(Received for publication, September 25, 1996, and in revised form, December 4, 1996)
From the Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Health Science Center, Houston, Texas 77225
Prostaglandin H synthase-1 (PGHS-1) is a
constitutively expressed key enzyme in the biosynthesis of
physiologically important prostanoids. The promoter of the human PGHS-1
gene lacks a TATA box, has a very GC-rich region, and contains multiple
transcription start sites. To identify the elements involved in the
constitutive expression of the PGHS-1 gene, we constructed a 2075-base
pair fragment (2095 to
21 relative to the translation start codon) and a series of 5
-deletion mutants into a promoterless luciferase expression vector, which was transfected in HUVEC. Two important regions were identified. DNase I footprinting identified a protected segment, which contains an Sp1 binding site proximal to the
transcription start sites. Band shift assays confirmed specific binding
of Sp1 to this segment. Band shift assays further revealed specific
binding of Sp1 to a distal region containing a canonical Sp1 site.
Mutation of either Sp1 binding site significantly reduced the promoter activity. When both sites were mutated, the activity was reduced to
29% of that of the wild type. Mutation of Sp1 sites did not abrogate
promoter activity stimulated by phorbol ester. These results indicate
that binding of Sp1 or its related proteins to two widely separated Sp1
sites on the promoter region activates the basal PGHS-1 gene
transcription.
Prostaglandin H synthase (PGHS,1 EC
1.14.99.1) is a bifunctional enzyme containing a cyclooxygenase
activity that catalyzes the bisoxygenation of arachidonic acid to form
the peroxide prostaglandin G2 (PGG2) and a
peroxidase activity that catalyzes the reduction of PGG2 to
PGH2 (for a review, see Ref. 1). PGH2 is the
common precursor of biologically active prostaglandins, thromboxane and prostacyclin. PGHS, hence, occupies a pivotal position in prostanoid biosynthesis. There are two isoforms of PGHS. The type 1 PGHS (PGHS-1)
is constitutively expressed in most mammalian cells, whereas the
expression of PGHS-2 is induced by cytokines and mitogenic factors (2).
Both isoforms of PGHS are important in catalyzing prostacyclin
synthesis in endothelial cells. PGHS-1 catalyzes the synthesis of basal
levels of prostacyclin, while prostacyclin productions by inflammatory
agents correlate with the induced expression of the PGHS-2 enzyme (3,
4). We have been interested in understanding the transcriptional
regulation of the constitutive PGHS-1 gene in endothelial cells. The
human PGHS-1 gene spans about 22 kb on chromosome 9 containing 11 exons
(5, 6). The coding region encodes a 599-amino acid protein including a 23-amino acid signal peptide (5). The 5-flanking region of the human
PGHS-1 gene has multiple transcription start sites (TSS), does not
possess the canonical TATA or CAAT box, and is GC-rich. These features
are consistent with those of a housekeeping gene. The 5
-flanking
region of PGHS-1 bears several putative binding sites for
transcriptional activators (6). However, the promoter activity of the
5
-flanking region has not been delineated. Furthermore, the mechanism
by which the basal promoter function is activated has not been
elucidated. By fusing the 5
-flanking region to luciferase cDNA, we
have recently demonstrated promoter activity in the 5
-flanking region
of the PGHS-1 gene, but the promoter activity was very weak (6). We
postulate that other cis-acting elements are important in
the basal promoter activation of the PGHS-1 gene. We report here that
binding of Sp1 and/or its closely related proteins to two Sp1 binding
sites are critical for activating the basal transcription of the PGHS-1
gene.
Reagents for cell cultures were obtained from
Sigma. Lipofectin, Optimen I, and kits for the
-galactosidase assay were obtained from Life Technologies, Inc.
Restriction enzymes and Klenow polymerase were obtained from New
England Biolabs. pSV-
-gal plasmids, DNase I, Taq
polymerase, purified Sp1, and kits for the luciferase assay and DNase I
protection assay were obtained from Promega. Radiolabeled nucleotides
were obtained from Amersham Corp. Consensus oligonucleotides containing
binding sites for Sp1 (ATTCGATCGGGGCGGGGCGAGC), AP-2 (GATCGAACTGACCGCCCGCGGCCCGT), and NF-
B (AGTTGAGGGGACTTTCCCAGGC) were
obtained from Promega.
