©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Characterization of the Promoter Region of the Platelet-activating Factor Receptor Gene
IDENTIFICATION OF AN INITIATOR ELEMENT ESSENTIAL FOR GENE EXPRESSION IN MYELOID CELLS (*)

Jong-Hwei S. Pang , Ru-Ying Hung , Chia-Jung Wu , Yaw-Yeu Fang , Lee-Young Chau (§)

From the (1) Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 MeSO 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 MeSO-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.


INTRODUCTION

Platelet-activating factor (PAF)() 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 MeSO-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.


EXPERIMENTAL PROCEDURES

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% MeSO 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 NaVO 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 NaVO, 1 mM NaPO, 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.


RESULTS

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 MeSO 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 MeSO 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% MeSO (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 MeSO-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.




DISCUSSION

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, YYANT/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, TCTTCACTTCTG, 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 MeSO, 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.

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 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.

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.


FOOTNOTES

*
This work was supported by grants from the Academia Sinica and Grant NSC-83-0203-B001-102-B5 from the National Science Council of Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U11032.

§
To whom correspondence should be addressed. Tel.: 886-2-789-9137; Fax: 886-2-785-3569/782-5573.

The abbreviations used are: PAF, platelet-activating factor; Inr, initiator; 5`-RACE, 5`-rapid amplification of cDNA ends; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).


ACKNOWLEDGEMENTS

We thank Dr. Young-Sun Lin for critical reading and Dr. Cathy Fletcher for editing of the manuscript.


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