From the
To understand the molecular mechanisms that direct the
expression of the gene encoding the platelet-activating factor (PAF)
receptor, the 5`-flanking region of the human PAF receptor gene was
cloned, and its promoter activity in myeloid cell lines was
characterized. By the 5`-rapid amplification of cDNA ends method and
primer extension, the transcription initiation site was mapped to an
adenosine residue 137 bases upstream of the ATG translation initiation
codon. The promoter region lacks a typical TATA or CCAAT box. However,
the sequence encompassing the transcription initiation site shows high
homology to the initiator (Inr) sequence. Transfection of promonocytic
U937 cells with recombinant plasmids containing a series of truncated
segments of the 5`-flanking region linked to the luciferase reporter
gene revealed that the sequence from nucleotides -44 to +27
relative to the transcription initiation site was sufficient to promote
a high level of gene expression. The promoter activity was much lower
in nonexpressing HeLa cells and promyelocytic HL-60 cells, which
express relatively low levels of the PAF receptor. Gel mobility shift
analysis demonstrated the binding of nuclear factors extracted from
myelocytic cells to the -16/+18 sequence containing the Inr
element. No binding activity was detected using the nuclear extracts
from the nonmyelocytic HeLa cells. The DNA-protein complexes were
sequence-specific since the binding was not significantly affected by
the mutated Inr sequences or the Inr sequence of the terminal
deoxynucleotidyltransferase gene. Furthermore, point mutations in the
Inr element significantly reduced promoter activity in both U937 and
THP-1 cell lines. When Me
Platelet-activating factor (PAF)
In this study, the promoter region of the human PAF receptor
gene encoding transcript I was cloned and characterized. As determined
by both 5`-RACE and primer extension methods, the transcription
initiation site was mapped to an adenosine residue located 137 bp
upstream of the ATG translation codon. The same initiation site was
also observed previously by Mutoh et al.(11) . However,
in their study, two additional initiation sites located 192 and 264 bp
upstream of the present initiation site were identified
(11) .
The reason for the discrepancy is not clear. Nevertheless, the
5`-flanking sequence of the PAF receptor gene contains no TATA or CCAAT
box. Like many TATA-less genes, the sequence at the transcription
initiation site resembles the Inr consensus sequence,
YYA
The functional significance of the Inr
element was clearly demonstrated by the fact that mutagenesis of this
element reduced the promoter activity by 50-60% in cultured
monocytic cell lines and abolished its ability to compete against the
Inr binding activity in gel mobility shift assay. Previous studies on
the Inr element of other genes have shown that Inr requires the
cooperation of the Sp1 site nearby to direct optimal
transcription
(13, 14, 22, 23, 24) .
However, in the case of the PAF receptor gene, the Sp1 site is located
quite far away (350 bp) from Inr and apparently is not required for the
promoter activity. Nevertheless, the present observation that mutation
of the Inr site did not completely inhibit the promoter activity raises
the possibility that other adjacent regulatory elements, although not
yet identified, act in concert with Inr to direct optimal
transcription.
A number of transcriptional factors, including USF,
YY1, TFII-1, and HIP1, have been shown to interact functionally with
unique Inr sequences around the transcription initiation sites of
specific genes and to impart transcriptional
activity
(16, 22, 25, 26, 27) .
Purification of these proteins from HeLa cell nuclear extracts revealed
that the molecular masses of USF, YY1, TFII-1, and HIP1 are 43, 68,
120, and 180 kDa, respectively. To further identify the proteins
binding to the Inr element of the PAF receptor gene, UV cross-linking
experiments were performed. SDS-PAGE analysis revealed that the
apparent molecular masses of the Inr-binding protein units in NP-1 and
NP-D complexes were
In summary, this study
clearly demonstrates that the sequence spanning nucleotides -44
to +27 of the PAF receptor gene is capable of directing the
transcription of the receptor in myeloid cells. The unique Inr element
overlapping with the transcription initiation site appears to play an
essential role in defining cell-specific expression of the PAF
receptor, although it is not involved in the differentiation-regulated
expression of the receptor gene. Disclosure of the molecular nature of
these nuclear proteins bound to the Inr sequence might provide insight
into the molecular mechanism of how the transcription of the PAF
receptor gene is initiated.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. Young-Sun Lin for critical reading and
Dr. Cathy Fletcher for editing of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
SO or retinoic acid was used to
induce granulocytic differentiation of HL-60 cells, a distinct
Inr-protein complex was induced concurrently, but the complex was not
observed in 12-O-tetradecanoylphorbol-13-acetate-induced
monocytic differentiated HL-60 cells or Me
SO-induced
differentiated U937 cells, indicating that the inducible Inr binding
activity is granulocyte-specific. These results suggest that distinct
nuclear factors interact with the unique Inr element and play a role in
the transcriptional regulation of the PAF receptor in various myeloid
cells.
