(Received for publication, May 23, 1995)
From
the
Platelet
glycoprotein (GP) Ib-IX-V is a multisubunit adhesion receptor that supports
platelet attachment to thrombogenic surfaces at sites of vascular
injury. The congenital absence of the receptor results in a bleeding
disorder associated with ``giant'' platelets, a condition linking the
expression of the complex to platelet morphogenesis. To understand better
the expression of the GP Ib-IX-V complex, studies were undertaken to define
the essential genetic elements supporting the expression of the -subunit of the complex (GP Ib
). GP Ib
promoter
activity was evaluated by transfection of human erythroleukemia cells with
reporter plasmids coding for the enzyme, luciferase. Studies were initiated
with a fragment extending 2,738 nucleotides 5` to the transcription start
site and lead to the identification of 253 nucleotides retaining full
promoter activity in human erythroleukemia cells. In cells of
nonhematopoietic lineage, human endothelial and HeLa cells, the GP Ib
promoter activity was no greater than background levels obtained with
promoterless constructs. Gel shift assays and site-directed mutagenesis
studies defined essential GATA and Ets binding motifs 93 and 150
nucleotides upstream of the transcription start site, a finding which
further substantiates these elements as important determinants of
megakaryocytic gene expression. The results define essential cis-acting
elements responsible for the expression of GP Ib
and provide insights
into molecular events coinciding with the release of normal platelets into
the bloodstream.
Basic information on the differentiation of megakaryocytes and the mechanisms responsible for the release of platelets into the bloodstream has significantly lagged behind basic studies of other cellular components of myeloid tissue. Major reasons for the lag have been the lack of suitable cell lines mimicking the unique properties of megakaryocytes in vitro. Certainly, the recently described c-Mpl ligand is essential for the differentiation of the pluripotent stem cell to a megakaryocyte(1, 2, 3) , but the late events of the process, specifically those that result in the release of platelets from the mature megakaryocyte are still poorly understood. To this end, the expression of megakaryocytic or platelet-specific antigens is one unique aspect of megakaryocytopoiesis and platelet production that can be examined and the importance of such studies has been validated by the recent work identifying the transcription factor, NF-E2, as an essential genetic factor for platelet production(4) .
A role for the platelet
membrane receptor, glycoprotein (GP) ()Ib-IX-V, in platelet morphogenesis is suggested by the
congenital absence of GP Ib-IX-V, a condition associated with the release
of abnormal or ``giant'' platelets and referred to as the Bernard-Soulier
syndrome(5, 6) . The platelet GP
Ib-IX-V complex is assembled from four distinct gene products, the
-
and
-subunits of GP Ib (GP Ib
and GP Ib
), GP
IX, and GP V(7, 8, 9, 10, 11)
. Considerable progress has been made defining the essential role of this
receptor in hemostasis(12, 13) ,
but little is known about the molecular events and/or factors associated
with the transcription and expression of the individual genes of the
complex. Moreover, nothing is known about the mechanisms which link the
expression of the complex and platelet morphogenesis. Recent studies have
supported the hypothesis that expression of the complex on the
megakaryocyte, and ultimately on the platelet surface, is dependent upon
the coordinated assembly of at least three individual gene products(14) . This implies that (i) similar genetic regulatory
elements exist within each of the three components, or (ii) the
megakaryocytic-specific expression of one, or perhaps two subunits,
controls the surface expression of the complete complex.
We previously
reported the generation of a transgenic mouse colony expressing the human
gene for GP Ib as part of a
human-murine chimeric GP Ib-IX-V receptor complex(15)
. Our results documented the expression of the human transgene and
established among bone marrow cells that in vivo gene expression is
dependent upon a 6-kilobase DNA sequence containing the entire human GP
Ib
gene. Thus, within this fragment are the promoter and
enhancer sequences necessary for the in vivo expression of GP
Ib
protein. In the present study we have extended the
characterization of the GP Ib
gene identifying the
transcription start site and the promoter elements responsible for
megakaryocytic gene expression. Using the megakaryocytic-like cell line,
HEL (human erythroleukemia cells), essential GATA and Ets consensus
sequences were identified, a finding which further documents these elements
as important determinants of megakaryocytic gene expression(16) . The results provide insights into the regulatory elements
involved in megakaryocytic expression of the GP Ib
gene and
provide a better understanding of the events leading to the commitment of
cells to the megakaryocytic lineage and the release of platelets.
