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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2777-2790
Human Integrin  3 Gene Expression: Evidence for a
Megakaryocytic Cell-Specific cis-Acting
Element
By
Ying Jin,
Calvin C. Wilhide,
Chi Dang,
Lu Li,
Su-Xia Li,
Manuel Villa-Garcia, and
Paul F. Bray
From the Division of Hematology, Department of Medicine, Johns
Hopkins University School of Medicine, Baltimore, MD; and Centro de
Desarrollo Biotechnologico, Monterrey, Mexico.
 |
ABSTRACT |
The human integrin  3 participates in a wide range of
adhesive biologic functions and is expressed in a selected subset of tissues, but little is known about the cis-acting DNA elements or trans-acting factors responsible for this regulation. Using cell lines characterized for 3 expression, a number of
upstream regulatory regions in the 3 gene were
identified. (1) The three regions from  1159 to 584, 290 to
146, and 126 to 115 demonstrated positive, negative, and
negative activity, respectively. (2) The region from 115 to +29 of
the 3 gene was sufficient for cell-specific activity.
Deletion of the sequence from 115 to 89 produced a 6- to 40-fold
reduction in reporter gene activity in 3-expressing megakaryocytic cell lines (K562, Dami, and HEL), but only a 1.7- and
2.7-fold reduction, respectively, in 3-expressing
endothelial and melanoma cell lines, and 1.3- and 2.8-fold reduction,
respectively, in non-3-expressing Chinese hamster ovary
and 293 cell lines. This sequence also bound nuclear proteins in a
cell-specific manner in electrophoretic mobility shift assays.
Mutational analysis indicated that the sequence GAGGGG (positions
113 to 108) is a megakaryocytic cell line-specific
cis-acting element. (3) The region from 89 to +29 promoted
lower activity in all cell lines. We also provide evidence that a
CCCACCC sequence at position 70 has transcriptional activity, most
likely through the Sp1 transcription factor. These data supply the
first detailed map of the transcriptional regulatory elements of the
5 region of the 3 gene, define positive regulatory sequences with potent megakaryocyte preferential activity, and indicate that the ubiquitous transcription factor, Sp1, may augment
3 gene expression.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE BIOLOGIC IMPORTANCE of the integrin
family of adhesive molecules is well-established, and these molecules
participate in such diverse biologic processes as thrombus formation,
angiogenesis, embryogenesis, inflammation, as well as tumor
metastases.1 Many of these processes require the ability to
upregulate or downregulate different integrin molecules. Integrins
consist of  heterodimers and integrin 3 is able to
pair with two subunits: IIb and v,
the classical fibrinogen and vitronectin receptors, respectively. When
paired with IIb, 3 expression is
restricted to the megakaryocyte/platelet lineage, whereas the classical
vitronectin receptor (VNR), v3, is
expressed in a number of tissues. The biologic importance of 3 protein is underscored by the inherited bleeding
disorder, Glanzmann thrombasthenia, which results when the gene for
3 is mutated and not expressed.2 To date,
nearly 20 3 mutations have been described in Glanzmann
thrombasthenia,3 but all have been in coding sequence or
have resulted in defective RNA splicing, so that no clues have been
provided as to transcriptional regulatory sequences.
3 expression is controlled at the level of
transcription, as demonstrated by the response to phorbol esters.
Increased 3 mRNA levels have been demonstrated by
Northern blot analysis and nuclear run-on experiments,4 and
increased 3 was observed in cell-free translations of
mRNA from K562 cells treated with phorbol 12-myristate 13-acetate
(PMA).5 Although 3 stability and surface
expression are in some measure dependent on a subunit expression (an
example being the rapid 3 degradation in the
nonexpressing IIb thrombasthenia mutations), a variety
of other factors and biologic processes are known to modulate
3 expression. For example, 3 is
upregulated during the second half of the menstrual cycle and during
pregnancy,6,7 and we have shown that this effect may be
mediated by sex hormones.8 In a variety of different cells
lines the expression of 3 is also regulated by basic
fibroblast growth factor,9 vitamin D,10
retinoic acid,11 the human homeobox gene
HOX4A,12 and factors that promote
angiogenesis.13,14 Although a vitamin D-responsive element
has been identified in the avian gene,10,15 none of the
necessary cis-acting elements for such regulatory effects have
been identified in the human gene.
Because IIb expression is restricted to the
megakaryocyte/platelet lineage, the IIb3
complex is expressed exclusively in these cells. A substantial body of
work exists on the transcriptional regulation of the IIb
gene,16-26 but comparisons to 3 regulatory sequences have been difficult due to limited information on the latter.
On the other hand, v3 is expressed in a
number of tissues, including human osteoclasts,27
endothelial cells,28,29 human placental syncytiotrophoblast
brush border,30,31 monocyte-derived macrophages,32-34 cultured human embryonic
fibroblasts,35,36 as well as a number of malignant cell
lines. Upregulation of v3 expression has
been associated with malignant potential; for example, during the
transition from dysplastic nevi to tumorigenic melanomas37 and the neovascularization of tumors.13 However, although
the tissue distribution of 3 has often been called
widespread, this has not been assessed in a formal fashion. Some older
studies used monoclonal antibodies (MoAbs) against v to
assess VNR expression and equated this with 3
expression. Because v can associate with
5,38,39 6,40,41
8,42 and 3, specific markers must be used to assess its tissue distribution. A large report on
platelet antigens used four different MoAbs to 3 and
found that more than half of the tissues and cell lines examined were negative for 3 expression.43 This
differential pattern of tissue expression for IIb and
3 suggests that independent factors control gene
transcription. On the other hand, unlike all other known integrin
pairs, the genes for IIb and 3 are
physically linked on 17q21.32,44,45 raising the possibility
of shared cis-acting elements coordinating gene expression in
megakaryocytes. Regardless, the mechanisms controlling independent,
shared, or even developmental expression of 3 are
unknown.
