functionally identified by DNase I footprinting or detected by sequence
analysis in the case of RIIb-817, are indicated by the
symbols: ( ) GATA, ( ) Ets, ( ) Sp1 family, ( ) STAT, and ( )
sites homologous to the GPIIb repressor. (B) On the left is a schematic
diagram of the different constructs: (upper) a control SV40 promoter
construct (pGL3/SV40) used to set the 100% standard activity; (middle)
progressive 5' to 3' deletions of the 1413/+25
construct; and (lower) an inverted construct (pGL3/1413R) of the
1413/+25 segment. On the right, the luciferase activities of the
constructs transfected into Dami (shaded histograms) or HeLa cells
(open histograms) are given as percentages of the control (pGL3/SV40)
activity. Values were corrected for transfection efficiency by
cotransfection with a sea pansy/luciferase construct under the control
of the thymidine kinase promoter. Points are the mean ± SEM of at
least 3 experiments performed in triplicate.
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The regulatory regions were mapped by creating a series of
5'-deletions. Deletions from position 1413 to position
903 resulted in a steep 73% decrease of promoter activity in
Dami cells, suggesting the removal of strong positive elements. This
region contains the GATA-989, GATA-984, Ets-960, and Ets-936 sites
identified by DNase I footprinting. A further deletion from position
903 to position 816 induced a 107% increase in promoter
activity relative to the 903/+25 construct (pGL3/903),
which is compatible with the presence of a negative regulatory element.
Sequence analysis showed a 5'-CTCATG-3' motif, homologous
in its reverse orientation to a repressor domain in the GPIIb
promoter,31 that was named RIIb-817.
Interestingly, the RIIb domain RIIb-610
localized by DNase I footprinting is located downstream and, hence, may
be excluded from this putative negative regulatory region.
The 103 to +1 region is sufficient for efficient transcription
of GPV.
Sequential deletions from 816 to 362 bp caused little
change in promoter activity. In contrast, removal of the 362 to
276 bp region was responsible for a 40% decrease in activity,
indicating the presence of a positive element, most likely the DNase
I-protected Sp1-292 motif. Deletion from 276 to 103 bp
produced hardly any further change in activity, despite the presence of
3 protected GATA sites. The 103/+25 region still supported
approximately 64% of full-length promoter activity and must therefore
play an important role in basal transcription of the GPV gene.
Subsequent deletions from position 103 to position +10
progressively suppressed promoter activity, indicating that the
elements required for minimal transcription of GPV are located in this
area. This domain includes the protected GATA-71, Ets-66, and Ets-42
motifs, and the 103/+25 region is also extremely well conserved
between the human, mouse, and rat genes.49
Further analysis of the 103/+25 domain
(Fig 9) showed that deletion of the
GATA-71/Ets-66 tandem left a construct (pGL3/53) still having
43% of full promoter activity. On the contrary, deletion of the Ets-42
site completely abolished this activity, suggesting a critical role of
Ets-42 in basal GPV promoter activity.
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| Fig 9.
Reporter deletion studies of the GPV 103/+25 region.
On the left is a schematic diagram of the GPV promoter/luciferase
constructs. Sequential deletions of pGL3/103 progressively removed
the GATA-71/Ets-66 and Ets-42 domains and the luciferase activity was
tested after transfection into Dami and HeLa cells. The pGL3/103
construct containing the 103-bp flanking region upstream of the firefly
luciferase gene is the same as in Fig 8B and had 64% activity as
compared with pGL3/SV40. The lower construct (pGL3/SV40-103), which
corresponds to the 103-bp flanking region of GPV linked to pGL3/SV40,
was used to search for enhancer activity. On the right, the luciferase
activities of the different constructs transfected into Dami and HeLa
cells are given as percentages of the control (pGL3/SV40) activity.
Cotransfection with a sea pansy/luciferase construct was used to
normalize for transfection efficiency, and points are each the mean ± SEM of at least 3 experiments performed in triplicate.