A
bacteriophage EMBL-3 human genomic library constructed from placental
DNA (Clontech) was screened with a 32P-labeled 0.7-kb
fragment at the 5-flanking region of human PGHS-1 genomic DNA, the
sequence of which had been previously reported (6). One positive clone
containing a 16-kb insert was isolated from 3 × 106
plaques. The positive clone was plaque-purified and mapped. This clone
included the first eight exons and a 2.5-kb 5
-flanking region. A
3.5-kb XhoI/EcoRI fragment of this clone
containing the 5
-flanking region, the first two exons, and part of the
second intron was subcloned into pGEM7-Zf (Promega) for sequencing.
Nucleotide sequences were determined by the chain termination method
using specific primers of PGHS-1 genomic DNA.
5-Deletion constructs of the PGHS-1 promoter were
generated by polymerase chain reaction (PCR), using the 3.5-kb
XhoI/EcoRI fragment (described above) as the
template. All of the upstream PCR amplification primers contained
either a BamHI or an XhoI restriction recognition
site, and the downstream PCR primers were linked to a
HindIII site. Primers were synthesized according to the
sequence relative to the ATG codon (A as +1): the upstream primers,
2095 to
2071,
1261 to
1237,
916 to
899,
744 to
728,
565 to
547,
257 to
240, and
137 to
117; the downstream primers,
41 to
21;
143 to
126; and
761 to
744. The PCR
products were purified from agarose gel, digested, and cloned into a
promoterless luciferase expression vector, pXP1 (7). The constructs
were designated according to their positions relative to the ATG codon as shown in Fig. 2. To generate the constructs of
916/
21, where the
two Sp1 sites were changed, a PCR-mediated site-directed mutagenesis was employed. The primers used for mutations were as follows (from 5
to 3
, with mutated bases underlined): primer A,
GGGCTGGCTC
G
CCTGAAGCCA; primer A
,
TGGCTTCAGG
C
GAGCCAGCCC; primer B,
GGAGGAGCGG
T
GAGCCCGGGGG; primer B
,
CCCCCGGGCTC
A
CCGCTCCTCC. The mutant
610/
604 (see Fig. 5, construct b) was obtained by first
amplifying the template
916/
21 with the primers A and
41/
21 to
generate a 0.6-kb fragment. In a separate tube, a 0.3-kb fragment was
generated by 30 cycles of PCR using primers A
and
916/
899. Both
0.6- and 0.3-kb fragments were gel-purified and combined for one cycle
of PCR. Subsequently, primers
916/
899 and
41/
21 were added for
another 30 cycles of PCR. The amplified fragment was digested and
subcloned into pXP1 vector. Each PCR cycle was 94 °C for 1 min,
52 °C for 1 min, and 72 °C for 1 min. The mutant
111/
105 (see
Fig. 5, construct c) was prepared by identical methods using
primers B and B
. The double mutant (construct d) was
obtained using the mutant
610/
604 as the template and primers B and
B
as mutagenized primers. The mutants were confirmed by nucleotide
sequencing.
Cell Culture, Transient Transfection, and Luciferase Assays
Human umbilical vein endothelial cells (HUVECs) were
cultured as described previously (8). To ensure consistent results, passage 1 cultured cells were used throughout the studies unless otherwise indicated. One day before transfection, cells were seeded at
30-40% confluence in a six-well dish. Liposome-mediated transient transfection was performed as described (9). Briefly, HUVECs were
transfected with a Lipofectin/DNA mixture containing 12 µg of
Lipofectin (Life Technologies, Inc.) and 2 µg of the promoter constructs with or without 0.3 µg of pSV--gal (Promega) in 1.2 ml
of Optimen I for 4 h. Lipofectin and DNA plasmids were
subsequently removed and replaced with complete medium. Cells were
harvested and lysed with 200 µl of reporter lysis buffer (Promega).
Cell extracts were centrifuged in a microcentrifuge for 5 s to
remove debris. 50 µl of the supernatant was removed for luciferase
assay in a luminometer (Monolight, model 2010) according to the
manufacturer's procedures. The protein content was determined by the
BCA protein assay kit (Pierce) using bovine serum albumin as a
standard.