We have previously cloned and partially characterized a portion of the
3 gene containing the 5 region and signal
peptide.46,47 We now report our characterization of the
transcriptional regulatory elements of the 5 region of the
3 gene and provide evidence for both significant
tissue-specific and tissue- nonspecific cis-acting elements.
These results provide detailed information on elements and a
transcription factor affecting 3 gene expression and may supply insights into the regulation of expression of other integrin genes.
| |
MATERIALS AND METHODS |
Reagents.
Purified Sp1 protein (Promega, Madison, WI), rabbit polyclonal IgG, 1C6
(anti-Sp1) MoAbs (Santa Cruz Biotechnology, Santa Cruz, CA), and the
irrelevant antibody, mouse  globulin (Pierce, Rockford, IL) were
purchased. PMA (Sigma, St Louis, MO) was diluted to 1 mg/mL in dimethyl
sulfoxide (Sigma) and frozen at 80°C. Radioisotopes were
from Amersham (Arlington Heights, IL). MoAb T10 is specific for the
human IIb3 complex48 and was
a generous gift of Dr Rodger McEver (Oklahoma Medical Research
Foundation, Oklahoma City, OK); MoAb AP3 is specific for human
349 and was a generous gift of Dr Peter
Newman (Blood Research Institute, Milwaukee, WI).
Cell lines and culture conditions.
The 3-expressing megakaryocytic cell lines
K562,50,51 Dami,52 and HEL53 and
the non-3-expressing HeLa cell line (American Type
Culture Collection [ATCC], Rockville, MD) were cultured as described
previously.54 The Dami cells were obtained from the ATCC in
1989 and demonstrate greater 3 expression than do our
HEL cells, despite the likelihood of being a subclone of HEL. In some
experiments, K562 cells were treated with PMA (100 nmol/L). The human
microvascular endothelial cell (HMEC-1) line that expresses
355 was cultured in endothelial basal medium
(MCDB 131; Clonetic Corp, San Diego, CA) supplemented with 10% fetal
bovine serum (GIBCO BRL, Grand Island, NY), 10 µg/mL hydrocortisone
(Sigma), and 10 ng/mL epidermal growth factor (EGF; Collaborative
Biomedical Products-Becton Dickinson, Bedford, MA). WM793 is a
3-expressing melanoma cell line obtained from Dr
Meenhard Herlyn (The Wistar Institute, Philadelphia, PA) that was grown
in Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) with 10%
fetal bovine serum. Cultures were maintained in a humidified atmosphere
of 5% CO2 at 37°C. Two
non-3-expressing cell lines were used as controls: Chinese hamster ovary (CHO) cells were cultured in -Minimal
Essential Media (MEM; GIBCO BRL) containing 10% vol/vol fetal bovine
serum (GIBCO BRL), and the transformed human embryonal kidney cell line 293 (ATCC) was cultured in Eagle's minimal essential medium (GIBCO BRL) supplemented with 10% fetal bovine serum (GIBCO BRL).
Flow cytometry.
Cells were washed and suspended in phosphate-buffered saline (PBS) at a
final concentration of 2 × 106/mL and incubated with
T10 or AP3 at a final concentration of 1 µg/mL. Samples were washed
twice in PBS and then incubated with fluorescein isothiocyanate
(FITC)-labeled goat antimouse antibody for 60 minutes at 4°C.
Samples were washed twice with PBS, centrifuged at 750g for 10 minutes, and then resuspended in 400 µL of PBS for analysis by flow
cytometry.
Northern analysis.
Total cellular RNA was isolated from cell lines according to the method
of Chomczynski and Sacchi.56 RNA samples (10 µg/lane) were electrophoresed in 1% agarose formaldehyde gels, transferred to
nylon membranes, and hybridized with radiolabeled probes for human
328 and human fetal
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes as
previously described.54
Plasmid constructs.
Various regions of the 3 gene were cloned upstream of
the luciferase reporter gene in the pGL2 Basic vector (Promega) or upstream of the CAT reporter gene in the PLCAT vector.46 In all 3 constructs, the 3 end of the
3 gene sequence immediately flanked the ATG translation
start codon (position +29 from the transcription start site). The
plasmid pGL2 Promoter vector (Promega) that has an SV40 promoter was
used as a positive luciferase control and for some normalization
studies. The CMV-Sp1 expression plasmid and its parental vector without
the Sp1 cDNA (CMV-empty) were the generous gifts of Robert Tjian
(University of California, Berkeley, CA).
The 1159 internal deletion construct was generated by
restriction digestion at the 146 Xba I and 31
Not I sites, blunting the ends with T4 DNA polymerase, and
religating. Mutations were introduced into the 146 wild-type
template using the Site-Directed Mutagenesis kit (Clontech, Palo Alto,
CA) according to the manufacturer's recommendation. Primers for
introducing mutations are listed in Table
1. The mPw1 primer was used to generate the 146 mPw1mut construct; primer mMS7 was used to generate the 146 Sp1mutant construct. All constructs were sequenced to confirm that the mutation had been properly introduced.