Figure
1:Identification of the transcription start site of
the human GP Ib gene. A, the
GP Ib
gene and its two known exons are schematically shown(19) . The coding sequence for the GP Ib
precursor is contained within exon II and is represented by the filled
black box. Primer extension analysis (panel B) used two
oligonucleotides corresponding to the antisense sequence within exon I
(RPEX1) and antisense sequence spanning exons I and II (RPEX2). An RNA
probe of approximately 200 nucleotides in length was synthesized in an
in vitro transcription reaction for the RNase protection assay
shown in panel C.B, each oligonucleotide was end-labeled with
[
-
P]ATP and annealed to 3 µg
of poly(A
) RNA purified from HEL
cells. After incubation with reverse transcriptase, the primer extension
products were analyzed on a 6% denaturing polyacrylamide gel. The results
are shown in the absence (-) or presence (+) of HEL poly(A
) RNA
(Template). A sequencing reaction using the M13 -40 universal
primer labeled with [
-
P]ATP and M13 mp19 was included
as a size marker and is shown on the left. Major extension products
of 82 nucleotides and 107 nucleotides in length were observed using RPEX1
and RPEX2, respectively. C, an RNase protection assay was performed
using a radioactive RNA probe generated from an in vitro
transcription reaction and subsequently purified as described under
``Experimental Procedures.'' The RNA probe was annealed overnight to
poly(A)
RNA from HEL cells and subsequently digested with RNase A and RNase T1. The
digested products were denatured and electrophoresed through a 6%
polyacrylamide-urea sequencing gel. The marker (left) is a dideoxy
sequencing reaction using M13 mp19 and predicts a protected RNA species of
92 nucleotides in length.
Human umbilical vein endothelial cells (HUVECs) were collected
from a human umbilical vein as described (21) and
cultured in EGM media (Clonetics, San Diego, CA) supplemented with 10%
fetal bovine serum. HUVECs were transfected using Lipofectin (Life
Technologies, Inc.) according to the manufacturer's recommended
conditions. Briefly, cells were seeded in 60-mm dishes to obtain 50%
confluency at the time of transfection. Six µg of each plasmid was
diluted in OPTI-MEM I (Life Technologies, Inc.) and mixed with 36 µg
of Lipofectin reagent. After a 30-min incubation, the mixture was added to
the cells for a 3-h (37 °C) incubation in a 5% CO
incubator. Following the 3-h incubation, the medium was replaced with
complete medium and left until time to harvest the cell lysate for
analysis.
HeLa cells (CCL 2, American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Transfection of HeLa cells was performed using a standard calcium phosphate procedure(22) .
Luciferase activity
produced by 20 µl of the cell lysate was determined by mixing with 100
µl of assay buffer (50 mM KHPO
, 50
mM KH
PO
, 15
mM MgSO
, 5 mM ATP, 1 mM
dithiothreitol) and analyzing with a Monolight 2010 luminometer in which
D-luciferin was automatically injected (Analytical Luminescence Laboratory,
San Diego, CA). A similar amount of cell extract was assayed for CAT
activity in an assay buffer composed of 132 mM Tris (pH 7.8), 0.64 mM
acetyl-CoA, and 0.1 µCi of [
C]chloramphenicol. The conversion of chloramphenicol to
acetylated chloramphenicol was allowed to proceed for 45 min (37 °C)
and then separated by thin layer chromatography. CAT activity was
determined by scintillation counting and by radioimaging using a
PhosphorImaging screen (Molecular Dynamics, Sunnyvale, CA).
Figure
4:Nucleotide sequence of the 5`-region of the GP
Ib gene. Studies presented in Fig. 2demonstrate the core promoter elements of the GP
Ib
gene to be within 253 nucleotides 5` to the transcription
start site. A portion of the sequence is shown and a number of potential
motifs are highlighted that may represent cis-acting elements within the GP
Ib
promoter. The numbering scheme is based on defining the
nucleotide immediately 5` to the transcription initiation site as
-1. Nine double-stranded oligonucleotides were generated for analysis
in gel shift assays and are schematically shown (labeled
1-9).
