From the Program of Cellular and Molecular Biology,
Allegheny University of the Health Sciences and the
§ Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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The Sp1 family of transcription
factors are often involved in the regulated expression of TATA-less
genes, frequently enhancing gene transcription. In this paper, we
demonstrate that an Sp1-binding element inhibits the expression of the
megakaryocyte-specific IIb gene in all cell lines
tested and that this inhibition is actively overcome only in
megakaryocyte-like cell lines. We had noted previously in primary
megakaryocytes that a 50-base pair (bp) deletion from
150 to
101 bp
in the rat
IIb promoter region resulted in increased
expression. We now show that deletion of this region markedly increased
expression in both megakaryocytic and non-megakaryocytic cell lines,
eliminating the tissue specificity of the
IIb promoter.
Electrophoretic mobility shift assays (EMSA) defined a single complex,
which bound to a
145 to
125 bp subregion. Point mutations within
this region, localized the critical point of binding around bases
136/
135, and expression studies showed that introduction of the
136/
135 mutation into the rat
IIb promoter had a
comparable result to that seen with the 50-bp deletion. EMSA studies
with the homologous human
IIb promoter region gave an
identical migrating band. Southwestern blots of HeLa nuclear proteins
with both the rat
145 to
125 DNA and its human homologue bound to a
single ~110-kDa protein, the known molecular weight of Sp1.
Confirmation that this region of the
IIb gene promoter bound Sp1 was accomplished using EMSA studies with an Sp1 consensus probe, anti-Sp1 and -Sp3 antibodies, and recombinant Sp1 protein. Further support for the role of Sp1 in the silencing of the
IIb promoter was obtained using a Gal4 binding site
substitution for the silencer region of
IIb and
co-expression of near full-length Sp1/Gal4 fusion protein expression
vectors. Ectopic reinsertion of the
150 to
101 bp region, back into
the
150 to
101 bp deleted promoter, enhanced rather than decreased
expression, suggesting that Sp1's inhibitory role at
136/
135
depends on its local interactions. In summary, we believe that we have
identified a cross-species, non-consensus Sp1-binding site that binds
Sp1 and that acts as a silencer of
IIb expression in
many cell lines. A model is presented as to how this Sp1-binding
silencer element contributes to the megakaryocyte-specific expression
of
IIb gene.
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INTRODUCTION |
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Platelets have a central role in thrombus formation. These
anuclear cytoplasmic fragments are derived from bone marrow
megakaryocytes and are highly differentiated (1). One of the
specialized features found on the platelet membrane is the
IIb/
3 (also known as glycoprotein IIb/IIIa or CD41b) integrin receptor (2). This receptor is densely
packed on the platelet surface. Following platelet activation, this
receptor binds fibrinogen and plays an important role in platelet
aggregation (2). Normally,
IIb/
3 is only
found on developing megakaryocytes and circulating platelets. This is
due to the tissue-specific nature of the
IIb subunit.
While
3 is expressed in several different cell types
(3), the
IIb gene is normally limited in its expression
to megakaryocytes (4). We have shown previously that the
IIb gene is a TATA-less gene comprising 30 exons,
extending over an 18-kilobase pair region of the long arm of chromosome
17 (5, 6).
The IIb gene is presently the best studied
megakaryocyte-specific gene. Better understanding of the regulation of
IIb transcription in a lineage-restricted fashion allows
us to learn more about the molecular mechanisms controlling
hematopoietic differentiation. It may also allows us to develop new
approaches for regulating gene expression in developing megakaryocytes
and modulating platelet thrombogenic tendency. Other investigators
defined four important elements in the 5'-flanking region of the human
IIb gene that promote tissue-specific expression: two
pairs of GATA- and Ets-binding sites, one proximal to the
transcriptional start site and one more distal (7-10). Deletion or
mutation of any of these GATA- or Ets-binding sites had a 2-3-fold
effect on the level of reporter gene expression in transient expression
studies in megakaryocytic cell lines. Studying the rat
IIb promoter in a primary rat marrow system, we found
the same four regulatory regions, but demonstrated a significant
quantitative difference in promoter activity; the distal GATA-binding
site at
454 bp1
(GATA454) had a 50-fold effect on expression and was
essential for observing any promoter activity (11). We have since shown that these quantitative differences in promoter strength were not
species-specific differences in the promoter region, but due to
differences in the cell systems studied. When the same rat
IIb promoter constructs were restudied in a
megakaryocytic cell line (HEL), the results seen were indistinguishable
from the human data (12).