CAT assays.
Several of the initial functional analyses were performed on the
3 gene by cloning various regions of the
3 genomic clone into plasmid PLCAT,46
transiently transfecting constructs into K562 cells, and performing CAT
assays as described previously.46 To account for variation
in transfection efficiency, results were normalized to the luciferase
activity obtained by cotransfecting 2 µg of pGL2-Promoter vector
(Promega).
Luciferase assays.
K562, Dami, and HEL cells were harvested at a density of 1.5 to 2.0 × 105 cells/mL and electroporated (1 × 107 cells/sample) using a gene pulser (Bio-Rad, Richmond,
CA) set at 500 µFD and 400 V with test (20 µg) and control
pSV40-CAT (2 µg) plasmids. Cells were then incubated on ice for 10 minutes, resuspended in 25 mL of complete media, and incubated at
37°C and 5% CO2 atmosphere. CHO cells were transfected
using a diethyl aminoethyl (DEAE) dextran method as
described previously.57 Two hundred ninety-three cells were
transfected using the CaCl2 method as described
previously.58 HMEC-1 and WM793 cells were plated in 60-mm
plates and transfected at 60% confluence with a total of 4 µg
plasmid DNA, using 15 µL Lipofectin (GIBCO BRL) for 3 hours.
Transfected cells were collected after 24 hours, washed twice with
media, lysed, and analyzed using the Luciferase Assay System
(Promega).
To account for variation in transfection efficiency, luciferase
activity was normalized to CAT activity by cotransfection of the
pSV40-CAT construct. CAT activity was measured as described previously.46 To assess the importance of various sequences within a given cell line, luciferase values that had been adjusted for
transfection efficiency with CAT were divided by either the CAT-normalized luciferase activity of the pGL2-Basic (promoterless) or
pGL2-Promoter (SV40 promoter) vectors (Promega). This fold increase
over the pGL2-Basic and pGL2-Promoter vectors was not used for
comparison between cell lines, because these arbitrary values were
relatively high or low due to the different activities of the
pGL2-Basic and pGL2-Promoter vectors in different cell lines. Rather,
comparisons between cell lines were made by the relative changes in
activities among different deletion constructs within a cell line.
Electrophoretic mobility shift assays (EMSAs).
Crude nuclear extracts were prepared from K562, Dami, HEL, HeLa, 293, HMEC-1, and CHO cells using the method of Andrews and Faller.59 Briefly, cells were lysed in buffer A (10 mmol/L
HEPES-KOH, pH 7.9, at 4°C, 1.5 mmol/L MgCl2, 10 mmol/L
KCl, 0.5 mmol/L dithiothreitol, 0.2 mmol/L phenylmethyl sulfonyl
fluoride [PMSF]) and centrifuged, and the pellets were resuspended in
buffer C (20 mmol/L HEPES-KOH, pH 7.9, 25% glycerol, 420 nmol/L NaCl,
1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L
dithiothreitol, 0.2 mmol/L PMSF). Cellular debris was removed by
centrifugation, and supernatants were stored at 80°C.
DNA probes were prepared as follows. Single-stranded DNA
oligonucleotides were slowly annealed to form double-stranded probes by
incubating the sense and antisense strands at 95°C followed by a
slow cool to room temperature. Double-stranded DNA probes (Table 1)
were labeled with 32P-deoxycytidine triphosphate (dCTP;
3,000 Ci/nmol) or 32P-deoxyguanidine triphosphate (dGTP;
3,000 Ci/nmol) using DNA polymerase (Klenow fragment; Boehringer
Mannheim, Indianapolis, IN). Crude nuclear extracts (5 µg
protein/sample) were incubated with double-stranded
32P-labeled oligonucleotide probes in binding buffer (10 mmol/L Tris-Cl, pH 7.4, 80 mmol/L KCl, 5% glycerol, 1 mmol/L
dithiothreitol) for 15 minutes at room temperature. Poly dI:dC was used
in gel shifts at 0.055 to 0.22 mg/mL to minimize background protein
binding to probe. Competition experiments were performed by the
simultaneous incubation with a 50-fold molar excess of unlabeled
specific or irrelevant DNA probes (Table 1). Supershifts were performed
using 2 µg of mouse Ig per reaction. Samples were electrophoresed on 6% polyacrylamide gels that were pre-run at 100 V for 1 hour and run
for 2.5 hours at 250 V and 4°C. Gels were dried for 1 hour at
80°C and exposed to film (Eastman Kodak, Rochester, NY) for 12 to
96 hours at 80°C.
To optimize the detection of any potential DNA-protein interactions,
several different mobility shift assay buffers were tested. In addition
to the buffer described above, a second buffer (Incubation buffer;
Hotfoot Buffers Kit; Stratagene, La Jolla, CA) was identified that was
more sensitive to some of the DNA-protein interactions. The composition
of this latter buffer is proprietary.
Computer search for DNA homologies.
We used the Basic Local Alignment Search Tool (BLAST) software through
the National Center for Biotechnology Information (NCBI; Bethesda, MD)
to search for nucleic acid homologies. We used the blastn program to
search all nonredundant sequences in all the GenBank databases as well
as those sequences in the eukaryotic promoter database (EPD).
| |
RESULTS |
Identification of megakaryocytic cell preferential positive activity in
the 5 region of the 3 gene.