Figure
2:Transient expression analysis of the GP Ib
promoter in HEL cells. On the left is a schematic representation of
a series of GP Ib
promoter-luciferase
constructs that were generated to evaluate promoter activity. Each
construct is identified by the number of base pairs 5` to the transcription
start site, as shown to the left of each promoter
fragment. p-1142inv/LUC contains the promoter fragment of
p-1142/LUC in an inverted orientation. On the right are the
relative luciferase activities of each plasmid as determined from at least
three independent experiments. Transfection efficiency into HEL cells was
determined by cotransfection of a plasmid generating chloramphenicol
acetyltransferase activity and the results were normalized for transfection
efficiency prior to determining luciferase activities (see ``Experimental
Procedures''). Parallel transfections were performed with a plasmid
containing the Rous sarcoma virus promoter driving the expression of
luciferase (pRSV/LUC). The luciferase activity generated by pRSV/LUC was
arbitrarily set at 100%. The activity of each GP Ib
promoter
construct is reported as the percent of the activity obtained in the
parallel experiment using pRSV/LUC.
HEL and HeLa cell nuclear extracts
were prepared by washing cells with phosphate-buffered saline followed by a
buffer composed of 10 mM Tris (pH 7.5), 130 mM NaCl, 5 mM KCl, 8 mM
MgCl, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride. The washed cell
pellet was resuspended in a hypotonic buffer (20 mM HEPES (pH 7.9), 5 mM
KCl, 0.5 mM MgCl
, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride) and allowed to sit
on ice for 10 min. Cells were then lysed in a Dounce homogenizer and the
nuclei were collected by centrifugation at 1500
g (10 min). The
nuclei were resuspended in an extraction buffer (20 mM HEPES (pH 7.9), 0.5
M NaCl, 1.5 mM MgCl
, 0.2 mM EDTA,
25% (v/v) glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and 5 µM spermidine) and incubated at 4 °C for 1
h. The extracts were again centrifuged (10,000
g, 10 min) to
pellet the cellular debris followed by dialysis against a binding buffer
composed of 25 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM EDTA, 10% (v/v)
glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride. Approximately 1.5 ml of nuclear extract was dialyzed against 1.5
liters of binding buffer for 16 h followed by a change of buffer and an
additional dialysis for 8 h.
Each labeled double-stranded fragment (2
10
cpm) was mixed
with 5 µg of nuclear extract, 2 µg of poly(dI-dC)
poly(dI-dC) (Pharmacia), and the binding buffer listed
above. The resultant mixture was incubated at room temperature for 30
min. Samples were analyzed by electrophoresis in 6% polyacrylamide gels
prepared in a running buffer composed of 50 mM Tris (pH 8.5), 0.38 M
glycine, 5 mM EDTA. After electrophoresis, gels were dried and exposed to
x-ray film.
Additionally, we performed an RNase protection assay to
confirm the primer extension results. A protected RNA fragment of 92
nucleotides in length was identified as the dominant protected fragment (Fig. 1C). This result mapped the 5` boundary
of the first GP Ib exon to be one
nucleotide further 5` as compared to the results from primer extension
analysis. However, the molecular weight determination of RNA by denaturing
gel electrophoresis may vary up to 10% given the anomalous mobilities
which have been confirmed using different electrophoretic conditions(24) . Thus, the DNA fragment lengths determined by primer
extension analysis are the more accurate determination since the DNA marker
is a similar primer extension product produced via a dideoxy sequencing
reaction. Cumulatively, these results establish that exon I is 91 base
pairs in length and the GP Ib
mRNA contains 97
nucleotides 5` to the initiating methionine codon.
Figure
3:Cell specificity of the GP Ib
promoter. As described in Fig. 2, the promoter
activities of three different constructs were evaluated by transient
transfection analysis of HEL, HeLa, and human umbilical vein endothelial
cells (HUVECs). As before, the results are presented as a percentage of the
activity obtained with parallel transfections using pRSV/LUC. Raw data from
individual experiments using pRSV/LUC ranged from 50,000 to 300,000 light
units from HEL cells; 35,000-100,000 light units from HeLa cells;
30,000-150,000 light units from HUVECs; and less than 300 light units from
nontransfected cells.