-Galactosidase activity was assayed by chemiluminescence
(Clontech) as described (10).
HUVEC nuclear extracts were
prepared as described previously (11) with the following modifications.
HUVECs were harvested by scraping, washed in cold phosphate-buffered
saline, and incubated in two packed cell volumes of buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 300 mM sucrose, 0.5% Nonidet P-40, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, 1 µg/ml each leupeptin and aprotinin) for 10 min on ice.
The crude nuclei released by lysis were collected by
microcentrifugation (9500 rpm, 20 s), rinsed once in buffer A, and
resuspended in 2/3 packed cell volume of buffer B (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1.0 mM
dithiothreitol, and 1.0 µg/ml each leupeptin and aprotinin). Nuclei
were disrupted by passing through a 23-gauge syringe 10 times. The
homogenate was gently stirred on ice for 30 min, and the debris was
removed by microcentrifugation for 2 min. The resulting supernatant was
diluted 1:1 with buffer C (20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml each leupeptin and aprotinin). Nuclear extracts
were frozen on dry ice and stored at 80 °C. The final protein
concentration of the nuclear extracts ranged from 5 to 8 mg/ml.
DNase I footprinting was performed
according to a method previously described (11). Labeled probe (476
to
21) was prepared by PstI/HindIII digestion
of construct pXP1/(
744 to
21), and the digested probe was isolated
and labeled with [
-32P]dATP by Klenow DNA polymerase.
The labeled probe (2.5 × 104 cpm) was incubated with
either purified Sp1 (Promega) or HUVEC nuclear extracts (30 µg) at
room temperature for 15 min in 50 µl of binding buffer containing 50 µg/ml bovine serum albumin, 10 µg/ml poly(dI:dC), and 0.03%
Nonidet P-40 to allow binding and then digested with 0.15 µg/ml DNase
I at room temperature for 1 min. The samples were analyzed on a
sequencing gel.
The shift assay was performed by a previously described procedure (12). The binding mixture (20 µl) contained 5 × 104 cpm of 32P-labeled Sp1 consensus oligonucleotide or DNA probe, 10 µg of HUVEC nuclear extracts, or 5 ng of purified Sp1 and 2.5 µg of poly(dI:dC) in a binding buffer (10 mM HEPES, pH 7.9, 50 mM KCl, 0.1 mM EDTA, 10% glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml each of leupeptin and aprotinin). After a 15-min incubation on ice, the samples were incubated at room temperature for 20 min. Band shift patterns were resolved by electrophoresis. In competition experiments, nuclear extracts or purified Sp1 were incubated for 5 min with the unlabeled oligonucleotide or DNA fragment in a 50-150-fold molar excess prior to the addition of the labeled probe. The gel supershift assay was performed by adding 2 µg of rabbit polyclonal anti-Sp1 antibody (PEP2, Santa Cruz Biotechnology) to the DNA/protein mixture for 30 min on ice, and the band formation was analyzed on gel electrophoresis as described above. An unrelated rabbit polyclonal anti-PGHS-1 antibody was included as negative control.
Primer Extension AnalysisPrimer extension using HUVEC
PGHS-1 mRNA as the template was performed by a procedure previously
described (6). Primer extension using the PGHS-1 promoter/luciferase
construct was done similarly. The primer used in the extension analysis
is shown in Fig. 6. The extension products were analyzed on a 6%
polyacrylamide sequencing gel.
Rapid Amplification of cDNA Ends (RACE)
The 5-end RACE
for determining the 5
-end of the PGHS-1 transcript was based on the
procedure previously described (13). 3 µg of cellular RNA prepared
from HUVECs was reverse transcribed to cDNA using an antisense
PGHS-1 oligonucleotide (+83/+107 relative to the translation start
site). The aliquoted cDNA products were used for amplification by
PCR using the following primers: primer a,
5
-GA
TCGACATCGATTTTTTTTTTTTTTTT-3
, which
contains an XhoI digestion site as underlined, and primer b,
5
-CC
CAGGACGGGGAGCGGC-3
, which also contains an
XhoI digestion site. This primer contains an antisense
sequence corresponding to PGHS-1 nucleotides +70 to +51. RACE reaction
products were digested with XhoI and cloned into the
XhoI sites of pGEM7. The transformed cells were screened with PGHS-1 genomic probe (
744/+163).