Our initial studies were performed in K562 cells, because they
expressed 3 (Fig 1) and had the highest
transfection efficiency of the megakaryocytic cell lines. A series of
deletion constructs from positions 1159 to 31 relative to
the transcription start site of 3 gene were used in
transient transfection assays for reporter gene activity. Positive
regulatory activity was shown in the region from 1159 to
584; negative activity was observed between 290 and
146 and between 126 and 115. The greatest positive
activity was observed in the region from 146 to the first exon.
There was substantial loss of activity when this region was deleted,
although the 89 to 31 region retained a modest positive
effect. The minimal promoter has arbitrarily been defined at the
31 position, because these sequences were still able to drive
transcription.46 Subsequent studies focused on regions of
positive activity downstream of 146.
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| Fig 1.
Functional analysis of the 5 region of the
3 gene in K562 cells. The insert at the lower left shows
flow cytometry analysis of K562 cells. Fluorescence (FL) is shown on a
logarithmic scale on the x-axis; cell number is shown on the y-axis.
Dashed lines are the results using MoAb T10, which is specific for
human IIb3. A restriction enzyme map with
positions relative to the transcription start site of the
3 gene is shown above. Reporter gene constructs
containing portions of the 1,159 bp upstream of the transcription start
site were transiently transfected into K562 cells and assayed for
reporter activity. The reporter gene used in these studies included
both CAT and luciferase, and equivalent results were obtained with both
reporters. Fold increase and the standard error of the mean over the
promoterless construct (background) are indicated. The number of times
each construct was tested is shown in parentheses. The Materials and
Methods describes how values were normalized, emphasizing the
importance of the relative changes, not the absolute fold increase.
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Because 3 is expressed in a variety of cell types, to
assess tissue-specificity we characterized its expression in a number of cell lines (Fig 2A). The megakaryocytic cell lines,
HEL and Dami, showed high levels of IIb3
surface expression, as did endothelial (HMEC-1) and melanoma (WM793)
cell lines; no expression was observed in CHO and 293 cells. K562 cells
showed increased expression with PMA induction (compare Figs 1 and 2A).
Several of these cell lines were also studied by Northern analysis (Fig 2B), which demonstrated undetectable levels of 3 mRNA in
293, CHO, and unstimulated K562 cells, but prominent expression in PMA-stimulated K562 cells and Dami cells and lower levels in HEL cells.
As we and others have previously observed, uninduced K562 cells, which
have megakaryocytic as well as erythroid properties, express only low
levels of 3 protein, and mRNA is not easily observed by
Northern blotting but can be detected by reverse
transcription-polymerase chain reaction (data not shown).
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| Fig 2.
Characterization of cell line 3
expression. (A) Flow cytometry analysis of CHO, 293, PMA-induced K562,
HEL, Dami, HMEC-1, and WM793 cells. Fluorescence (FL) is shown on a
logarithmic scale on the x-axis; cell number is shown on the y-axis.
Dashed or dotted lines are the results using MoAb T10, which is
specific for human IIb3 on CHO,
PMA-induced K562, HEL, and Dami cells, or 3-specific
MoAb AP-3 for 293, HMEC-1, and WM793 cells; solid lines are the
negative control using mouse Ig as the primary antibody. FACS analyses
were performed at different times such that baseline fluorescence
differed among cell lines. The key finding was the shift in
fluorescence using MoAbs specific for 3 or
IIb3. (B) Northern blot analysis of 10 µg total RNA from selected cell lines probed with a full-length
2.6-kb 3 cDNA and exposed to film at 80°C for 14 hours (upper panel). The 3 mRNA signal in HEL cells is
more easily seen on 24 hours of exposure. The filter was stripped and
rehybridized with a GAPDH cDNA to assess loading equivalency (lower
panel). Note that HEL and Dami cell 3 RNA levels
correlate well with protein expression, whereas PMA-stimulated K562
cells do not, suggesting that PMA-stimulation has complex effects on
3 protein expression.
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Cell line specificity of the 146 region was assessed using this
spectrum of 3-expressing cell lines
(Fig 3A). Because the background
(promoterless) vector produced variable activity in the different cell
lines, the absolute fold over background values cannot be compared
across the different cell lines; rather, the relative changes in
activities among different deletion constructs within a cell line were
examined. The largest construct showed variable activity in both the
megakaryocytic lines and in the nonmegakaryocytic lines. A mutant was
generated in which the 146 to 31 sequence was removed
from the 1159 construct. This mutant had greatly reduced
activity in all cell lines, with the greatest decrease in the
megakaryocytic cell lines, suggesting that the 146 to 31
sequence contained cell-specific activity not requiring the presence of
the upstream negative and positive regulatory sequences. We observed
modest and similar changes in activities in both megakaryocytic and
nonmegakaryocytic cells upon deleting from 1159 to 146
and from 146 to 115. However, deletion from 115 to
89 resulted in a 13.0-, 9.8-, and 40-fold reduction in the
megakaryocytic cell lines K562, Dami, and HEL, respectively. The
3-expressing endothelial and melanoma cell lines
displayed a modest 1.3- and 2.8-fold reduction, respectively, which was similar to that seen the in non-3-expressing CHO and
293 cell lines (1.7- and 2.7-fold reduction, respectively), suggesting megakaryocytic cell-preferential activity in the 26 bp between 115 to 89. To address whether the use of the promoterless
construct as background might affect the results shown in Fig 3A,
several of the key experiments were repeated, but this time normalized to a luciferase construct driven by a promoter of moderate activity (Fig 3B). In this case, the pSV2 construct showed variable and overall
high activity in the different cell lines, leading to much lower fold
changes. However, the key finding was unchanged: that deletion of the
26 bp between 115 and 89 caused a greater reduction in
activity (6.2-fold) in the megakaryocytic HEL cell line as compared
with a 1.4-, 1.7-, and 2.7-fold reduction in CHO, HMEC-1, and WM793
cells, respectively.