Fig. 5is a representative
gel shift analysis using oligonucleotides 1 through 3 which correspond to
nucleotides -128 to -64 of the GP Ib promoter (Fig. 4). Four major bands, their relative mobility positions
referred to as A through D, are considered specific DNA-protein complexes
since they disappear in the presence of cold competitor (Fig. 5, panels A and B). The bands at
position A were generated with oligonucleotide 1 and are present in both
HEL and HeLa nuclear extracts. However, HeLa nuclear extracts are slightly
different exhibiting a doublet band as compared to a single band observed
with HEL nuclear extracts. Bands at positions C and D are observed using
oligonucleotide 2 and are specific for HEL nuclear extracts (a comparison
of panels A and B, Fig. 5). It is
noteworthy that bands at positions C and D are always present together
suggesting that band D may be a proteolytic product of the larger complex
observed at position C. Bands at position B appear with oligonucleotides 1,
2, and 3 (Fig. 5A). In similar experiments
using oligonucleotides 4-9, several bands were observed that would
disappear upon competition with cold oligonucleotides (Fig. 6, A and B). However, we observed no
HEL-specific complexes using oligonucleotides 4-9 (comparing panels
A and B, Fig. 6).
Figure
5:Gel shift analyses using synthetic DNA fragments
spanning -64 to -128 of the GP Ib promoter and nuclear
extracts from HEL and HeLa cells. HEL (A) and HeLa (B)
nuclear extracts were mixed with radiolabeled double-stranded
oligonucleotides (Probe) 1 through 3 (Fig. 4) and
electrophoresed through native polyacrylamide gels. After electrophoresis
the gel was dried and exposed to x-ray film. A photograph of the resultant
autoradiograph is presented. The samples were applied at the position
labeled Origin. To evaluate specificity of the DNA-protein
complexes, the same unlabeled oligonucleotide (Competitor) was added to the
mixture at 10- (+) or 100-fold (++) molar excess. The migratory positions
of the major ``specific'' complexes are labeled A-D.
Figure
6:Gel shift analyses using synthetic DNA fragments
spanning -124 to -243 of the GP Ib promoter and nuclear
extracts from HEL and HeLa cells. As described in Fig. 5, HEL (A) and HeLa (B) nuclear extracts
were mixed with radiolabeled DNA fragments 4 through 9 (Fig. 4) and electrophoresed through native polyacrylamide
gels.
The observation that oligonucleotide 2 produced protein-DNA complexes using nuclear extracts from HEL cells, but not HeLa cells, prompted further investigation into the binding sequence(s) within oligonucleotide 2. In particular, we tested a potential GATA recognition sequence by generating a mutant oligonucleotide 2 containing the consensus 5`-GATAA-3` sequence changed to 5`-GAGCA-3` (Fig. 8A). As shown in Fig. 7, the mutant oligonucleotide 2 was unable to compete with the labeled normal oligonucleotide 2 for the DNA-protein complexes at positions C and D produced using HEL cell nuclear extracts. Thus, these bands most likely represent a specific GATA binding protein interacting with oligonucleotide 2.
Figure 8:Promoter activity of GATA and Ets mutant promoter constructs. A, site-directed mutagenesis was performed to alter the GATA and/or Ets motifs within the luciferase construct designated p-253/LUC (Fig. 2). The mutated sequences are shown below the native promoter sequence. The resultant mutant plasmids were designated p-253/LUC+GATAmut and p-253/LUC+Etsmut indicating the alteration of the potential GATA or Ets binding site, respectively. The single plasmid containing both GATA and Ets substitutions was designated p-253/LUC+GATA+Etsmut. B, the transient promoter activity in HEL cells for the wild type (p-253/LUC) and mutant plasmids is shown. The experimental methods are as described in the legend to Fig. 2and under ``Experimental Procedures.'' A background or negative control plasmid, p-6/LUC, is included for comparison.
Figure 7:Gel shift analysis using oligonucleotides with potential GATA or Ets binding motifs and HEL nuclear extracts. A mutant oligonucleotide 2 (mut. #2) was prepared and evaluated for its ability to be a competitive inhibitor of a radioactive oligonucleotide 2. Mutant oligonucleotide 2 is identical to the native oligonucleotide 2 except that it contains an altered GATA binding site (5`-GATAA-3` to 5`-GAGCA-3` illustrated in Fig. 8A). A second oligonucleotide (#5, see Fig. 4and Fig. 6), contains two potential Ets binding motifs (Fig. 8A). The mutant oligonucleotide 5 was tested for its ability to be a competitive inhibitor of a radioactive oligonucleotide 5 containing normal sequence in a similar gel shift experiment.
