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

Kato K, Yokomizo T, Izumi T, Shimizu T - J. Exp. Med. (2000)

Bottom Line: Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis.Shimizu. 2000.Med. 192:421-431).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2193224&req=5

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.

Mentions: There are many CpG sites surrounding the promoter region of the BLT1 gene. To determine the methylation state at these CpG sites, Southern blotting was performed using genomic DNAs from various cell lines. Although both MspI and HpaII recognize and digest 5′-CCGG-3′ sequences, MspI but not HpaII can cleave these sequences when the second cytosine residue is methylated. Capitalizing on this difference, methylation in the promoter region was detected. Using probe A, a 5′ flanking region of the promoter, a band of 1.1 kb was detected by MspI digestion in all the cells examined (Fig. 5 A). However, this band of 1.1 kb was detected only in HL-60 and U937 cells by HpaII digestion, whereas a partial digested band of 2.5 or 5.5 kb was detected in HL-60, U937, and THP-1 cells or HL-60 cells, respectively. In HeLa and HepG2 cells, neither the 1.1 kb nor the 2.5 kb band was observed, whereas longer bands (∼3.0 kb and 7.0 kb, respectively) were detected (Fig. 5 A). Similar results were observed in Southern blotting using probe B, a sequence 3′ to the promoter region (Fig. 5 B). Considering the size of the detected bands, the region surrounding the BLT1 promoter is not methylated in U937 and THP-1 cells, and is almost completely methylated in HeLa and HepG2 cells. In HL-60 cells (both differentiated and nondifferentiated), this region appears to be partially methylated. Northern blotting of these cell lines showed that HL-60, U937, and THP-1 cells express BLT1 mRNA, whereas HeLa and HepG2 cells do not (Fig. 5 D). These results led us to the conclusion that methylation inhibits BLT1 transcription. Thus, the effect of methylation on the promoter activity was investigated. The insert of p(−123/+91) was treated with SssI methylase, which methylates cytosine residues at the CpG sites, and a luciferase assay was performed. The activity of methylated construct was decreased to ∼15% of that of the unmethylated construct (Fig. 6), supporting the conclusion that methylation of the CpG sites inhibits the BLT1 promoter activity.


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

Kato K, Yokomizo T, Izumi T, Shimizu T - J. Exp. Med. (2000)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193224&req=5

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.
Mentions: There are many CpG sites surrounding the promoter region of the BLT1 gene. To determine the methylation state at these CpG sites, Southern blotting was performed using genomic DNAs from various cell lines. Although both MspI and HpaII recognize and digest 5′-CCGG-3′ sequences, MspI but not HpaII can cleave these sequences when the second cytosine residue is methylated. Capitalizing on this difference, methylation in the promoter region was detected. Using probe A, a 5′ flanking region of the promoter, a band of 1.1 kb was detected by MspI digestion in all the cells examined (Fig. 5 A). However, this band of 1.1 kb was detected only in HL-60 and U937 cells by HpaII digestion, whereas a partial digested band of 2.5 or 5.5 kb was detected in HL-60, U937, and THP-1 cells or HL-60 cells, respectively. In HeLa and HepG2 cells, neither the 1.1 kb nor the 2.5 kb band was observed, whereas longer bands (∼3.0 kb and 7.0 kb, respectively) were detected (Fig. 5 A). Similar results were observed in Southern blotting using probe B, a sequence 3′ to the promoter region (Fig. 5 B). Considering the size of the detected bands, the region surrounding the BLT1 promoter is not methylated in U937 and THP-1 cells, and is almost completely methylated in HeLa and HepG2 cells. In HL-60 cells (both differentiated and nondifferentiated), this region appears to be partially methylated. Northern blotting of these cell lines showed that HL-60, U937, and THP-1 cells express BLT1 mRNA, whereas HeLa and HepG2 cells do not (Fig. 5 D). These results led us to the conclusion that methylation inhibits BLT1 transcription. Thus, the effect of methylation on the promoter activity was investigated. The insert of p(−123/+91) was treated with SssI methylase, which methylates cytosine residues at the CpG sites, and a luciferase assay was performed. The activity of methylated construct was decreased to ∼15% of that of the unmethylated construct (Fig. 6), supporting the conclusion that methylation of the CpG sites inhibits the BLT1 promoter activity.

Bottom Line: Sp1 was found to be a major activator of basal transcription by electrophoretic mobility shift assays and site-directed mutagenesis.Shimizu. 2000.Med. 192:421-431).

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan.

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.

Show MeSH
Related in: MedlinePlus