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| Fig 3.
Cell line specificity of regulatory regions of the
3 gene. (A) The indicated constructs were transiently
transfected into the 3-expressing megakaryocytic cells
K562, Dami, and HEL, into the 3-expressing but
nonmegakaryocytic cells HMEC-1 and WM793, and into
non-3expressing cell lines CHO and 293 and
assayed for luciferase activity. Fold increase and the standard error
of the mean over the promoterless construct (background) are indicated.
The number of times each construct was tested is shown in parentheses.
The Materials and Methods describes how values were normalized. (B)
Additional reporter gene assays were performed four times and are
presented as the fold increase over the pGL-2-Promoter with the
standard error of the mean.
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Identification of a cell-specific DNA-protein interaction associated
with the 115 to 89 positive regulatory region of the
3 gene.
A series of EMSAs (probes listed in Table 1) identified DNA-protein
interactions in the region of positive regulatory activity from
115 to 89. A 26-bp probe called MSPw
(Fig 4A),
which spanned the entire 115 to 89 region, demonstrated a
complex pattern of shifted bands in EMSAs with crude nuclear extracts
from K562 cells (Fig 4B, lane 2). Although these bands were competed by unlabeled MSPw (lane 3), the band labeled P1 was not competed by an
irrelevant DNA probe (lane 4) and was only seen with nuclear extracts
from K562, Dami, and HEL cells (lanes 2, 5, 6, and 7) but not in
HMEC-1, HeLa, 293, or CHO cells (lanes 8 through 11). Much greater
specificity was observed using probe Pw containing sequence shifted in
a 5 direction (Fig 4C). The Pw-generated megakaryocytic cell
complex was presumed to be P1, because it comigrated with the complex
seen using the MSPw probe when run on the same gel (data not shown).
The lack of the P1 complex with HMEC-1 cells, which express
3, supported the functional studies and suggested that
the 115 to 89 element functions in a
megakaryocyte-specific fashion.
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| Fig 4.
Cell-specific protein-DNA complex associated
with a strong positive regulatory region (115 to 89) of the
3 regulatory unit. (A) Location of the probes spanning a
portion of the 3 regulatory unit that has cell-specific
and potent positive regulatory function. In (B) and (C), cold specific
and irrelevant oligonucleotide competitors (Table 1) were used in a
50-fold molar excess. Prefixes: c, unlabeled (cold) oligonucleotide; m,
mutant oligonucleotide; cm, cold mutant oligonucleotide. (B) EMSA using
32P-labeled MSPw oligonucleotide probe incubated with the
indicated nuclear extracts. Nuclear extracts used in lane 5 were from
K562 cells treated with PMA, which usually, but inconsistently, showed
the P1 complex. The megakaryocytic cell-specific band is indicated as
P1. The intensity of the P1 signal from PMA-treated K562 cells did not
correlate with 3 RNA (see Fig 1B), suggesting that the
115 to 89 sequence is not the sole contributor to transcription.
Irr., irrelevant DNA competitor (Table 1). (C) EMSA using
32P-labeled Pw, mPu1, or mPu2 oligonucleotide probes
incubated with the indicated nuclear extracts. The megakaryocytic
cell-specific band is again shown as P1, because it comigrates with the
MSPw complex (not shown). Radiolabeled probes are Pw in lanes 1 through
10, mPw1 in lanes 11 and 13, and mPw2 in lanes 12 and 14. (*)
32P-labeled DNA probe. (D) EMSA using
32P-labeled wild-type (MS4) and mutant (mMS4)
oligonucleotide probes and K562 nuclear extracts. cMS4 is unlabeled
MS4. Irr., irrelevant DNA competitor.
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As a first attempt at identifying the necessary sequences required for
the P1 cell-specific DNA-protein interaction, two variations of the Pw
probe (mPw1 and mPw2; Table 1 and Fig 4A) were synthesized that
contained nucleotide substitutions around position 115. As seen
in Fig 4C, mPw2 (lane 14), but not mPw1 (lane 13), resulted in the P1
shifted band seen with Pw (lane 2), indicating the mutations in mPw1
abrogated P1 binding. Competition experiments indicated that unlabeled
mPw1 was unable to compete away the Pw-generated P1 band, whereas mPw2
did (Fig 4C, compare lanes 4 and 5). In data not shown, the short probe
Pws (Table 1 and Fig 4A) bound no nuclear proteins, suggesting that
non-tissue-specific factors bound to the 3-most portion of
probes MSPw and Pw. These data indicate that the tissue-specific shift
required sequence around position 115 (present in the 5
regions of MSPw and Pw). Because the short Pws probe contained the
115 region but did not bind either the specific or nonspecific
proteins, it is possible that the tissue-specific shift either required
the additional concomitant binding of the 3 non-tissue-specific
factor or a conformation of the DNA not present in the probe shortened
at the 3 end.