Based on studies using other
platelet-expressed genes which suggest that GATA proteins interact with
other regulatory complexes, such as Ets motifs(16, 25, 26) , we also examined the role
of two potential Ets motifs (5`-GGAA-3`) within the sequence of
oligonucleotide 5 by generating a mutant oligonucleotide 5 with the
substitutions depicted in Fig. 8A. In
similar experiments to those described above, the mutant oligonucleotide 5
was unable to compete for a DNA-protein complex identified at position E
(Fig. 7). Thus, the possibility for a DNAEts-related protein complex exists in HEL cells, but based on
the results shown in Fig. 5B this complex
is not HEL cell-specific.
To test the role of the potential Ets motif,
we prepared a mutant construct and performed transfection assays using the
same parent plasmid, p-253/LUC (Fig. 8). The
disruption of the potential Ets motifs reduced the promoter strength from
21 to 6%, similar to that observed with the GATA mutations alone (Fig. 8). However, the coordinated effects of the GATA and
Ets motifs were evident when the double mutation, containing both GATA and
Ets mutant sequences, was evaluated in a similar experiment. This construct
resulted in luciferase activity of less than 0.5%, a level
indistinguishable from background (Fig. 8). Thus,
the luciferase activity driven by the plasmid containing both GATA and Ets
mutations was completely diminished identifying these motifs as essential
elements of the GP Ib core
promoter.
Our parent plasmid, p-2738/LUC, contained
2,738 nucleotides 5` to the transcription start site (Fig. 2). Additionally, this plasmid contained all the DNA
sequence preceding the initiating methionine codon of the precursor
protein, which includes the sequence of exon I, sequence of the only
intron, and the first 6 nucleotides of exon II (Fig. 2). Thus, p-2738/LUC contained sequences downstream of
the transcription start site in the event that these sequences contributed
to the activity of the promoter. Unidirectional deletion analysis of
p-2738/LUC identified the shortest construct containing full promoter
activity (p-253/LUC) as one containing 253 nucleotides 5` to the
transcription start site. The progressive deletion from -2738 to
-253 did result in 2-fold, yet reproducible, differences in promoter
activity (Fig. 2). Thus, the possibility exists
for positive and negative enhancer sequences outside the 253 nucleotides
containing the core elements of the GP Ib promoter.
Our
studies suggest for endothelial and HeLa cells that the GP Ib promoter
is only capable of a basal level of transcription that is indistinguishable
from background levels. Indeed, the in vivo expression of GP Ib
has not been systematically evaluated in nonhematopoietic cells, but a
number of in vitro observations suggest that GP Ib
mRNA
and/or protein expression may be induced by cytokines to detectable levels
in endothelial cells and smooth muscle cells(27, 28) . Additionally, some indirect evidence for GP Ib
antigen on the surface of activated endothelial cells has been shown by the
ability of these cells to bind von Willebrand factor in a
ristocetin-dependent manner(29) , a characteristic
property of platelet GP Ib-IX-V and its soluble ligand, von Willebrand
factor(30, 31) . The only in
vivo corollary for these observations is an immunological
identification of GP Ib
protein in inflamed
tonsillar endothelium(27) . Indeed, we have observed
that unstimulated endothelial cells contain a very low, yet detectable,
level of GP Ib
mRNA as seen by
Northern blot analysis (data not shown), a finding originally reported by
Rajagopalan et al.(28) . With respect to our
presented results (Fig. 3), it must be recognized
that each of the three cell types (HEL, HUVECs, and HeLa) were transfected
via different procedures in order to obtain the highest transfection
efficiency for each cell type. Additionally, the determination of GP Ib
promoter activity within each cell type is based on the activity, assumed
to be maximal, generated from a transfection with the viral promoter in
pRSV/LUC. It is possible that each cell type responds to this viral
promoter in a slightly different manner which would invalidate comparative
conclusions among the three cell types (Fig. 3). If fact, a more likely explanation is that the GP
Ib
promoter is somewhat ``leaky'' and capable of a low level of
transcription in cells of nonhematopoietic lineage. However, in light of
the subunit requirements for efficient surface expression of a GP Ib-IX
complex(14) , the in vivo relevance of such
low levels of GP Ib
expression is
questionable.