In addition, we used the same rat primary marrow transient expression
system to define a GA-rich Sp1-binding site at 14 bp (Sp114) (13). We showed that the complex bound to this site interacted with the complex bound at the proximal Ets-binding site at
35 bp (Ets35). The Ets35 site appears to
tether Sp1 to its binding site, and we proposed that this tethered Sp1
is important in mooring the transcriptional initiation complex to the
TATA-less
IIb gene.
While carrying out these studies, we noted that, when a series of 50-bp
deletions were made in a 912-bp IIb 5'-flanking region reporter construct, leaving the GATA454 intact and
beginning the deletions at
450 bp, all of the constructs had
decreased activity compared with the wild-type construct, except for
one construct that expressed twice as well as the wild type. We
proposed that either this region between
150 bp and
101 bp
contained a silencer element or the increase in expression was due to
architectural disruption of the promoter region by the deletion (11).
Since then, two other groups have defined silencer elements near this region in the human
IIb promoter region (14, 15). The
common element between these two studies suggests that there is a
region at
120 to
116 bp in the human
IIb 5'-flanking
region 5'-ATGAG-3' (corresponding to the rat
113 to
109 bp region)
that binds a silencer complex. In this paper, we demonstrate a
different site ~30 bp further upstream as being important in
silencing
IIb expression. We show that an Sp1-binding
site that is conserved across species is the center of this silencer
element and that this site appears to bind Sp1 (16). Mutation of this
site leads to high levels of expression in both megakaryocytic and
non-megakaryocytic cell lines, and therefore eliminates the tissue
specificity of the
IIb promoter. This site does not
appear to bind to Sp3, a known negative regulator in the Sp1 family
(17-19). Thus, it appears that the silencer domain of the
IIb gene involves an increasingly recognized role of Sp1
as a negative regulator. We show that ectopic reintroduction of the
silencer domain into the silencer-deleted
IIb promoter
enhanced rather than decreased expression, suggesting that Sp1
silencing effect may depend on local interactions with other bound
nuclear proteins. A model is presented as to how the silencer element
may function in
IIb expression.
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MATERIALS AND METHODS |
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Cell Culture-- CHRF-288-11 (called CHRF) (20), a megakaryoblastic cell line generously provided by Dr. Michael Lieberman (University of Cincinnati, Cincinnati, OH), was maintained in RPMI medium 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma). All other cell lines used in this study were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). K562 (chronic myelogenous leukemia, ATCC CCL-243), HEL (erythroleukemia, ATCC TIB-180), and HL-60 (promyelocytic leukemia, ATCC CCL-240) cells were grown in RPMI medium 1640 containing 10% FBS. HeLa (cervical epithelioid carcinoma, ATCC CCL-2) and NIH 3T3 cells (murine embryonic fibroblast, ATCC CRL-1658) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS. All media contain 100 units/ml penicillin, 100 units/ml streptomycin, and 200 mM L-glutamine. K562 cells were incubated with 40 nM phorbol myristate acetate (PMA) for 5 days to induce megakaryocytic differentiation (14).
Plasmid Construction--
Both 912 and 453 base pairs of rat
IIb 5'-flanking region were PCR-amplified from a
2.2-kilobase pair SstI fragment of the rat
5'-
IIb gene that was isolated from a partial
Sau3A rat genomic library (5) and subcloned into
single-stranded M13mp18 (11). The sense primers for PCR were designed
according to published sequence (5) with a BamHI site
flanking their 5' ends. The antisense primer
5'-AAGCTTCTTCCTTCTCCCCAAATGT-3', includes the untranslated
region of rat
IIb gene and a HindIII site
(underlined). PCR products were cut with both BamHI and
HindIII, and subcloned into
BglII/HindIII-digested promoter-less luciferase
reporter vector pGL3-basic (Promega Corp., Madison, WI).
150 to
101
bp deletion, and CC
AA substitutions at
140/
141 bp,
135/
136 bp, and
130/
131 bp, were then created by overlapping PCR (11) using
912bp-pGL3 or 453bp-pGL3 construct as templates. All the PCR-based
constructs were sequenced to exclude any PCR-induced mutations.
Transfection and Reporter Gene Assays--
For CHRF cell
transfection, cells were seeded at 0.3 × 106/ml.