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To analyze precisely the respective contribution of GATA-71 and Ets-66
on the activity of the 103/+25 region, these sites were deleted
or mutated by performing G to A replacements. Reporter assays
(Fig 10) showed that double mutation of
GATA-71 and Ets-66 sites or that a single mutation or deletion of
GATA-71 completely abolished promoter activity in Dami cells. Mutation
of the Ets-66 site decreased activity by 80% as compared with the
103/+25 construct. These results suggest a critical role of both
sites of the GATA/Ets tandem in GPV promoter activity, which was not
shown by exonuclease III deletion analysis.
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| Fig 10.
Reporter mutation analysis of the GPV GATA-71/Ets-66
tandem. On the left is a schematic diagram of the GPV
promoter/luciferase constructs. Crossed-out GATA-71 or Ets-66
corresponds to mutation of these sites and is noted as "mut" (see
Materials and Methods). " " corresponds to deletion of the
GATA-71 site. On the right are the luciferase activities of the
constructs after transfection into Dami and HeLa cells. Cotransfection
with a sea pansy/luciferase construct was used to normalize for
transfection efficiency. Each point is the mean ± SEM of at least 3 experiments performed in triplicate.
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The 103-bp fragment was also tested for possible enhancer/repressor
activity by placing it upstream of a weak SV40 promoter in the
pGL3-SV40 vector (pGL3/SV40-103; Fig 9). A modest 25% increase in
activity was observed on comparison of the effects of pGL3/SV40 and
pGL3/SV40-103 in transfected Dami cells. On the contrary, the same
construct yielded a 70% decrease in SV40 promoter activity in HeLa cells.
GPV promoter activity in Dami, HEL, and nonmegakaryocytic cell lines.
The 1413 promoter displayed no activity in HeLa cells (Figs 8B
and 9) or in the erythroid K562 or myeloid HL60 cell lines (Fig 11). Surprisingly, 194% ± 3%
of pGL3/SV40 activity was observed in transfected HEL cells, which do
not express GPV, as compared with 81% ± 4% of pGL3/SV40 activity
in Dami cells. Progressive deletion from 1413 to 816 bp
led to a parallel decrease and then increase in promoter activity in
Dami and HEL cells, with maintenance in the pGL3/816 construct
of an approximately 100% greater activity in HEL as compared with Dami
cells. A different pattern of evolution appeared after removal of the
region containing RIIb-610 and ETF-566. This deletion
induced a significant decrease in activity in HEL cells without
affecting the activity in Dami cells, whereas from 362 to +10
bp, the 2 cell lines displayed comparable promoter activities.
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| Fig 11.
Comparison of the promoter activities of pGL3/1413
and its deletion constructs in Dami, HEL, K562, and HL60 cells. On the
left is a schematic diagram of the GPV promoter/luciferase constructs
(see Fig 8). On the right are the luciferase activities of the
constructs after transfection into Dami, HEL, K562, and HL60 cells.
Cotransfection with a sea pansy/luciferase construct was used to
normalize for transfection efficiency, and points are each the mean ± SEM of at least 3 experiments.
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DISCUSSION |
In this first functional study of the GPV gene promoter, we showed in
gene reporter and 5'-deletion experiments that the region extending from +1 to 1413 behaves like a cell-specific promoter and identified several subdomains that act positively or negatively on
its transcriptional activity. An exhaustive analysis of this region by
DNase I footprinting further allowed the identification of binding
sites for cell-specific and ubiquitous nuclear factors.
One feature of interest of the GPV gene is its restricted cell
distribution to megakaryocytes. Previous studies of the promoters of
other megakaryocyte-specific genes were often performed in leukemic
cell lines displaying megakaryocytic features such as HEL or phorbol
myristate acetate (PMA)-induced K562 cells. The problem of
different cell lines that we have tested is that they are probably
blocked at early stages of differentiation, because they express both
erythroid and megakaryocytic markers. In fact, none of these cells
express GPV, even at the mRNA level, whereas they express early markers
such as GPIIb, with the exception of Dami cells, despite recent
demonstration that present stocks of Dami cells are subclones of HEL
cells. Our current Dami stock has been verified and confirmed to be a
HEL derivative (H.G. Drexler, personal communication).
These cells have developed a different phenotype, possibly as a result
of modulation of their growth or transcription fact |
TOP
ABSTRACT
INTRODUCTION