(
)
is a
potent inflammatory lipid mediator with a broad spectrum of biological
activities
(1, 2, 3) . In addition to its
originally described activity of inducing platelet aggregation, PAF is
capable of inducing chemotaxis, aggregation, and degranulation of
neutrophils, eosinophils, and
monocytes
(1, 2, 3) . Specific receptors are
present on target cells to mediate the biological actions of PAF. A
cDNA for the human PAF receptor (transcript I) has been cloned from
human neutrophils and promonocytic U937
cells
(4, 5, 6) . The deduced amino acid sequence
revealed that it is a G-protein-coupled receptor with a
seven-transmembrane
-helix structure. Although the PAF receptor is
not restricted to myelocytic cells, the expression of the receptor on
promyelocytic HL-60 cells has been shown to be developmentally
regulated
(7, 8, 9) . Significant increases in
PAF receptor gene expression and function were observed in HL-60 cells
following Me
SO-induced granulocytic or vitamin
D
-induced monocytic differentiation. Recently, a PAF
receptor cDNA with a different 5`-noncoding sequence (transcript II)
was isolated from human heart
(10) , indicating that at least two
distinct promoters are involved in the transcriptional regulation of
PAF receptor gene expression in various tissues and cells
(11) .
Nevertheless, it has been shown that only transcript I of the PAF
receptor is present in peripheral leukocytes
(11) . In an attempt
to understand the fundamental mechanisms that direct gene expression of
the PAF receptor in myeloid cells, we isolated the 5`-flanking region
of the transcript I human PAF receptor gene for further
characterization. In this study, we constructed recombinant plasmids
containing the 5`-flanking DNA linked to the reporter luciferase gene.
Analysis of the promoter activity of the recombinant constructs in
transiently transfected myeloid cell lines allowed us to identify the
regulatory sequences that are essential for the promoter activity.
Interestingly, the DNA sequence from nucleotides -44 to +27
was sufficient for a high level of reporter gene expression. The
binding of nuclear factors from myeloid cells to the possible
regulatory sequence within this region was further characterized by gel
mobility shift assay. We show that an Inr
element
(12, 13, 14, 15, 16) located at the transcription initiation site of the PAF
receptor gene interacts with distinct nuclear proteins from myelocytic
cells of different lineages and is involved in the transcriptional
regulation of gene expression in these cells.
Materials
Cell culture reagents, yeast RNA, and
restriction enzymes were purchased from Life Technologies, Inc.
AmpliTaq DNA polymerase was from Perkin-Elmer.
[-
P]dCTP and
[
-
P]dGTP were obtained from Amersham Corp.
Synthetic oligonucleotides were from Cruachem.
Cell Culture
Human U937, THP-1, and HL-60 cells
were subcultured in RPMI 1640 medium supplemented with 10% fetal calf
serum. HeLa cells were subcultured in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum. After
incubation in a humidified atmosphere of 95% air, 5% CO at
37 °C for 2 days, cells were harvested for experiments. In some
experiments, HL-60 cells were cultured in the presence of 1.3%
Me
SO for 5 days to induce granulocytic differentiation
prior to harvest.
5`-Rapid Amplification of cDNA Ends
(5`-RACE)
5`-RACE of the PAF receptor transcript was performed
using a CLONTECH 5`-AmpliFINDER RACE kit. Briefly, first strand cDNA
was synthesized using 2 µg of poly(A) RNA isolated
from U937 cells, 10 pmol of the PAF receptor gene-specific primer
(P
) that is complementary to nucleotides +121 to
+144 relative to the ATG translation initiation codon, and 10
units of avian myeloblastosis virus reverse transcriptase (Promega).