After growing for 2 days, cells were collected and resuspended in
electrophoresis buffer (30.8 mmol/liter NaCl, 120.7 mmol/liter KCl, 8.1 mmol/liter Na2HPO4, 1.46 mmol/liter
KH2PO4, 5 mmol/liter MgCl2) at a
concentration of 30 × 106 cells/ml. Thirty micrograms
of assay plasmid DNA and 20 µg of pCMV were added to 0.5 ml of
cells in 0.4-cm electroporation cuvettes. After a 15-min incubation on
ice, these cells were electroporated by Cell-Porator (Life
Technologies, Inc.) at 230 V and 800 millifarads. Cells were then
allowed to recover on ice for 10 min and at room temperature for 15 min. After washing with complete growth medium, cells were resuspended
in 3 ml of growth medium and grown in six-well tissue culture plates
for 24 h before being assayed for luciferase activity. HEL, HeLa,
NIH 3T3, and HL-60 cells were transfected using LipofectAMINETM reagent
from Life Technologies, Inc. Briefly, 2.5 µg of assay plasmid DNA and
0.5 µg of internal control DNA pCMV
, were incubated with 10 µl
of LipofectAMINETM reagent in Opti-MEM I reduced serum medium (Life
Technologies, Inc.) for 30 min. The formed DNA-liposome complexes were
added to either exponentially growing HEL and HL-60 cells with 1.5 × 106 cells/sample or 60-70% confluent HeLa and NIH 3T3
cells grown in six-well tissue culture plates. All cells were washed
with and then suspended in Opti-MEM medium before the addition of
DNA-liposome mixture. A 5-h incubation at 37 °C in a CO2
incubator followed. After being washed with the appropriate complete
growth medium, cells were grown in 3 ml (HEL and HL-60) or 2 ml (HeLa
and NIH 3T3) of the same medium in six-well tissue culture plates for 48 h before the reporter gene assays. For Gal4/Sp1 cotransfection assays in HeLa cells, 2 µg of the 453Gal4-pGL3 vector (having the
Gal4 binding site substituted into the
145 to
125 region of the
453-bp rat
IIb promoter), 1 µg of the various Gal4/Sp1 expression vectors, and 0.5 µg of pCMV
, were cotransfected to each
well of cells using 12.5 µl of LipofectAMINETM reagent.
Electrophoretic Mobility Shift Assays (EMSA)--
Nuclear
extracts were prepared as described previously (11) from HeLa, CHRF,
and K562 cells with or without PMA induction. The single-stranded
oligonucleotides were synthesized by Integrated DNA Technologies
(Coralville, IA), and the complementary sense and antisense strands
were then annealed into double-stranded DNAs used for EMSA. The Sp1
consensus binding sequence was purchased from Promega Corp. These
double-stranded DNAs were end-labeled by T4 polynucleotide
kinase and [-32P]ATP. Then, 0.1-0.2 ng of probes
(~2 × 105 cpm) were incubated with 5 µg of
nuclear extract on ice for 20 min in a 20 µl binding reaction that
contained 18 mM HEPES, pH 7.8, 40 mM KCl, 4 mM MgCl2, 0.5 mM dithiothreitol, 3 µg of poly(dI-dC), 2 µg of bovine serum albumin, and 10% (v/v)
glycerol. For competition studies, prior to the addition of radioactive
probes, unlabeled competitors were added to binding reaction and
incubated with nuclear extract for 10 min on ice. Samples were then
electrophoresed at 4 °C on a 4% (v/v) polyacrylamide,
non-denaturing gel in 0.5× Tris-boric acid-electrophoresis (TBE)
buffer. Rabbit anti-human Sp1, Sp3, and YY1 polyclonal antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For gel
supershift assays, 0.5-2 µl (1 µg/µl) of antibody were added to
the binding reaction and incubated on ice for additional 15 min before
loading. A total of 0.5 footprint units of recombinant human Sp1
protein (Promega Corp.) was used instead of nuclear extracts in
EMSA.