There is a consensus ets/PU.1 binding site from 108 to
103 in the 3 5 region. Probe MS4 from this
region specifically bound nuclear protein (Fig 4D), but when the
consensus ets site was mutated (probe mMS4), the DNA-protein complex
was still observed (lane 1), suggesting that ets/PU.1 protein does not
bind this region. This conclusion is supported further by the fact that the mutant sequences in mPw1 abolished the DNA-protein binding despite
retaining the intact ets site.
Functional analysis of the 113 region.
To test the functional significance of the DNA-protein interactions
seen in Fig 4 and to define the extent of the megakaryocytic cell
preferential activity, mutations were introduced between positions
115 to 107 and studied in reporter gene assays.
Nucleotide substitutions corresponding to those in mPw1 resulted in an
average 2.93-fold lower activity in the megakaryocytic cell lines K562, Dami, and HEL (Fig 5). No decrease was
observed in 293 cells, consistent with the cell-preferential nature of
the sequence mutated in mPw1. Point mutations at positions 115,
114, and 107 produced no significant change in reporter
gene activity from the 146 wild-type construct (data not shown).
These functional studies, together with the EMSAs discussed above,
indicated that the minimal sequence conferring cell preferential
activity was the 6-bp sequence from 113 to 108: GAGGGG.
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| Fig 5.
The 112 to 109 sequence demonstrates cell
preferential activity. The 146 construct was mutated at those
positions that were altered in EMSA probe mPw1 and called 146
mPw1mut. Wild-type and mutant constructs were analyzed in the indicated
cell lines for their luciferase activity and are displayed as fold
activation over background with standard error bars. The number of
times each experiment was performed is indicated in parentheses. The
activity of the 146mPw1mut is only compared with the wild-type
activity in those experiments when both constructs were used. Because
there are some variations between experiments and because Fig 3
summarizes numerous different experiments with the wild-type 146
construct, there are some differences in the activities of this
construct between Figs 3 and 5.
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The 3 promoter may interact with the Sp1 transcription
factor.
As shown in Fig 3, the sequence from 89 to +29 possesses
substantial activity in seven different cell lines. Using EMSAs to
study this region, no specific protein-DNA complexes were detected with
the MS6 probe (Fig 6A) and nuclear extracts
of K562 cells (data not shown). Five bands emerged in EMSAs using the
MS7 probe and K562 nuclear extracts, and were named 7a through 7e (Fig
6B). Bands 7a and 7b were most clearly seen as a doublet when the gel was run longer (see Fig 7, lanes 9 and 10). Bands 7d and 7e represent nonspecific protein-DNA complexes, because they were competed away with
an irrelevant DNA probe. Bands 7a, 7b, and 7c were not competed with
unlabeled irrelevant DNA probe but were competed away with unlabeled
MS7, demonstrating that they were specific DNA-protein interactions.
Because of their substantially greater intensity, we focused our
attention on bands 7a and 7b (7c was not consistently observed). Using
additional nuclear extracts (lanes 5 through 10), we also observed that
bands 7a and 7b were most abundant in the megakaryocytic cell lines.
View larger version (47K):
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| Fig 6.
Demonstration of a DNA-protein complex in the MS7 region
of 3. (A) Location of the MS6 and MS7 probes and the
putative binding site for the Sp1 transcriptional factor. (B) EMSA
using 32P-labeled MS7 DNA probe and the indicated nuclear
extracts. Cold specific or irrelevant oligonucleotide competitors
(Table 1) were used at a 50-fold molar excess. Irr., irrelevant DNA
competitor; c, unlabeled oligonucleotide.
|
|
A binding site for the Sp1 transcription factor was present in the MS7
DNA probe, and we performed a series of studies to determine if this
site (between 73 and 66 bp) was functional (Fig 7). Several experiments were highly
suggestive that bands 7a and 7b contained Sp1. (1) Bands 7a and 7b
disappeared when the probe was mutated (mMS7, Table 1) at sites known
to destroy Sp1 binding60 (Fig 7, lane 3). (2) Bands 7a and
7b were supershifted with an MoAb specific for Sp1 (lanes 5 and 9) and
not with an irrelevant mouse IgG (lane 6). (3) A comigrating band was
seen when purified Sp1 protein was used in an EMSA with MS7 (compare lanes 7 and 10, Fig 7); the experiment in lane 8 confirms the position
of the supershift as well as authentic Sp1 reactivity with probe MS7.
View larger version (56K):
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| Fig 7.
Interaction of Sp1 with the 3 promoter.
EMSA using 32P-labeled mMS7 or MS7 oligonucleotide probes
incubated with K562 nuclear protein or purified Sp1 protein as
indicated above by the "+." Sp1-purified protein was used at
1.5 µg/sample. Lanes 7 through 10 are from a different gel that was
run longer. The appearance of the 7b band was inconsistently observed
with purified Sp1 and this probe. Anti-Sp1 antibody or the irrelevant
mouse Ig (irrel. Ab) was added to samples as indicated by the
"+" above. Prefix "m" indicates mutant oligonucleotide,
as described in Table 1.
|
|
Functional evidence for Sp1 activity.