After incubation at 52 °C for 30 min, excess primer was removed by
a Chroma Spin +TE-100 column (CLONTECH), and the RNA was
hydrolyzed with 0.3 N NaOH. A single strand oligonucleotide
anchor (5`-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG-3`) was ligated to
the 3`-end of the first strand cDNA with T4 RNA ligase. After
incubation at room temperature for 20 h, the cDNA was subjected to
polymerase chain reaction (PCR) using a nested specific PAF receptor
primer (P
) (complementary to nucleotides -21 to
-1) and anchor primer (5`-CCTCTGAAGGTTCCAGAATCGATAG-3`). PCR
amplification was carried out by 35 cycles of denaturation at 94 °C
for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C
for 1 min. The PCR product was electrophoresed on a 1.2% agarose gel
and purified from the gel with a gel extraction kit (QIAGEN Inc.).
Primer Extension
A single oligonucleotide
(P), 5`-TCAGCTGCAGTGACCGTGTGTC-3`, which is complementary
to nucleotides -80 to -58 relative to the ATG translation
initiation site, was end-labeled with
P using T4
polynucleotide kinase (Life Technologies, Inc.) and purified on an 8
M urea, 20% polyacrylamide sequencing gel. Two µg of
poly(A
) RNA from U937 cells was incubated with 100,000
cpm of
P-labeled P
at 68 °C for 10 min and
then placed at room temperature for 5 min. Primer extension was carried
out with 5 units of avian myeloblastosis virus reverse transcriptase at
42 °C for 1 h. The products were analyzed on a 6 M urea,
6% polyacrylamide sequencing gel.
Isolation of Genomic Clones
Approximately 2
10
bacteriophage plaques from a human leukocyte
genomic DNA library in EMBL3 SP6/T7 (CLONTECH) were screened with
P-labeled cDNAs. The product of 5`-RACE was used as probe
and labeled with [
-
P]dCTP by random
priming. Hybridization was performed at 42 °C for 20 h in 50%
formamide, 2
SSC, 10% dextran sulfate, and 1% SDS. The filters
were washed twice with 2
SSC containing 1% SDS at room
temperature for 30 min and twice with 0.2
SSC containing 1% SDS
at 37 °C for 30 min. Positive plaques were detected by
autoradiography using Kodak X-Omat AR films and intensifying screens.
DNA Sequencing
Dideoxynucleotide sequencing was
performed on an Applied Biosystems 373A automated DNA sequencer by
dyedeoxy terminator cycle sequencing methods according to the
manufacturer's instructions. Sequence analysis was performed
using the PC/Gene software program (IntelliGenetics, Inc./Betagen,
Mountain View, CA).
PAF Receptor Gene/Luciferase Fusion Plasmid
Construction
The various lengths of the DNA fragments from the
PAF receptor gene were obtained by PCR using the 5`-flanking region of
the PAF receptor gene. A HindIII restriction site was created
in both sense and antisense primers used in the PCR. The sequences of
the sense oligonucleotides used for generating the various constructs
are summarized as follows: p(-1099/+27)Luc,
5`-TCACCTGCAAGCTTCCATGAACTCATAC-3`; p(-610/+27)Luc,
5`-GCTCTTCCAAGCTTCTCTCTTCTCCCTC-3`; p(-246/+27)-Luc,
5`-TCACTCTCAAGCTTCCTTTTTCGCTC-3`; p(-122/+27)Luc,
5`-TCTTTGAGAAGCTTCTGTGCAGTGCC-3`; and p(-44/+27)Luc,
5`-TAGGGCCAAAGCTTTTCCTCCCAGGGGT-3`. The sequence of the antisense
oligonucleotide used for the above constructs was
5`-CCCAGCCTAAGCTTCTATGCTGTCT-3`. For constructing the
p(-1099/-176)Luc plasmid, the sequences of the sense and
antisense primers were 5`-TCACCTGCAAGCTTCCATGAACTCATAC-3` and
5`-GGCCAGAAGCTTAGGTGAGAAAG-3`, respectively. The PCR products were then
subjected to HindIII restriction enzyme digestion followed by
subcloning into the HindIII site of the pGL2-basic vector
(Promega). To construct the m(-44/+23)Luc plasmid, which
contains point mutations in the Inr site, PCR was performed with the
sense primer 5`-TAGGGCCAACGCGTTTCCTCCCA-3` and the antisense primer
5`-CAGCCTCGAGTGCTGTCTGGCAATTGCAGTAGTTAAGA-3`, which contains the
mutated nucleotides. The PCR product was digested with MluI
and XhoI restriction enzymes and subcloned into the
MluI-XhoI site of the pGL2-basic vector. The identity
and orientation of the subcloned DNA fragments were verified by DNA
sequencing. Plasmid DNA was purified with a QIAGEN plasmid purification
kit and used for transfection experiments.