Southwestern Blotting Analysis--
Thirty micrograms of HeLa
nuclear extract was electrophoresed on a 6% sodium dodecyl sulfate
(SDS)-polyacrylamide gel with prestained protein molecular weight
standards (14,300-200,000 molecular range) from Life Technologies,
Inc. The proteins were then electroblotted to nitrocellulose filters
(Schleicher & Schuell BA85, 0.45 mm), and renatured on the filters by
serial dilution from 6 M guanidine hydrochloride to Z'
buffer (25 mM Hepes-KOH, pH 7.6, 12.5 mM
MgCl2, 20% glycerol, 0.1% Nonidet P-40, 0.1 M KCl, 10 µM ZnSO4, 1 mM
dithiothreitol). The membrane was then incubated for 30 min in blocking
solution, containing 3% nonfat dried milk in Z' buffer, and probed
with 32P-end-labeled double-stranded rat 145 to
125 bp
sequence, its human homologue, and M2 mutant in binding buffer (Z'
buffer containing 0.25% nonfat dried milk) for 1 h at room
temperature. The filter was then washed three times with Z' buffer for
a total time of 15 min and exposed to autoradiographic film. A parallel
gel containing the same amount of HeLa nuclear extract was also stained
with Coomassie Brilliant Blue.
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RESULTS |
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Functional Studies of the Potential Silencer Element in Both
Megakaryocytic and Non-megakaryocytic Cell Lines--
Previous studies
have shown that 150 to
101 bp region upstream of the
transcriptional start site of rat
IIb gene, when deleted, caused a 2-fold increase in the reporter gene, human growth
hormone expression in a rat primary marrow transient expression system
(23). To confirm this finding in other expression systems and to
examine the tissue-specific nature of this deletion on expression, we
shuttled the 453-bp and 912-bp 5'-flanking region of the rat
IIb gene into the pGL3-basic vector, which contains a
luciferase reporter gene. The
150 to
101 bp region was then deleted
from the wild-type constructs. The important positive regulator
GATA454 element is present in 912-bp constructs but not in
453-bp constructs. Transient transfection studies of all these
constructs were carried out both in the
IIb-expressing CHRF and HEL cells, and in the
IIb-non-expressing cell
lines, epithelioid HeLa, fibroblastic NIH 3T3, and myeloid HL60
cells.
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DNA-Protein Interaction in the 150 to
101 bp Region by
EMSA--
To examine if there are any nuclear proteins binding to the
150 to
101 bp region, EMSA was carried out using nuclear extracts prepared from CHRF, K562 (another megakaryocyte-like cell line that may
represent a less differentiated megakaryocyte-stage; Ref. 14) with or
without PMA induction, and HeLa cells. The results were very similar
with all three cell lines. Because the silencer effect was seen with
non-megakaryocytic as well as megakaryocytic cell lines, the data
presented below focus on the HeLa mobility shift studies. As seen in
Fig. 2A, three initial
double-stranded DNA probes were made, two overlapping the
150 to
101 bp region, and one corresponding to the human silencer region
(14, 15) spanning the rat sequence from
114 to
88 bp (5). The rat
114 to
88 bp region is the homologue of human
124 to
98 bp sequence, used by Fong et al. (14) as a probe in EMSA. The
rat
114 to
88 bp double-stranded DNA did not complex with nuclear extracts from all three cell lines tested (data not shown). The other
two double-stranded DNAs were from
150 to
116 bp and from
135 to
101 bp, and overlap by 15 bp. Only the
150 to
116 bp DNA formed a
complex (Fig. 2B), and this complex could be cold competed
away by non-radiolabeled
150 to
101 DNA (lanes 3 and 4). The complex could not be competed away by the
135 to
101 bp region (lane 5) or by an unrelated Ets sequence
(lane 6), indicating specific binding to the
150 to
116
bp region. The
135 to
101 bp double-stranded probe, containing the
homologous human silencer region, failed to give rise to any
significant complex formation (Fig. 2C). For the
135 to
101 bp double-stranded probe, varying salt conditions (from 20 to 100 mM), Mg2+ concentration (from 0 to 10 mM, Fig. 2C), amount of nonspecific competitor
(from 1 to 3 µg of poly(dI-dC) per reaction), and buffering capacity
(from 5 mM to 25 mM HEPES, pH 7.8) did not lead
to a detectable band.
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Further Characterization of the Protein Binding Sequence in the
145 to
125 bp Region--
To identify the base pairs that are
important for protein binding in the
145 to
125 bp region, three CC
AA substitutions were made at
140/
139 bp (M1),
136/
135 bp
(M2), and
131/
130 bp (M3) as shown in Fig. 2A. These
mutated double-stranded DNAs were used in EMSA as cold competitors
against wild-type
145 to
125 bp binding (Fig.