A genomic clone containing a 2.5-kb fragment of the
5-flanking region of the human PGHS-1 gene was isolated and sequenced. The sequence reveals that this region bears several putative binding sites for transcriptional activators (Fig. 1). The
adenine residue of the ATG is designated as +1. A GC box containing a
canonical Sp1 binding site (GGGGCGG) is located at nucleotides
610 to
604. Three shear stress-responsive elements (SSRE) are
localized near nucleotides
395,
625, and
1810, respectively. In
addition, two Sp1 sites are located at nucleotides
83 to
89 and
105 to
111 proximal to the ATG site. To characterize
cis-acting elements in the human PGHS-1 promoter region, we
constructed a 2075-bp fragment (
2095/
21) and a series of
5
-deletion mutants into a promoterless luciferase expression vector,
pXP1. These constructs were transiently transfected into the cultured
HUVECs by lipofection according to the methods described under
"Experimental Procedures." The parental construct containing
nucleotides from
2095 to
21 of the PGHS-1 gene conferred strong
constitutive luciferase expression. The promoter activity of this
parental construct was about 22% of that of the pSV2-luc construct,
which utilizes the SV40 early promoter and enhancer to drive luciferase
expression (Fig. 2). The promoter activity remained
unchanged by progressive 5
-deletion until reaching nucleotide
565,
where the activity of construct
565/
21 dropped to about 50% of the
parental construct activity (Fig. 2). There was a small gradual decline
in the promoter activity with further progressive deletion. However,
even a short fragment
137/
21 still conferred about 100-fold higher
activity than the promoterless vector, pXP1. To demonstrate that the
basal promoter activity resides between
137 and
21, we constructed
two 3
-deletion mutants,
916/
126 and
257/
126, into the
luciferase reporter gene and carried out transient transfection
experiments. As shown in Fig. 2, both constructs almost entirely lost
the basal promoter activity. To normalize the variation of transfection
efficiency among these constructs, we also performed co-transfection
experiments. The
-galactosidase reporter gene driven by the SV40
early promoter was co-transfected with each construct described above
into the HUVECs. The promoter activities of these constructs were
compared by determining the ratios of the luciferase activity to the
-galactosidase activity. The results of the co-transfection
experiments are consistent with those of the experiments with
luciferase alone (Fig. 2). These results strongly suggest that two
regions located between nucleotides
744 and
565 and between
137
and
21 are critical for PGHS-1 promoter activity.
Binding of HUVEC nuclear proteins to these two regions were
investigated. Fig. 3A shows band shift when
nuclear extracts of HUVECs were incubated with a 744/
569 probe
containing the distal activator region. Two bands were noted with this
probe. Since this region comprises putative binding sites for Sp1 and
AP-2, a 150-fold molar excess of consensus oligonucleotide containing Sp1 or AP-2 sequence was preincubated with nuclear extracts prior to
the addition of the probe. Both bands were specifically competed by Sp1
or unlabeled probes but not by AP-2 oligonucleotide. Furthermore, a
50-fold molar excess of
744/
569 in which the canonical Sp1 site,
610GGGCGG
604 was mutated to
G
C
G failed to inhibit the formation of
these two DNA-protein complexes (Fig. 3A, lane
5). These results indicate that Sp1 and/or closely related
proteins bind to the canonical Sp1 site. This is further confirmed by
the formation of two bands when labeled consensus Sp1 oligonucleotides
were incubated with HUVEC nuclear extracts (Fig. 3B). Both
bands were competed by unlabeled Sp1 oligonucleotides (lane
2) and
744/
569 fragment (lane 5) but not by AP-2 or
NF-
B oligonucleotides (lanes 3 and 4). In all
of the experiments, nuclear extracts formed two bands with the
canonical Sp1 site. Since both bands are specifically competed by Sp1
sequences but not by mutated Sp1 sequence, both bands are complexes
formed between Sp1 and/or Sp1-related protein and the Sp1 sequence. Our
results are in keeping with several reports of the formation of two
distinct complexes between nuclear extracts and Sp1 binding sites
(14-17).