Transient Expression
Transfection was performed as
described
(17) with some modification. Briefly, cells were
washed twice with RPMI 1640 medium and then resuspended in RPMI 1640
medium at a density of 4 10
/ml. Cells (1
10
) were preincubated with 10 µg of tested plasmid DNA
and 2 µg of pCMVB (CLONTECH) at room temperature for 5 min.
Transfections of U937, THP-1, and HL-60 cells were carried out by
electroporation with a Bio-Rad Gene Pulser at 300 V and 960
microfarads. Transfection of HeLa cells was conducted at 250 V and 960
microfarads. Cells were chilled on ice for 10 min and then transferred
to 5 ml of prewarmed culture medium. After 6 h in culture, cells were
harvested, and cell lysates were prepared. Both the luciferase and
-galactosidase activities were measured using assay kits from
Promega.
Preparation of Nuclear Extracts
Cells were
harvested by centrifugation at 1000 g for 5 min,
washed once with ice-cold phosphate-buffered saline containing 1
mM Na
VO
and 5 mM NaF, and
centrifuged again. Cell pellets were resuspended in ice-cold hypotonic
buffer composed of 20 mM HEPES, pH 7.9, 20 mM NaF, 1
mM Na
VO
, 1 mM
Na
P
O
, 1 mM EDTA, 1
mM EGTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin and
centrifuged in a microcentrifuge at full speed for 30 s. Hypotonic
buffer containing 0.2% Nonidet P-40 was then added directly to the cell
pellets to lyse the cells. After placing on ice for 5 min, nuclei were
pelleted by centrifugation in a microcentrifuge at full speed for 5 min
at 4 °C. Nuclei were resuspended in hypotonic buffer containing 420
mM NaCl and 20% glycerol (high salt buffer), and the
extraction of nuclear proteins was carried out on ice for 30 min.
Nuclear extracts were cleared by centrifugation at 300,000
g for 5 min at 4 °C. Excess salt was removed by dialysis
against buffer containing 20 mM HEPES, pH 7.9, 100 mM
KCl, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl
fluoride, 0.5 mM dithiothreitol, and 20% glycerol at 4 °C
for 5 h. Protein concentration was determined by the Bio-Rad protein
assay using bovine serum albumin as a standard.
Gel Mobility Shift Assay
PCR-75, which contained
the DNA fragment spanning nucleotides -44 to +27 of the PAF
receptor gene, was prepared by PCR using two primers,
5`-TAGGGCCAACGCGTTTCCTCCCA-3` and 5`-CCCAGCCTAAGCTTCTATGCTGTCT-3`. The
PCR product was digested with MluI and HindIII
restriction enzymes to create the recessed 3`-end. The other
oligonucleotides used as probe or competitor were chemically
synthesized. The annealing of two complementary oligonucleotides was
carried out in 10 mM Tris-HCl, pH 8.0, 100 mM NaCl,
and 1 mM EDTA at 80 °C for 1 h, and the mixture was then
slowly cooled down to room temperature. The duplex DNA was end-labeled
at room temperature for 30 min with
[-
P]dGTP in a reaction mixture containing
50 mM Tris-HCl, pH 7.8, 5 mM MgCl
, 0.1
mM dithiothreitol, 0.5 mg/ml nuclease-free bovine serum
albumin, and 1 unit of the Klenow fragment to a specific activity of
10
cpm/µg DNA. The
P-labeled DNA was
separated from the free nucleotides by Sephadex G-50 chromatography.
Nuclear extracts (5-10 µg) were incubated with 2 µg of
poly(dI-dC)
poly(dI-dC) in 20 µl of binding buffer containing
10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM
dithiothreitol, and 5% glycerol for 10 min at room temperature.
P-Labeled oligonucleotides (5000-10,000 cpm) were
then added to the reaction mixture, and incubation was continued for
another 20 min. The DNA-protein complexes were analyzed on a 6%
polyacrylamide gel at 170 V using 0.25
TBE (1
TBE
= 89 mM Tris borate, 89 mM boric acid, and 0.2
mM EDTA) as electrophoresis buffer. Gels were dried and
exposed to Kodak X-Omat AR films overnight at -70 °C.
