Cell-specific transcriptional regulation of human leukotriene B(4) receptor gene

Abstract

Leukotriene B(4) (LTB(4)) is a lipid mediator that activates leukocytes and is involved in host defense and inflammation. BLT1, a high-affinity receptor for LTB(4) (originally termed BLT), is expressed exclusively in inflammatory cells and is inducible in macrophages upon activation. The mechanisms of tissue-specific expression and induction of BLT1 are important for the understanding of mechanism of onset and the potential treatment of inflammatory disorders. Here, we report the genomic structure and a promoter analysis of the human BLT1 gene, with an emphasis on the mechanism of cell-specific transcription. No TATA or CAAT elements exist around the transcription initiation sites, but a GC-rich sequence is observed in this region. A reporter gene assay revealed that a region approximately 80 basepair upstream from the initiator sequence is required for the basal transcription of the BLT1 gene. Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis. The CpG sites of the BLT1 promoter region were highly methylated in BLT1-nonexpressing cells, but not methylated in BLT1-expressing cells. Further, methylation of this region in vitro inhibited the promoter activity to approximately 15% of the control. Thus, methylation at CpG sites in the promoter region is important for cell-specific transcription of the BLT1 gene. The promoter region of the BLT1 gene is localized within the open reading frame (ORF) of the BLT2 gene, which encodes a low-affinity receptor for LTB(4) (Yokomizo, T., K. Kato, K. Terawaki, T. Izumi, and T. Shimizu. 2000. J. Exp. Med. 192:421-431). To our knowledge, this is the first example of "promoter in ORF" in higher eukaryotes.

Figure 1

Genomic structure of the human BLT1 gene. The ORF (light gray box), promoter region (dark gray box), and UTRs (open boxes) of the BLT1 gene and the ORF of the BLT2 gene (hatched box) are indicated. Restriction enzyme sites are indicated as follows: B, BamHI; E, EcoRI; H, HindIII; K, KpnI; and S, SacI. These sequence data (LambdaNOK) are available from EMBL/GenBank/DDBJ under accession no. AB008193. Note that the BLT2 ORF is overlapped with the BLT1 promoter region.

Figure 2

Identification of the transcription initiation site of the BLT1 gene. (A) The sites of primers used for 5′ RACE are shown. Arrows indicate the synthetic oligonucleotides. (B) Nucleotide sequences of the 5′ flanking region of the BLT1 gene. Asterisks show the transcription initiation sites determined by 5′ RACE. GC boxes (consensus sequences of Sp1) are shown in open boxes. The consensus sequences of various transcription factors are underlined.

Figure 2

Identification of the transcription initiation site of the BLT1 gene. (A) The sites of primers used for 5′ RACE are shown. Arrows indicate the synthetic oligonucleotides. (B) Nucleotide sequences of the 5′ flanking region of the BLT1 gene. Asterisks show the transcription initiation sites determined by 5′ RACE. GC boxes (consensus sequences of Sp1) are shown in open boxes. The consensus sequences of various transcription factors are underlined.

Figure 3

Serial deletion mutant analysis of the BLT1 promoter. Promoter activities are shown as the luciferase activity relative to that of the pGL3 basic vector (a promoterless vector). The activities of THP-1 cells (white bars) and HeLa cells (black bars) are shown as the mean ± SD from three independent experiments performed in triplicate.

Figure 4

Sp1 binds and activates the BLT1 promoter. (A) EMSAs were performed with ³²P-labeled −76/−33 using nuclear extracts prepared from THP-1 and HeLa cells. Two DNA–protein complexes were detected in both cells (lanes 2 and 8; shown as b and c). They were competed by 200-fold molar excess of unlabeled −76/−33 (−76/−33 WT, lanes 3 and 9), and not by 200-fold molar excess of unlabeled −76/−33 with mutations in the GC box (−52/−47) (−76/−33 Sp1 m, lanes 4 and 10). The band b was supershifted by incubation with 1 μg of anti-Sp1 antibody (lanes 6 and 12; shown as a) but not by the control IgG (lanes 5 and 11). (B) The effect of mutagenesis of Sp1 binding site on the promoter activities. Luciferase activities of the wild-type construct, p(−123/+91) (white columns) and the construct mutated at GC box (black columns) are indicated as mean ± SD from three independent experiments performed in triplicate. Mutation sites are shown in bold at right.

Figure 4

Sp1 binds and activates the BLT1 promoter. (A) EMSAs were performed with ³²P-labeled −76/−33 using nuclear extracts prepared from THP-1 and HeLa cells. Two DNA–protein complexes were detected in both cells (lanes 2 and 8; shown as b and c). They were competed by 200-fold molar excess of unlabeled −76/−33 (−76/−33 WT, lanes 3 and 9), and not by 200-fold molar excess of unlabeled −76/−33 with mutations in the GC box (−52/−47) (−76/−33 Sp1 m, lanes 4 and 10). The band b was supershifted by incubation with 1 μg of anti-Sp1 antibody (lanes 6 and 12; shown as a) but not by the control IgG (lanes 5 and 11). (B) The effect of mutagenesis of Sp1 binding site on the promoter activities. Luciferase activities of the wild-type construct, p(−123/+91) (white columns) and the construct mutated at GC box (black columns) are indicated as mean ± SD from three independent experiments performed in triplicate. Mutation sites are shown in bold at right.

Figure 5

Methylation of CpG sites in the BLT1 promoter region. (A and B) Genomic DNAs isolated from various cell lines were digested with HpaII or MspI, followed by digestion with EcoRI. The digested DNAs were electrophoresed in 1% agarose gels, transferred to nylon membranes, and blotted with (A) probe A and (B) probe B. The positions of the probes A and B are shown in C. HL-60 (RA) means HL-60 cells differentiated by 1 μM retinoic acid for 48 h. (C) Methylation sites and genomic organization. (D) Northern blotting of various cells for BLT1 ORF. 3 μg of poly(A)⁺ RNA was used for each lane.

Figure 5

Methylation of CpG sites in the BLT1 promoter region. (A and B) Genomic DNAs isolated from various cell lines were digested with HpaII or MspI, followed by digestion with EcoRI. The digested DNAs were electrophoresed in 1% agarose gels, transferred to nylon membranes, and blotted with (A) probe A and (B) probe B. The positions of the probes A and B are shown in C. HL-60 (RA) means HL-60 cells differentiated by 1 μM retinoic acid for 48 h. (C) Methylation sites and genomic organization. (D) Northern blotting of various cells for BLT1 ORF. 3 μg of poly(A)⁺ RNA was used for each lane.

Figure 6

Effect of the methylation at CpG sites on the promoter activity. The insert of p(−123/+91) was incubated with or without SssI methylase, ligated into pGL3 basic vector, and transfected into HeLa cells. The relative luciferase activities are shown as mean ± SD from three independent experiments performed in triplicate.