The 5-lipoxygenase promoter is regulated by DNA methylation.
5-lipoxygenase (5-LO), the key enzyme in leukotriene biosynthesis, is expressed in a tissue- and cell differentiation-specific manner. The 5-LO core promoter required for basal promoter activity has a unique (G+C)-rich sequence that contains five tandem Sp1 consensus sequences. The mechanisms involved in the regulation of cell type-specific 5-LO expression are unknown. Here we show that 5-LO expression is regulated by DNA methylation. Treatment of the 5-LO-negative cell lines U937 and HL-60TB with the demethylating agent 5-aza-2'-deoxycytidine (AdC) up-regulated expression of 5-LO primary transcripts and mature mRNA in a similar fashion, indicating that AdC stimulates 5-LO gene transcription. Analysis of the methylation status of the 5-LO promoter revealed that the core promoter region was methylated in U937 and HL-60TB cells, whereas it was unmethylated in the 5-LO-positive parent HL-60 cell line. Reporter gene assays with 5-LO promoter constructs gave up to 68- and 655-fold repression of 5-LO promoter activity in HeLa and Mono Mac 6 cells by methylation. 1,25-dihydroxyvitamin D(3) and transforming growth factor-beta (TGFbeta), potent inducers of the 5-LO pathway in myeloid cell lines, increased 5-LO RNA expression in HL-60TB and U937 cells, but co-treatment with AdC was required to achieve 5-LO expression levels in HL-60TB cells that were comparable with wild-type HL-60 cells. In reporter gene assays, 1,25-dihydroxyvitamin D(3) and TGFbeta were unable to induce promoter activity when the 5-LO promoter constructs were methylated, which suggests that 5-LO promoter demethylation is a prerequisite for the high level induction of 5-LO gene expression by 1,25-dihydroxyvitamin D(3) and TGFbeta and that the effects of both agents on 5-LO mRNA expression are not related to DNA methylation.
Uhl
Johannes
J
Institutes of Pharmaceutical Chemistry and Microbiology, University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany.
Klan
Niko
N
Rose
Matthias
M
Entian
Karl-Dieter
KD
Werz
Oliver
O
Steinhilber
Dieter
D
D001094:Arachidonate 5-Lipoxygenase / Q000096:biosynthesis / Q000235:genetics*; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001483:Base Sequence; D018899:CpG Islands; D019175:DNA Methylation; D017931:DNA Primers; D000077209:Decitabine; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D016859:Lipoxygenase Inhibitors; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020298:U937 Cells
D017931:DNA Primers; D004791:Enzyme Inhibitors; D016859:Lipoxygenase Inhibitors; D000077209:Decitabine; D001094:Arachidonate 5-Lipoxygenase; D001374:Azacitidine