UV Cross-linking of Inr-Protein Complexes
A gel
mobility shift experiment was performed as described above using 20
µg of nuclear proteins and P-labeled
bromodeoxyuridine-substituted Inr DNA probe. After electrophoresis, the
wet gel was UV-irradiated at a distance of 12 cm under a 254-nm UV lamp
at 4 °C for 20 min. The region containing the DNA-protein complex
was revealed by autoradiography and excised. The DNA-protein complex
was eluted from the gel piece by 250 µl of denaturing buffer
containing 50 mM Tris-HCl, pH 7.0, 4% SDS, 0.1%
-mercaptoethanol, and 10% glycerol at room temperature overnight.
The residue of the gel piece was removed by centrifugation, and the
DNA-protein complex was recovered by adding 15 µg of bovine serum
albumin and 1.25 ml of ice-cold acetone, followed by centrifugation in
a microcentrifuge at full speed for 5 min. The pellet was redissolved
in 10 µl of denaturing buffer and subjected to electrophoresis on a
12% SDS-polyacrylamide gel. In some experiments, the DNA-protein
complex was eluted by denaturing buffer without
-mercaptoethanol
and analyzed by SDS-PAGE under nonreducing conditions.
Identification of the Transcription Initiation Site of
the Human PAF Receptor Gene
As a first step to identify the PAF
receptor gene transcription start site, we used the 5`-RACE method to
obtain the 5`-end region of the PAF receptor cDNA. An antisense
oligonucleotide primer complementary to nucleotides +144 to
+121 relative to the ATG translation initiation codon of the PAF
receptor cDNA sequence was used to extend poly(A) RNA
from U937 cells. The 3`-end of the first strand cDNA was ligated with
an anchor primer. Amplification by PCR of the cDNA using a second
nested primer complementary to nucleotides -21 to -1 of the
PAF receptor cDNA sequence and an anchor primer resulted in a major
product of
160 bp (Fig. 1B). DNA sequencing of the
PCR product revealed that the 5`-end of the PAF receptor transcript is
an adenosine residue located 137 bp upstream of the ATG translation
initiation site. When primer extension was performed with
poly(A
) RNA from U937 cells, again only one major
extension product with the expected size was observed
(Fig. 1C), further confirming the location of the
transcription start site of the PAF receptor gene.
Figure 1:
Identification of the transcription
start site of the human PAF receptor gene. A, the nucleotide
sequence of the 5`-end of the PAF receptor cDNA (transcript I). The ATG
translation initiation codon is double-underlined. The primers
used for 5`-RACE (P and P
) and primer extension
(P
) are underlined. The Inr sequence is
boxed. The asterisk indicates the transcription
initiation site. B, the PCR product obtained from 5`-RACE.
C, the primer extension product.
Characterization of the Promoter Activity of the
5`-Flanking Region in Transient Transfections
The 5`-flanking
region of the PAF receptor gene was isolated through screening of a
human leukocyte genomic library using the PCR product from the 5`-RACE
as probe. Two clones containing the 99 bp of the 5`-end sequence of the
PAF receptor cDNA and the first intron sequence, which intervenes 38 bp
upstream of the ATG translation initiation codon
(11) , were
obtained. Sequence analysis revealed that the 5`-flanking region
upstream of the transcription start site of the PAF receptor gene
contains no typical TATA box. However, the sequence at the
transcription initiation site TCTTCACTTCTG is highly
homologous to the Inr consensus sequence, YYANT/AYY
(where Y is a pyrimidine nucleotide and N is a
nucleotide)
(12, 13, 14, 15, 16) .
To test the promoter activity of the 5`-flanking region of the PAF
receptor gene, a series of truncated segments of the 5`-flanking
sequence inserted before a promoterless luciferase gene in the
pGL2-basic plasmid were prepared. As shown in Fig. 2,
transfection of U937 cells with p(-1099/+27)Luc, which
contains
1 kilobase pair of sequence upstream of the transcription
initiation site of the PAF receptor gene, resulted in luciferase
activity
30-fold above the background level (promoterless
pGL2-basic). The promoter activity was only
20 times the
background level in cells transfected with the construct
p(-610/+27)Luc, but was at a level comparable to that for
p(-1099/+27)Luc with the shorter constructs. The shortest
construct, p(-44/+27)Luc, was sufficient to direct a high
level of reporter gene expression (28-fold above the background level).