4, lanes 4-6) as well as
probes to assay their own binding activity (Fig. 4, lanes
7-12). As a cold competitor, M1 (lane 4) showed
moderate competition against the wild-type
145 to
125 bp probe,
while M2 (lane 5) did not appear to be an effective
competitor, and M3 (lane 6) appeared to compete as well as
the wild-type cold competitor (lane 3). Consistent with these findings, labeled M1 formed a slight amount of complex
(lane 8), M2 formed no observable complex (lane
10), and M3 readily formed a complex (lane 12)
identical to the wild-type complex in intensity (lane
2).
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Expression Studies with the Three CC AA Mutations--
We then
asked whether the EMSA findings would be reflected in expression
studies. We introduced the three CC
AA mutations into the wild-type
rat 453bp-pGL3 construct, and tested these new constructs in HeLa, NIH
3T3, and HL60 cells. All three cell lines gave similar results (Fig.
4B). In agreement with the EMSA data, M1 had a modest
increase in expression (3-16% of the 453
construct), M2 had a
significant increase (40-80% of the 453
construct), and M3 had the
same low level of expression as the wild-type 453 construct
(0.12-1.2% of the 453
construct). Thus, elimination of binding of
a single complex to this region appears to correlate with lost of
silencing of the
IIb promoter.
EMSA with the Human Homologue of the Rat 145 to
125 bp
Region--
To examine whether this site was species-specific or more
universally applicable, we then tested whether the human homologue to
this region, which has 7/21 nucleotide substitutions, would also bind
to the same complex in EMSA studies. As can be seen in Fig.
5A (lanes 2-6),
this human homologue formed a similar size complex that can be
effectively competed away by itself and by the rat
145 to
125 bp
double-stranded DNA. Conversely, the human homologue was equally
effective at competing away binding to the rat
145 to
125 bp probe
(Fig. 5A, lanes 8-12). M1, M2, and M3 cold
competition studies (Fig. 5B, lanes 7-12) were
identical to those shown in Fig. 4A for the rat wild-type
probe. These findings suggest that the same complex formed with both
the human and rat sequence in this region.
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Sp1-related Protein(s) Are Involved in the Binding of 145 to
125 Region--
In an attempt to identify the silencer-binding
protein, we searched the Transfac data base by Transcription Element
Search Software (TESS),2
looking for known transcription factors that have consensus binding sequence homologous to our silencer region around bases
136/
135. The search suggested MAZ (Myc-associated zinc-finger protein) (24-26),
Yi (27), and NF-1 (28) as potential candidates. Mela1 (24-26) and
YiMT3 (27), known MAZ- and Yi-binding elements, respectively, and the
NF-1 consensus binding sequence (Santa Cruz Biotechnology, Inc.), were
used as cold competitors in EMSA for the binding to the
145 to
125
bp region using HeLa nuclear extract. While YiMT3 and NF-1 consensus
did not show any effect on the binding, Mela1 did cold compete to the
same extent as unlabeled wild-type
145 to
125 bp DNA, suggesting
that a MAZ-related protein might be involved (data not shown).
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Further Confirmation of the Role of Sp1 in the Silencing of
IIb Gene Expression by Gal4/Sp1 Cotransfection
Studies--
To further confirm that Sp1 is important in
IIb silencing, we substituted a Gal4 binding sequence
from nucleotides
145 to
125 in the 453-bp
IIb
promoter region. As expected, this substitution in the silencer region
(453Gal4-pGL3) markedly increased expression of the 453bp-pGL3, leading
to levels of expression comparable to the SV40-driven positive control
(40% of SV40-pGL3) and nearly twice as high as 453M2-pGL3 (Fig.
8A). More importantly, when this construct is coexpressed
with a series of Sp1/Gal4 fusion proteins, containing the DNA binding
domain of the yeast transcription factor Gal4 (21), silencing activity
is seen with the two longest fusion proteins, pSG-Sp1WT and pSG-Sp1N
(see "Materials and Methods") (Fig. 8B). While pSG-Sp1A
and pSG-Sp1A&B increased the expression of 453Gal4-pGL3 2.3- and
1.7-fold, respectively, pSG-Sp1N and pSG-Sp1WT both decreased its
expression to 50% and 60%, respectively, compared with the pSG424
control (Gal4 only vector). In addition to providing further support
for our findings that Sp1 is involved in silencing, these data suggest
that near full-length Sp1 is necessary to achieve silencing of the
promoter and that other constructs that may not be able to interact in
a specific fashion with regulatory domains in this promoter actually
lead to a further increase in expression.