The choice of HEL cells as a model for evaluating gene
expression for components of the GP Ib-IX-V complex is based on the fact
that cDNAs for 3 subunits of the complex were synthesized from HEL mRNA,
the exception being GP V. It can argued that HEL cells are not a perfect
``model'' megakaryocytic cell. In fact, studies evaluating
megakaryocytic-specific promoters have lagged behind similar studies for
other hematopoietic cells simply because the ``perfect'' cell line has not
been identified. It is well known that HEL cells express genes of both the
erythroid and megakaryocytic lineages, and the evidence is quite strong
that erythrocytic and megakaryocytic lineages derive from a common
progenitor cell(17, 32)
. However, studies of other platelet-specific promoters, specifically gene
promoters for rat platelet factor 4 and the human platelet GP IIb receptor
( integrin), have utilized HEL
cells and have been meritorious(16, 26, 33, 34)
. Interestingly, Block et al.(26) have
determined promoter strength for the rat GP IIb promoter by transfecting
rat megkaryocytes. Comparing their results to that obtained by others
studying the human GP IIb gene transfected into HEL cells corroborated the
same promoter elements originally identified using HEL cells, but the
actual promoter strength or quantitative conclusions were significantly
different for the two cell types. One explanation for the conflicting
quantitative results is that HEL cells express the same transcription
factors as megakaryocytes, but the levels of individual transcription
factors differ resulting in different quantitative conclusions.
The
identification of essential GATA and Ets binding elements within the GP
Ib promoter is consistent with previous reports identifying
these elements as major regulatory regions in megakaryocytic-specific
genes(16) . The two previously studied
platelet-specific genes, platelet factor 4 and the platelet integrin
subunit, GP IIb (
), have confirmed
the important role of GATA-binding proteins for megakaryocytic-specific
gene regulation(16, 26, 33, 34) . The GATA family of proteins are
related by a high degree of sequence similarity throughout their zinc
finger-binding domains(35) . GATA-1 is primarily
expressed by mature erythroid cells, megakaryocytes and mast cells; GATA-2
is expressed by a wide variety of cell types; GATA-3 is most abundant in T
lymphocytes; and GATA-4 is expressed in endodermally-derived tissues and
heart(35, 36, 37) . Our choice of mutating the 5`-GATAA-3` site to 5`-GAGCA-3`
was based on the canonical binding site for GATA-1 protein
(5`-GAT(A/T)-3`)(38) . However, the expression of an
individual GATA binding factor cannot be the sole factor for
megakaryocytic-specific expression; in fact, the situation may be much more
complex(39) . Aird et al.(39) have proposed for platelet factor 4 that some GATA proteins,
such as GATA-2, may actually inhibit megakaryocytic transcription by being
a competitive inhibitory of GATA-1 protein. Thus, by this scenario the cell
specificity for the platelet factor 4 promoter is not within the promoter
sequence, but rather in the balance of transcription factors that are
expressed by the megakaryocyte.
Our results also identified an essential
binding site for an Ets-related protein. The Ets family of transcription
factors recognize a 5`-GGA(A/T)-3` motif(25) . The GP
Ib promoter contains two potential Ets motifs between -138
and -153 (Fig. 4). Our mutagenesis targetted
both potential binding sites by substituting the consensus 5`-GGAA-3` with
5`-CCAA-3` (Fig. 7A). However, GATA and Ets
motifs alone cannot explain cell specificity as evidenced by their
importance for expression of P-selectin, an endothelial cell and
megakaryocytic gene product containing functional GATA and Ets motifs (40) . Indeed, the requirements for lineage specificity of
these genes will only be understood after the positive identification of
the specific GATA and Ets protein interacting with a specific promoter
element.
Identifying the transcription factors which interact with the
GP Ib promoter along with similar studies on the other subunits of
the GP Ib-IX complex will provide the complete description of the molecular
factors regulating the expression of this critical platelet receptor. As
depicted in Fig. 4, the GP Ib
promoter
contains a number of additional binding motifs for previously characterized
transcription factors. Certainly, our results establishing GATA and Ets
motifs as the important core elements of the promoter do not exclude a
potential role for these other motifs, but they do provide definitive proof
for two of the key elements of gene expression.