On the other hand, the 3`-deletion construct,
p(-1099/-176)Luc, retained only
15% of the maximal
activity obtained with p(-1099/+27)Luc. This result suggests
that the cis-elements present from nucleotides -44 to
+27 are essential for controlling the promoter activity of the PAF
receptor gene in U937 cells. Transfection of promyelocytic HL-60 cells,
which express relatively low levels of PAF
receptor
(7, 8, 9) , revealed a similar profile,
but much lower promoter activity. When the transfection experiments
were conducted with the nonmyelocytic HeLa cells, which do not express
detectable levels of PAF receptor, the promoter activity gradually
decreased with increasing truncation of the promoter sequence. The
activity of p(-44/+27)Luc in HeLa cells was only
5-fold
above the background level, considerably less than that in U937 cells.
Figure 2:
Functional activity of the PAF receptor
promoter region. U937, HL-60, and HeLa cells were transfected with 10
µg of DNA in each construct by electroporation along with 2 µg
of a cytomegalovirus/-galactosidase construct as internal control
for transfection efficiency. Luciferase activity was measured and is
expressed as the activity relative to that of the pGL2-basic construct.
Data shown are the means ± S.D. of at least three independent
experiments with two DNA preparations. ND, not
determined.
Identification of Nuclear Proteins Interacting with the
Regulatory DNA Sequence
To assess the interactions of putative
nuclear factors with the regulatory DNA sequence, gel mobility shift
assays were performed. As shown in Fig. 3, nuclear proteins
extracted from U937 cells bound to the DNA sequence from nucleotides
-44 to +27 of the PAF receptor gene to form a DNA-protein
complex. This complex, designated NP-1, was completely blocked by
excess amounts of the unlabeled probe or the DNA fragment containing
sequence from nucleotides -16 to +18, but not by the DNA
fragment from nucleotides -46 to -28, indicating that the
DNA-binding site is located within the sequence from nucleotides
-16 to +18. To further examine whether the Inr element
located within the -16/+18 sequence is responsible for the
binding activity, a DNA sequence with two point mutations in the Inr
site was used in the competition experiment. As shown in Fig. 3,
mutated Inr did not effectively block NP-1 formation. Furthermore, the
Inr sequence of the terminal deoxynucleotidyltransferase gene
(12) was also ineffective, indicating that NP-1 is
sequence-specific. The binding of nuclear factors to the Inr probe was
also examined using nuclear extracts from peripheral monocytes isolated
from blood, THP-1 cells, and HL-60 cells (Fig. 4). In addition to
NP-1, two minor complexes with slower electrophoretic mobility were
observed in nuclear extracts from THP-1 and HL-60 cells. In some
experiments with HL-60 cell nuclear extracts, an additional larger
complex was detected (Fig. 5A). However, this complex is
nonspecific and not consistently observed. These Inr-protein complexes
appeared to be myelocytic cell-specific since no complex was detected
in nuclear extract prepared from HeLa cells. It has been suggested that
the Inr element is involved in the differentiation- or
development-regulated terminal deoxynucleotidyltransferase gene
expression in lymphocytes
(12) . Since the expression of the PAF
receptor has been shown to be up-regulated following
MeSO-induced granulocytic differentiation of HL-60 cells
(7), we examined whether the binding of the nuclear proteins from the
differentiated HL-60 cells to the Inr element would be altered. As
shown in Fig. 5B, granulocytic differentiation of HL-60
cells induced by Me
SO resulted in diminution of the NP-1
complex with a concomitant increase in a larger complex. The
differentiation-induced complex, designated NP-D, also appeared
following retinoic acid-induced granulocytic differentiation of HL-60
cells, but not following
12-O-tetradecanoylphorbol-13-acetate-induced monocytic
differentiation of HL-60 cells (Fig. 6A). Furthermore,
treatment of U937 cells with either Me
SO or
12-O-tetradecanoylphorbol-13-acetate did not induce the NP-D
complex (Fig. 6B), suggesting that the induction of NP-D
is cell lineage-specific. Nevertheless, there was no significant
alteration in the promoter activity of p(-44/+27)Luc in
transiently transfected granulocytic differentiated HL-60 cells (data
not shown), indicating that the induction of NP-D is irrelevant to the
up-regulation of PAF receptor gene expression following
differentiation.
Figure 3:
Binding of nuclear proteins from U937
cells to the regulatory sequence. The sequences of the double-stranded
oligonucleotides used as probe and competitors are listed. The
asterisk indicates mutated nucleotides. P-Labeled
PCR-75 was incubated with U937 nuclear extract in the absence
(Control) or presence of the indicated amounts of unlabeled
PCR-75 or a 100-fold excess of the other competitors. The formation of
the DNA-protein complex was then analyzed by electrophoresis.
