DNA Methylation Regulates 5-Lipoxygenase Promoter Activity
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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 an unique G + C-rich sequence which contains five tandem Spl consensus sequences . Previously, we could show that the 5-lipoxygenase promoter 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) upregulated 5-LO activity and the expression of 5-LO primary transcripts and mature mRNA indicating that AdC stimulates 5-LO expression at the level of gene transcription. Analysis of the methylation status by methylation-sensitive sequencing 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 wild-type HL-60 cell line. In transient reporter gene assays, there was a dramatic reduction of 5-LO promoter activity when the reporter gene constructs were methylated in-vitro by Sss I methylase before transfection, indicating that 5-LO promoter activity is strongly inhibited when the promoter is methylated.
KeywordsPromoter Activity Methylation Status U937 Cell Reporter Gene Construct Core Promoter Region
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