We introduced nucleotide substitutions into the 146 luciferase
construct that destroyed the Sp1 binding site and observed 2.2-fold
less activity compared with wild-type (Fig
8A), suggesting that this region binds a positive regulatory factor in
vivo. A series of cotransfection experiments were also performed to
assess whether the Sp1 transcription factor interacted with the
113 region (Fig 8B). Overexpression of Sp1 resulted in increased
expression, further implicating an Sp1 function in megakaryocytic
cells. The Sp1 stimulated activity did not require the wild-type
sequence at positions 113 to 109 (note the increase when
Sp1 is overexpressed with the 146mPw1 construct) and merely had
an additive effect.
View larger version (30K):
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| Fig 8.
Sp1 functions at the 70 position of the
3 promoter. (A) Sp1 mutational analysis. HEL cells were
transfected with equivalent amounts of 146 wild-type and Sp1 mutant
constructs, assayed for luciferase activity, and expressed as fold
activation over background. The mutant construct contained the 2-bp
substitution in probe mMS7 that destroyed the Sp1 site at position
70. Each experiment was performed five times. (B) Cotransfection of
Sp1 enhances expression. HEL cells were transfected with the indicated
plasmids and assayed for luciferase activity. Each experiment was
performed four times. Twenty micrograms of 146 wild-type or 146
mPw1mut and 4 µg of CMV-Sp1 or CMV-empty (total plasmid DNA, 24 µg)
were transfected in each experiment.
|
|
| |
DISCUSSION |
Although integrin 3 is expressed in numerous tissues,
our studies have identified a sequence in the 5 regulatory
portion of the 3 gene modulating expression in a
megakaryocytic-specific manner. We found that the GAGGGG sequence at
positions 113 to 108 augmented reporter gene expression
and bound nuclear proteins to a greater degree in megakaryocytic cells
than in nonmegakaryocytic cells, regardless of whether they expressed
3. We also obtained evidence suggesting that the Sp1
transcription factor binds to and upregulates expression through the
70 position of the 3 transcriptional regulatory
unit and that this interaction appeared especially prominent in
megakaryocytic cells. Thus, 3 expression is regulated by
specific and nonspecific factors. The latter, when bound in particular
combinations and specific locations, may perhaps contribute to
tissue-specific gene expression. Our data also provide
rationale for a significant degree of low-to-modest levels of
3 expression in a spectrum of different tissues and at
the same time support a hypothesis that megakaryocytes express higher
concentrations than other tissues.
Functional studies identified distal sequences upstream of the
3 gene with positive and negative regulatory activity,
and the region from 115 to 89 displayed megakaryocytic
cell- specific activity. K562, Dami, and HEL cells showed 6- to 40-fold
reduced activity when this sequence was deleted (Fig 3), whereas there was only a 1.7- and 2.7-fold reduction, respectively, in
3-expressing endothelial and melanoma cell lines; CHO
and 293 cells showed a 1.3- and 2.8-fold reduction, respectively. The
minimal sequence required for a megakaryocytic cell-specific
DNA-protein interaction was positions 113 to 99
(GAGGGGAGGAAGCGC), and the GAGGGG sequence behaved as a
megakaryocyte-specific element (MSE), because mutations in this
sequence affected activity in only the megakaryocytic lines. In
addition, there was no detectable binding of HMEC-1 nuclear proteins to
the GAGGGG sequence (Fig 4C). There are at least several possible
explanations for the minimal activity of the MSE in
3-expressing endothelial and melanoma cell lines, such
as (1) these cells contain cell specific factors that bind to enhancers
not contained within our constructs, and (2) 3
expression could be regulated in these cells by nontranscriptional
mechanisms. Although our data indicate that a nuclear protein expressed
at high levels in megakaryocytic cell lines and not in
nonmegakaryocytic cells binds to the MSE of the 3 gene,
non-tissue-specific factors also bound to several other adjacent
sequences, and additional studies are required to determine whether
these are involved in binding to the MSE.
We performed extensive searches of DNA databases for homologies to the
GAGGGG sequence and the best matches were with genes matching via their
CpG islands (none of which had any other relation to transcriptional
activity or to megakaryocyte genes) and with three genes in the
promoter database: wheat histone H3, the human c-sis/platelet
derived growth factor 2 (PDGF2), and a human skeletal actin. Of these,
only the region of the c-sis gene had been shown to affect gene
expression, although the specific sequence identical to the
3 gene had not been studied.61,62 To assess
whether this homologous sequence may be important in the context of
other genes, we prepared a 28-bp DNA probe from the c-sis/PDGF2
gene corresponding to probe Pw in the 3 gene that
contained the homologous region in its center. However, this probe did
not gel shift any specific bands with K562 nuclear extracts (data not
shown), suggesting coincidental homology or that other cell extracts or
binding conditions will be required to detect a protein interaction.
Functional studies showed that the 146 construct displayed
overall positive and megakaryocytic-preferential expression, and those
experiments shown in Figs 1 and 3A suggested a negative element in the
126 to 115 region. EMSAs using this region showed very
complex patterns, perhaps because of a 20-bp direct repeat at position
146, and mutations demonstrate that it has negative activity in
both HEL and 293 cells (data not shown). A tissue-specific, negative
regulatory sequence has been described with the -globin gene,63 but further studies will be required to determine
if this is present in the 3 gene. Another example of the
complexity of DNA-protein interactions at the MSE was the consistently
observed reduction in the P1 protein complex with probe Pw and nuclear extracts from PMA-stimulated K562 cells (Fig 4C). Thus, although PMA
treatment of K562 cells induces 3 RNA and protein
expression (Fig 2), such overall positive effects on expression must
operate through another site, while possibly suppressing the
DNA-protein interaction at 113 to 108. Taken together,
this transcriptional regulatory unit of the 3 gene
contains both positive and negative elements and multiple nuclear
factors are likely acting on them to regulate the complex pattern of
3 expression.