NC, sequence from nucleotides -46 to -28 of the
PAF receptor gene; mInr, mutated Inr; TdT, terminal
deoxynucleotidyltransferase.
Figure 4:
Binding of nuclear proteins from
peripheral monocytes and from THP-1, HL-60, and HeLa cells to the
regulatory sequence containing the Inr element. The nuclear extracts
were prepared from the indicated cells and used for mobility gel shift
assay with the P-labeled Inr probe with sequence -16
to +18.
Figure 5:
Alteration in Inr binding activity during
granulocytic differentiation of HL-60 cells. A, binding of
nuclear extracts from HL-60 cells and MeSO-treated HL-60
cells to the
P-labeled Inr probe in the absence or
presence of a 100-fold excess of unlabeled oligonucleotides as
indicated. The asterisks indicate additional specific
complexes absent in U937 cells. B, time course for the
appearance of the differentiation-induced Inr-protein complex. HL-60
cells were treated with 1.3% Me
SO (DMSO) for the
indicated number of days. The nuclear extracts were then prepared, and
the binding to Inr was analyzed. PAFR, PAF receptor;
TdT, terminal
deoxynucleotidyltransferase.
Figure 6:
Effects of various
differentiation-inducing agents on nuclear factor binding to the Inr
element. HL-60 cells (A) and U937 cells (B) were
treated with various agents for the indicated number of days
(d). The nuclear extracts were then prepared, and the binding
to Inr was analyzed. DMSO, MeSO; TPA,
12-O-tetradecanoylphorbol-13-acetate; RA, retinoic
acid.
Effect of Mutations in the Inr Element on the Promoter
Activity
To further test the functional role of the Inr element,
we prepared the construct m(-44/+23)Luc, which contains
point mutations in the Inr element. Transfection of U937 and THP-1
cells with the wild-type p(-44/+27)Luc and mutant
recombinant reporter constructs revealed that the mutations in Inr,
which suppress the DNA binding activity (Fig. 7A),
caused a 50-60% decrease in the promoter activity
(Fig. 7B). This result clearly indicates that the Inr
element contributes significantly to the basal promoter activity of the
PAF receptor gene in myeloid cells.
Figure 7:
Effect of mutations in the Inr site on the
promoter activity. A, competition of the P-Inr
binding to nuclear proteins from U937 and THP-1 cells by the indicated
amounts of unlabeled Inr or mutated Inr (mInr) sequence shown
in Fig. 3. B, effect of mutations in Inr on luciferase
expression. A reporter gene construct containing the mutated Inr
sequence as shown in Fig. 3 was prepared. Wild-type
p(-44/+27)Luc and the mutant construct
m(-44/+23)Luc were transfected into U937 and THP-1 cells,
and the luciferase activities were measured. The activity of the mutant
construct (Inr mt) is reported relative to that of the wild
type, which was set to 100. The data presented are the means ±
S.D. of four independent experiments.
Identification of Inr-binding Proteins by UV
Cross-linking
To further identify the Inr-binding proteins, we
performed UV cross-linking of the nuclear proteins bound to the Inr
element. SDS-PAGE analysis of the cross-linked NP-1 complexes prepared
from THP-1 and HL-60 cells and peripheral monocytes revealed a
DNA-protein band with a molecular mass of 30 kDa (Fig. 8).
After subtraction of the molecular size of the oligonucleotide (27 bp),
the apparent molecular mass of the protein was estimated to be
22
kDa. Likewise, cross-linking of the NP-D complex from
Me
SO-induced differentiated HL-60 cells followed by
SDS-PAGE revealed an Inr-binding protein of
43 kDa, indicating
that the Inr element interacts with distinct nuclear proteins to form
NP-1 and NP-D complexes.
Figure 8:
UV cross-linking analysis of the
Inr-binding proteins. After the gel mobility shift experiment, the
Inr-protein complexes, NP-1 and NP-D, prepared from different cell
nuclear extracts were UV-cross-linked, excised from the gel, and
analyzed by SDS-PAGE under reducing (lanes 1-4) or
nonreducing (lane 5) conditions. Lane1,
THP-1 cells; lane2, monocytes; lane3, HL-60 cells; lane4,
MeSO-induced differentiated cells; lane5, HL-60 cells.
NT/AYY
(12, 13, 14, 15, 16) .