Identification of the Proximal Sp1 Element
The proximal
activating region (nucleotides 137 to
21) is GC-rich and contains
at least two Sp1 sites (Fig. 1). The DNase I footprinting assay
revealed a protected area from nucleotide
114 to
98 when labeled
probes were incubated with Sp1 (Fig. 4A) or
HUVEC nuclear extracts (data not shown). This protected area bears an
Sp1 site (GGGGTGG) (Fig. 4A). When labeled probes (
137 to
21) containing the protected region were incubated with HUVEC nuclear
extracts, two bands were formed (Fig. 4B, lane
2), and both bands were competitively inhibited by unlabeled
probes, Sp1 consensus sequence, and/or an Sp1-containing
oligonucleotide (Fig. 4B, lanes 3, 5,
and 6, respectively). More importantly, both bands were not
competed by a 50-fold molar excess of the parental probe where only the
111GGGGTGG
105 site had been mutated (Fig.
4B, lane 4). The results clearly demonstrated the
binding of Sp1 and/or its related proteins to the
111/
105 site but
not to the
89/
83 site. A single band shift was noted when purified
Sp1 proteins were incubated with labeled probe containing the protected
area (Fig. 4C, lane 2), and this band was
competitively inhibited by Sp1 oligonucleotides (lane 5).
This band was supershifted with specific antibody directed against Sp1
(lane 3) but not with unrelated antibody such as anti-PGHS-1 antibody (lane 4). Incubation of HUVEC nuclear extracts with
this probe resulted in the formation of two complexes (Fig.
4C, lane 7). Both bands were competitively
inhibited by Sp1 oligonucleotides (Fig. 4C, lane 7 versus lane 8) and supershifted with anti-Sp1 antibodies
(lane 6). Hence, Sp1 and/or Sp1-related proteins bind to a
Sp1 site at
111 to
105.
Effects of Mutation of Sp1 Binding Sites on PGHS-1 Promoter Activity
To ascertain that these two separate Sp1 binding sites
are functionally important in enhancing the PGHS-1 basal promoter
activity, one or both Sp1 binding sites in the 5-flanking promoter
(
916/
21) of the PGHS-1 gene were altered by site-directed
mutagenesis. These mutants were constructed in pXP1 luciferase
expression vectors and transfected in HUVECs. Alteration of the distal
Sp1 (
610GGGGCGG
604 to GTTTCAG) reduced the
promoter activity to 53% of that of the wild-type promoter (Fig.
5, b versus a). Alteration of the proximal Sp1 binding site from
111GGGGTGG
105 to
GTTTTAG reduced the promoter activity to 38% of the wild-type promoter
(Fig. 5, c versus a). When both Sp1 binding sites were simultaneously mutated, the promoter activity was reduced to 29% of
that of the wild-type promoter. This reduction is statistically significantly larger (p < 0.05) than the reduction
caused by individual mutation of the proximal or distal site.
The extent of promoter activity reduction caused by the distal Sp1 site
mutation is comparable with that of the 5-deletion mutants in which
the distal Sp1 site was deleted (
565/
21 in Fig. 2 versus
Fig. 5b). To determine whether mutation of the Sp1 site in
the construct (
257/
21) would lead to a comparable reduction in the
promoter activity, we mutated the proximal Sp1 site located in the
257/
21 fragment and expressed it in HUVECs. The promoter activity
expressed by this was only 27% of that of the wild-type promoter
(
916/
21) (Fig. 5f). This value was essentially identical to that of the double Sp1 site mutations in
916/
21, confirming that
these two Sp1 binding sites contribute to 73% of the basal PGHS-1
promoter activity.
Experiments were then carried out to determine the impact of Sp1 site
mutations on PMA-stimulated promoter activity conferred by the
916/
21 region. PMA treatment (50 nM, 4-h incubation) increased the promoter activity of the wild-type
916/
21 by 1.8-fold (Fig. 5a). This result was comparable with that previously
reported (18). Mutations of either or both Sp1 sites were accompanied by a marked decrease in the basal promoter activity as described above.