Ectopic Reinsertion of the 150 to
101 bp Region Immediately
Upstream of the 453bp
-pGL3 Construct and Its Effect on Expression in
HeLa Cells--
To examine whether or not the Sp1-binding silencer
element functions in a position- and orientation-independent fashion,
we deleted the
150 to
101 bp region from 453 bp of rat
IIb promoter and reintroduced this 50-bp region back
into the same promoter, but upstream from the 453-bp region. A single
copy was inserted in two different constructs, one in forward and one
in reverse orientation. These constructs were transfected into HeLa
cells to examine the relocation effect on
expression. As we have shown above, the
453
-pGL3 construct gave rise to a significant level of expression.
However, the reintroduction of this 50-bp region into a different
position in either forward or reverse orientation did not suppress
expression. Instead, these constructs resulted in a 1.7 ± 0.23-fold and 2.4 ± 0.33-fold further increase in expression, respectively, when compared with the 453
-pGL3 construct. Therefore, it appears that the inhibitory activity of this silencer element is
dependent upon its physical location in the
IIb
promoter.
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DISCUSSION |
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We have found that the previously described effect of deleting the
150 to
101 region upstream of the transcriptional start site of the
rat
IIb gene (11) was not due to a physical disruption of the promoter region, but rather due to the lost of a cross-species conserved, Sp1-binding site. This site centers on nucleotides
136/
135, and we have named it the Sp1135 site. Complex
binding to this site silences
IIb promoter-driven
expression in both megakaryocytic and non-megakaryocytic cell
lines.
Our data also suggest that this silencer site binds Sp1, a member of a
multigene family of zinc finger transcription factors (36, 37). Sp1 is
ubiquitously expressed in all tissues, and its ubiquity is consistent
with our finding that Sp1135 complex suppresses
IIb expression in all tested cell lines. Sp1 binds to GC
boxes and similar motifs (38-41), but also binds to a number of
non-consensus sequences as well (42-44). The
145 to
125 bp region
is a CT-rich region that is consistent with such non-consensus Sp1-binding sites.
Other Sp1 family members include Sp2, Sp3, and Sp4 (36, 37). Sp2 binds a GT box in the promoter of a T-cell receptor gene and seems to have divergent nucleotide recognition sequence (36), while Sp3 (36, 37) and Sp4 (37) have binding specificity and affinity similar to those of Sp1.
Sp3 also appears to be ubiquitously expressed, while Sp4 appears to be limited in vivo to neural tissues (36). The zinc finger DNA-binding domains and glutamine- and serine/threonine-rich activation domains are highly conserved between Sp1, Sp3, and Sp4 (17, 45-47). However, while Sp1 and Sp4 appear to mostly promote transcription, Sp3 has often been shown to be a negative regulator of expression (17, 33, 47). The DNA-binding domains of Sp1 and Sp3 appear to be functionally interchangeable, but the activation domain of Sp3 is not functional when chimerically linked to the Sp1 DNA binding domain (17). This suggests that the negative regulatory activity of Sp3 may be due to its competition with Sp1 for a common binding site. Indeed, Sp3 suppression of Sp1-mediated transcriptional activation has been described in many genes, including both basal and Tat-activated expression of the human immunodeficiency virus promoter (33). Differential expression of Sp1 and Sp3 in different tissues and altered Sp1/Sp3 ratio during cell differentiation and transformation have been shown to be responsible for the regulation of several epithelial-specific promoters (44, 48). Therefore, Sp3 binding would have provided an explanation for the involvement of an Sp1-binding element in negative regulation of gene transcription.
We have examined whether or not Sp3 is involved in Sp1135
binding by EMSA studies using an anti-Sp3-specific antibody. Neither supershift nor blocking was observed. Furthermore, the
anti-Sp1-specific antibody can supershift virtually all of the complex
(Fig. 7B, lane 5), suggesting that the bound
protein is Sp1. However, it is still possible that in the intact cell,
the Sp1135 site, in the context of the entire proximal
IIb promoter region, binds Sp3. Perhaps the silencer
elements proposed by others just upstream or downstream of this site
(14, 15), interact with the Sp1135 site to allow it to bind
specifically to Sp3 and inhibit transcription.