Sp1 is a zinc finger transcription factor that has been shown to
regulate expression in other integrin genes, including
IIb,25 2,64 and
four of the leukocyte integrins, CD11a,65
CD11b,66 CD11c,65 and CD18.67 The
3 gene has at least four potential Sp1 binding sites and
the promoter is GC-rich, TATA-less and lacks a consensus Inr element,
which are often features of Sp1-responsive genes. Typically, the most
important Sp1 element is 40 to 70 bp upstream of the transcription
start site,68 so we focused on the only site in this region
of the 3 gene, ie, that at position 70. This
binding site does not conform to the classical Sp1 consensus binding
site (GGGCGG), but rather has a sequence observed in a variety of other
genes (CNCACCC [N = A, T, or C]). In addition to Sp1, this
sequence will also bind CACD or EKLF, which is felt to be an
erythroid-specific transcription factor related to the Kruppel family
of nuclear proteins.60,69 There are several lines of
evidence against EKLF binding to this site. (1) The MoAb used in the
gel shift assay reacts specifically with the carboxyl terminus of Sp1
and does not cross-react with Sp2, Sp3, Sp4, or EKLF. (2) Sp1 is
expressed in K562 cells,70 whereas EKLF is not.71 (3) An EKLF knock-out mouse had no megakaryocyte
phenotype.72 In addition, the data in Figs 7 and 8 provide
strong functional, biochemical, and immunologic evidence that Sp1 is
most likely the major factor binding to this sequence at position
70 of the 3 gene. This site is in a
region of modest transcriptional activity (Fig 3) and does not require
or cooperate with the MSE, although the two sites have additive effects
(Fig 8).
Sp1 is ubiquitously expressed and as such might not, by itself, be
expected to demonstrate the increased abundance of complexes that we
observed with DNA in megakaryocytic cell lines compared with
nonmegakaryocytic lines (Fig 6B). In the case of CD11c, Sp1 has been
shown to participate in tissue-restricted expression,65,73 but there are other possible explanations for the more abundant Sp1-DNA
complexes observed with megakaryocytic cell lines, such as partial
degradation of nuclear proteins in the nonmegakaryocytic cells or a
protein complexed to Sp1 in these cells that enhances (in
megakaryocytic cells) or prevents (in non-3-expressing
cells) Sp1 binding to the CCCACCC sequence. Sp1 can also mediate
transcription through the CCACCC sequence via an indirect stimulation
with the retinoblastoma (RB) gene product.74 Although we
cannot exclude the possibility that there are proteins positively or
negatively modulating Sp1 binding at position 70, we presently
have no evidence that either occurs.
Few general principles regarding integrin gene expression have been
elucidated,75 although the promoters of most integrin genes
studied to date tend to be GC-rich and lack TATA and CAAT boxes, and
only a few have Inrs.46,76-80 Repressor elements are features common to integrins,16,81,82 and the
3 gene contains at least two: one between 290 and
146 and another between 126 and 115. The
2 promoter has been studied in some detail: two members
of the Ets family (PU.1 and GABP) are required for cell-specific expression and both Sp1 and retinoic acid receptor binding sites have
been identified.79,83-85 Sequence analysis of the 5
regions of integrin genes has shown binding sites for Sp1, GATA-1,
AP1, AP2, and Ets, although few functional studies have been
performed.77,78,80,86,87 The IIb gene is
expressed solely in megakaryocytic cells, and this expression is
controlled at least in part by Sp1 and factors binding to GATA-1 and
Ets sites.25,88 The 3 gene has an ets/PU.1 site at 105, but our EMSA studies suggest there is most likely no ets/PU.1 binding (Fig 4C and D). There are several GATA-1 sites in
the 5 region of the 3 gene (in studies not shown,
a GATA-1 expression construct was not able to transactivate through
these), but none in sequences that showed the greatest cell-specificity in these studies. Thus, as would be expected, some aspects of 3 expression are mediated via different mechanisms than
IIb. On the other hand, a 22-bp sequence of the
IIb gene containing the GAGG portion of this sequence
has been shown to bind to megakaryocytic cell nuclear proteins by
DNaseI footprinting and EMSAs.23 Prandini et
al20 mutated the last 2 bp of this sequence (GAGG to GATT) and demonstrated a marked decrease in promoter activity in HEL and K562
cells, although nonmegakaryocytic cells were not tested. Thus, it will
be interesting in the future to determine whether the same
megakaryocyte-specific transcription factor acts on these sites in both
the IIb and 3 genes. Such a mechanism
could contribute to some degree of coordinated gene regulation.
| |
FOOTNOTES |
Submitted September 8, 1997;
accepted June 12, 1998.
Supported by Grant No. HL51457 from the National Institutes of Health
(Bethesda, MD) and by the Rogers-Wilbur Foundation.
Address reprint requests to Paul F. Bray, MD, Ross 1015, Johns Hopkins
University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205.
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.
| |
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Abstract
Introduction
Abstract