However, the level of stimulation by PMA treatment was not
significantly altered by the mutations (Fig. 5, b-f). These results suggest that PMA stimulation of PGHS-1 promoter activity depends on additional activators.
Multiple TSS for PGHS-1 were identified by
primer extension and S1 nuclease mapping in our previous study (6). A
major TSS was identified as adenine 135, relative to the ATG
translation start codon. TSS were situated upstream from the proximal
Sp1 cognate site (
111/
105). However, primers used in those
experiments were corresponding to nucleotide sequence
22 to
48,
which might mask TSS downstream from the proximal Sp1 site. Additional
primer extension experiments were, therefore, carried out to reevaluate the TSS. A P2 primer corresponding to nucleotides +115 to +137 (Fig.
6A) was used as an antisense primer in
extension experiments. The results from one experiment are shown in
Fig. 7A. Multiple bands corresponding to
A
31, G
33, G
37, and
A
135 were noted on the primer extension gel. Of these
four TSS, A
135 is in accord with that detected by using
nucleotides
22/
48 as the primer. Results from two other experiments
revealed four bands corresponding to A
31,
G
33, G
37, G
111, or
A
135. Hence, TSS at A
31, G
33,
and G
37 are consistent in all three experiments.
A
135 and G
111 are, on the other hand,
alternative TSS. We observed a similar alternative extension between
these two TSS when nucleotides
22 to
48 were used as the primer. In
all three experiments, A
31 and G
33 had the
highest density and were considered to be major TSS. Transcription
start sites for the PGHS-1 promoter-luciferase construct were
determined using PLuc primer corresponding to nucleotides +44 to +70
downstream from the luciferase start codon (Fig. 6B). Multiple bands were detected (Fig. 7B). Four bands with
higher densities are mapped to G
37, A
43,
G
78, and G
111 of the PGHS-1 promoter
region. Several less dense bands are scattered between
A
43 and G
111 (Fig. 7B). Judging
by the density of the primer extension bands, the major TSS for native
PGHS-1 transcript resides in the region at nucleotides
A
31 and G
33, whereas the major TSS for
luciferase fusion transcript resides in the region at
A
43. Another region that serves as TSS from both
transcripts resides at G
111. 5
-End RACE experiments were
carried out to further determine TSS. The 3
-primer used in the 5
-end
RACE experiments corresponds to the nucleotide sequence from +51 to
+70. Two TSS were identified by the RACE procedure, G
33
and A
17. Taken together, the results indicate that a
promoter region proximal to the Sp1 enhancer elements contains multiple
TSS including A
43, G
37, G
33,
A
31, and A
17. G
111 is a TSS
identified by primer extension of native and luciferase fusion
transcripts but is not detected as a TSS by 5
-end RACE. A
135 was identified as an alternative TSS to
G
111 only when primer extension was performed on native
PGHS-1 transcript. G
78 and several less dense bands were
noted only in luciferase fusion transcripts. The importance of
G
111, A
135, or G
78 as TSS for
the PGHS-1 gene in vivo is unclear. It is intriguing to note
that these nucleotides are localized upstream from the proximal Sp1
recognition site. A similar spatial relationship has been reported for
the promoter of the human cyclin-dependent kinase-2 gene
(19), in which among multiple TSS identified, one TSS was located
upstream from an Sp1 site functionally important in basal transcription
for this gene.
Results from this study demonstrate that two Sp1 elements are
essential for basal transcription of the human PGHS-1 gene. Evidence to
support this consists of specific binding of purified Sp1 and nuclear
extract proteins to these two regions and a marked loss of promoter
activity by Sp1 site mutations. Sp1 is a sequence-specific, ubiquitously expressed nuclear factor essential for basal expression of
a variety of eukaryotic genes (for review, see Ref. 20). It confers
transcriptional activation by interacting with transcription-associated factors, thereby facilitating the assembly of the basal transcription machinery (21). Reported data suggest that for activation of constitutive transcription of mammalian TATA-less gene, Sp1 is required
to bind to a spatially defined region, within 100 base pairs upstream
from the TSS (22-26). Hence, Sp1 involvement in basal transcription of
the PGHS-1 gene is different from that of other reported mammalian
housekeeping genes in that two Sp1 sites required for basal
transcription are separated by about 500 bp on the 5-flanking region.