Several recent reports (49-51) also suggest that a negative regulatory
protein, whose DNA binding site overlaps a Sp1 site, may competitively
interfere with Sp1 binding and inhibit transcription. Since our
mobility gel shift studies and Southwestern analysis only detected one
single protein complex at the Sp1135 site in all cell lines
tested, it is less likely that multiple proteins interact with this
site. Activation by Sp1 can also be repressed by the formation of
inactive (non-DNA-binding) complexes between Sp1 and other nuclear
proteins such as Sp1-I and p107 (52, 53). However, interference with
Sp1 activation, an established mechanism by which gene transcription
can be altered, obviously cannot explain the activity of
Sp1135 site in IIb promoter, because the
silencing function of this site correlates directly rather than
inversely with its binding of Sp1-related proteins.
Therefore, our study raises the possibility that Sp1 may itself decrease transcription when bound to certain Sp1 elements. Several recent reports have suggested a similar negative regulatory role for bound Sp1 (53-55). How this occurs is unclear, but one mechanism may involve its interactions with other nuclear elements. Sp1 has been shown previously to interact with other nuclear factors such as GATA-1 and Ets proteins (57-60). Sp1 is also described to be a tethering factor to recruit the transcription initiation complex to TATA-less promoters by physically interacting with components of general transcriptional machinery (61, 62).
Interestingly, we have shown previously that the Sp114 site
is a positive regulator of IIb expression (13). The
complex at this site appears to interact with the complex bound to the Ets35 site. It is suggested that the Sp114
complex promotes transcription by tethering the transcriptional
initiation complex to this TATA-less promoter. Our present finding is
that another Sp1-binding site is a negative regulator of
IIb expression. Therefore, depending on which site Sp1
is bound to, this nuclear factor appears to have both a positive and
negative role in the regulation of
IIb expression. A
similar dual effect of Sp1 has been described in the proximal promoter
of human adenine nucleotide translocase 2 (ANT2) gene (56). For the
TATA-containing ANT2 gene, the more proximal Sp1 site (from
7 bp to
2 bp) partially inhibits transcription, probably by disrupting the
recruitment and assembly of transcriptional initiation complex. A more
distal site, containing two adjacent Sp1-binding sites at
87 bp to
58 bp, activates expression.
Given the fact that the effect of Sp1 on transcriptional activity is
context-dependent, it is not surprising that only specific Gal4/Sp1 fusion protein shown in Fig. 8 inhibit expression, and that
other fusion constructs actually increase expression. The context
dependence of Sp1 binding in this region is also consistent with what
was seen following ectopic reinsertion of the 150 to
101 bp region,
which did not return silencer activity, but rather resulted in
increased expression. These finding support the idea that the complex
bound to the Sp1135 site interacts with other bound nuclear
factors in the
IIb promoter region. Physical disruption of the structural organization between Sp1 and these other regulators, therefore, may alter the regulatory activity of Sp1135 in
IIb transcription.
Silencer elements have been proposed in the regulated expression of
several other megakaryocyte-specific genes. A silencer element was
identified for the rat platelet factor 4 (PF4) promoter (23). However,
whether this region actually contains a silencer element was questioned
in studies of the human PF4 promoter, which suggest that the homologous
region is actually a strong promoter of PF4 expression (63). The
2 integrin gene is also TATA-less, and its regulated
expression in megakaryocytic cell lines has also been suggested to
involve a silencer region (64). In addition to a core promoter (
92 bp
to the transcriptional start site) that is active both in
megakaryocytic and non-megakaryocytic cells, a silencer element (
92
to
351 bp) region was defined that showed tissue specificity. It is
inactive in non-hematopoietic cells but active in megakaryocytic cells.
Located further upstream is a strong megakaryocyte-specific enhancer
that overcomes the silencer effect and restores megakaryocyte-specific
expression of the
2 gene. The tissue specificity of the
2 gene silencer contrasts with the Sp1135
site, which appears to be an active silencer of
IIb
expression in all cell lines tested. However, the two genes share a
common mechanism in that the silencer element is overcome by a distal
megakaryocyte-specific enhancer, leading to tissue-specific expression.
Two studies of the human IIb gene have defined a
silencer domain (14, 15). The exact site(s) of the involved silencer element varied between the two studies. EMSA studies by Fang and Santoro (14) suggested that there may be two sites involved in the
silencer effect seen in the human
IIb promoter,
198 to
178 bp and
124 to
99 bp. Their studies focused on K562 cells and
suggested that PMA induction markedly increased
IIb
expression and led to a inverse decrease in EMSA complex formation at
both sites. Preliminary data that was not shown by the authors
suggested that the protein that bound to the silencer element had a
molecular mass of ~30 kDa. Prandini et al. (15) also
detected a silencer element that was in part consistent with the above
studies, suggesting that there were two silencer elements at
120 to
116 bp and at
102 to
93 bp, whose mutation increased promoter
activity of the human promoter region 4- and 8-fold, respectively, and
20-fold in combination. No EMSA studies were done, but DNase I
footprinting showed a protected region between
120 bp and
116 bp.