A survey of 502 unrelated promoter sequences has revealed that the
cytidine at nucleotide -1, the adenosine at nucleotide +1,
and the thymidine at nucleotide +3 are the three most conserved
nucleotides among these genes
(28) . The Inr sequence of the PAF
receptor gene, TCTTCA
CTTCTG, apparently
perfectly matches this feature. The Inr element has been shown to be
critical in positioning RNA polymerase II and in directing
transcription in the absence of binding sites for other transcriptional
factors in either TATA-containing or TATA-less
genes
(12, 13, 22, 29) . Furthermore,
studies on a number of genes, including the terminal
deoxynucleotidyltransferase gene
(12) , CD45 gene
(30) ,
c-mos protooncogene
(31) , myelin basic protein
gene
(32) , and FE65 gene
(33) , have demonstrated the
involvement of the Inr sequence in tissue-specific or
development-dependent gene expression. It is of great importance to
unravel the function of the Inr element in the regulation of PAF
receptor gene expression. A GC box, which is a potential binding site
for the universal transcriptional factor Sp1 (18), is located at
nucleotides -350 to -345 proximal to the initiation site.
Another potential regulatory element, CCCCACCCC, which is commonly
observed in the promoters of many myeloid-specific genes (19-21),
is present at nucleotides -153 to -144. To determine
whether the 5`-flanking region of the PAF receptor gene can function as
a promoter to direct transcription, the 1-kilobase pair genomic
fragment spanning nucleotides -1099 to +27 was inserted in
front of a luciferase gene in a luciferase reporter vector
(p(-1099/+27)Luc). The promoter activity was determined by
transient transfection of the p(-1099/+27)Luc construct into
myelocytic U937 and HL-60 cells. As shown in Fig. 2, the
p(-1099/+27)Luc construct produced
30- and 10-fold
higher promoter activities than the promoterless pBL2-basic construct
in U937 and HL-60 cells, respectively. The lower promoter activity
detected in HL-60 cells was consistent with the relatively low
expression level of the PAF receptor in these cells. To further define
the sequences required for the promoter activity, a series of
5`-deletion mutants of p(-1099/+27)Luc were prepared for
transient transfections. As shown in Fig. 2, the shortest
construct, p(-44/+27), retained promoter activity comparable
to p(-1099/+27)Luc, indicating that the DNA sequence within
the -44/+27 domain is sufficient for directing basal
transcription of the PAF receptor gene in the myeloid cells. Since the
Inr element is present in this domain, it is important to know whether
it is the key cis-element responsible for the promoter
activity. As the first step to identify the possible
trans-factor(s) that interacts with the Inr element, the
binding of nuclear proteins to the Inr sequence was examined. As
revealed by gel mobility shift assay, a DNA-protein complex, NP-1, was
detected in nuclear extracts from myelocytic U937, THP-1, and HL-60
cells, but not in that from HeLa cells, suggesting that the binding is
cell type-specific. Competition experiments with a mutated sequence or
the Inr sequence of the terminal deoxynucleotidyltransferase gene
further demonstrated that the binding is sequence-specific. In addition
to NP-1, two larger complexes in smaller amounts were also present in
nuclear extracts from THP-1 and HL-60 cells. However, it is not clear
if the larger complexes are derived from the association of NP-1 with
other nuclear proteins. Nevertheless, when HL-60 cells underwent
granulocytic differentiation induced by Me
SO, the amount of
NP-1 decreased, and this was accompanied by the appearance of a larger
complex, NP-D. The changes in Inr-nuclear protein interactions
following granulocytic differentiation, however, do not significantly
alter the promoter activity of p(-44/+27)Luc, as detected in
transiently transfected differentiated HL-60 cells (data not shown).
This result indicates that the up-regulation of PAF receptor gene
expression observed in differentiated HL-60 cells may involve other
regulatory elements that are not yet identified. Nevertheless, NP-D was
only observed in HL-60 cells during granulocytic differentiation,
suggesting the involvement of a cell lineage-specific nuclear factor in
the formation of the complex.
22 and 43 kDa, respectively. Although these
two Inr-binding proteins are distinct, whether they share a common
DNA-binding domain remains to be clarified. However, based on the
difference in molecular mass as well as the restricted cell-type
origin, these two Inr-binding proteins are distinguishable from the
previously identified Inr-binding proteins. These observations suggest
that the transcription of the PAF receptor gene is imparted, at least
in part, through the interaction of distinct Inr-binding proteins with
the Inr element in a cell-specific fashion.
/EMBL Data Bank with accession number(s) U11032.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.