To our knowledge, this is the first instance of critical involvement of
two distantly located Sp1 sites in the activation of basal mammalian
housekeeping gene transcription.
It has been reported that the activity of the thymidine kinase promoter of the herpes simplex virus and an artificially constructed promoter is enhanced by interaction of two spatially widely separated Sp1 proteins to form Sp1 multimers (27-29). Detailed analysis of the promoter region by electron microscopy revealed DNA looping whereby the separated Sp1 proteins were brought into contact to form a tetramer followed by multiple tetramer formation (30). It is conceivable that basal PGHS-1 transcription regulated by two Sp1 sites may be mediated by a similar mechanism.
It is estimated from the mutation experiments that these two Sp1
enhancer elements contribute to about 70-75% of the promoter activity
conferred by the 2.0-kb promoter/enhancer fragment of PGHS-1 gene. Full
basal promoter activity may require the involvement of additional
enhancer element(s). Comparison of the promoter activity conferred by
the construct shown in Fig. 5d versus Fig. 5f
suggests that the region between nucleotides 257 and
21 bears additional enhancer elements important in full PGHS-1 gene expression. In this region, besides the functionally active Sp1 site at
105 to
111, there is another Sp1 site located at
83 to
89. The 3
-mutant
fragment
257/
126 (Fig. 2) in which both Sp1 sites are removed
exhibited almost no promoter activity. This result implies that the Sp1
site at
83 to
89 may be important in PGHS-1 basal transcription
in vivo. However, this 3
-deletion mutant is probably devoid
of the binding site for the transcription initiation complex and
consequently is expected to confer minimal (if any) promoter activity,
even when enhancer elements are present distally such as the
916/
126 mutant (Fig. 2). Since Sp1 did not bind to the Sp1 site at
83 to
89 by DNase I footprinting, it would be highly unlikely that
this Sp1 site is functionally active. Other sites in this region
including a putative PEA3 binding site (
155AGGAAG
150) may be the potential
enhancer element for a full PGHS-1 promoter activity. This is now being
investigated.
It has recently been reported in different types of cells that PGHS-1
gene expression is stimulated by serum, cytokines, or growth factors
(for a review, see Ref. 2). We have shown that endothelial PGHS-1
expression is stimulated approximately 2-fold over the basal level by
PMA and interleukin-1 (18). In this study, our results indicate that
PMA increased the promoter activity conferred by the 5
-flanking
promoter/enhancer of the PGHS-1 gene in HUVECs. Stimulation of the
promoter activity by PMA is not entirely dependent on the two Sp1 sites
but requires additional elements. Sp1 is involved in stimulation of
gene expression by interacting with other transcriptional activators
such as GATA, NF-
B, Egr-1, YY1, and Rb (31-37). It is possible that
PMA stimulates PGHS-1 transcription by a similar mechanism. Further
studies are needed to elucidate the mechanism by which PMA stimulates
PGHS-1 promoter activity.
Nuclear extracts of HUVECs form two distinct bands with distal or
proximal probes and with consensus Sp1 recognition sequences. Both
bands are specifically competed by unlabeled Sp1 cognate oligonucleotides but not by Sp1 mutant, AP-2, or NF-B sequences. These two bands are complexes of DNA with Sp1 and/or Sp1-related proteins. Purified Sp1, on the other hand, forms only a single DNA·Sp1 complex with fragment
119 to
94 (Fig. 4C).
These results are similar to those reported in several recent studies
(13-16). The reason for the double band formation with nuclear
extracts is unclear. It has been attributed to binding of Sp1 and a
closely related protein to Sp1 recognition sites (38). Three
Sp1-related proteins, Sp2, Sp3, and Sp4, have been identified (39-42).
These isoforms of Sp1 bind to Sp1 recognition sites and are
antigenically very close to Sp1. Hence, despite specific inhibition of
binding by Sp1 cognate sequences and supershift by specific Sp1
antibodies, the additional band is probably formed as a result of
binding of a Sp1-related protein to the Sp1 binding site.
We thank San Li for excellent technical assistance.