Thus, these two studies suggest a common silencer site centered at
human
120 to
116 bp with the sequence 5'-ATGAG-3' (corresponding to
rat
113 to
109 bp) (Fig. 2A).
Our studies suggest another site as being critical for the observed
silencing, ~30 bp upstream from the proposed human silencer site.
Prandini et al. (15) suggested that the sequence of
5'-ATGAG-3', which is found in the 5'-flanking region of several
platelet-specific promoters is a common silencer element for all of
these genes. However, this region is not conserved in the rat and mouse
IIb promoter regions, being 5'-GTGTG-3' in the rat and
5'-G-ACG-3' in the mouse, thereby having 2/5 and 4/5 mismatches (Fig.
2A). Indeed, the rat homologue (probe
114 to
88 in Fig.
2A) of the human
IIb silencer region did not
form a complex in EMSA studies with HeLa cell nuclear extract. We
focused our analysis on the
145 to
125 bp region that did form a
complex. It turned out that this region has a well localized site that
binds Sp1 in both the rat and human sequences, and that the human
homologue of this region is protected on the DNase I footprinting
studies by Prandini et al. (15). It may be that silencing of
the
IIb gene is more complex than any of these studies
suggest. Interactions between a number of sites may be necessary to
achieve
IIb gene silencing. Perturbations of any of
these sites may then have the same effect of relieving the silencer
effect.
In summary, we present two important new findings: 1) that there is an
important Sp1135 silencer domain involved in the regulation of IIb gene expression, and 2) that despite the presence
of the Sp1135 silencer domain in megakaryocytes, sequences
further 5' to the
453 bp can overcome this silencer effect in the
developing megakaryocytes. Our previous studies suggest that this
upstream megakaryocyte-specific element involves the
GATA454 site. Thus, we present in Fig.
9 a simplified model of how the
IIb gene is regulated in agreement with our data. In
non-megakaryocytic tissues, a silencer complex forms around the
Sp1135 site. This complex interacts with the proximal
promoter/initiation complex of this TATA-less gene to prevent
IIb expression. In megakaryocytes, this silencer complex
still interacts and inhibits expression, but a second more distal
complex involving the GATA454 site forms, and this complex
can overcome the silencer effect. The fact that the silencer domain
still interacts with the proximal promoter/initiation complex is based
on the consistent doubling in expression seen in transient expression
studies of both primary rat marrow cells (11) and megakaryocyte-like
cell lines. Since GATA proteins are not uniquely expressed in
megakaryocytes, the critical GATA454 element is not likely
to be solely responsible for the megakaryocyte-specific nature of the
upstream enhancer complex. This complex may, in addition, involve the
Ets element around
512 bp. It has been proposed by others (15, 65)
that, while GATA and Ets proteins are not individually
megakaryocyte-specific, they may act in concert to confer tissue
specificity. In fact, physically paired GATA- and Ets-binding sites
have been found in most megakaryocyte-specific gene promoters so far
characterized (11, 65-67).
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One caveat with these studies on the Sp1135 silencer domain
and with studies by us and others on other regulatory elements 5' to
the IIb gene is that they are all based on analysis of relatively short stretches of the 5'-flanking region of this gene. Whether or not a ubiquitous silencer element will be demonstrated in
studies with the intact gene and whether the upstream enhancer elements
maintains their role as a dominant, tissue-specific regulator remain to
be tested. It is possible that a targeted mutation of the
Sp1135 site in transgenic mice may have a number of
different outcomes, varying from having no effect on
IIb
expression to having an effect only in megakaryocytes to having a wider
affect of expression on other hematological or non-hematological
lineages.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL-40387, a grant from the Schulman Foundation, and Grant 3152 from the Council for Tobacco Research-USA, Inc.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: The Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-3896; Fax: 215-590-4834; E-mail: poncz{at}email.chop.edu.
1 The abbreviations used are: bp, base pair(s); EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; PMA, phorbol 12-myristate 13-acetate; PCR, polymerase chain reaction; CMV, cytomegalovirus.
2 TESS software is available via the World Wide Web (http://www.cbil.upenn.edu//cbil-home/index.html).
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