Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene

Lysyl oxidase is an extracellular enzyme involved in connective tissue maturation that also acts as a phenotypic suppressor of the ras oncogene. To understand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays indicated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcription. Interferon-mediated phenotypic reversion of ras transformed cells, in which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transfected mouse lysyl oxidase promoter indicated that it was active in the transformed background, despite the silencing of the endogenous lysyl oxidase promoter. The detection of comparable amounts of mRNA for transcription factors IRF-1 and IRF-2 in normal and ras-transformed cell lines suggests that the differential transcription of lysyl oxidase was not due to regulation of IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region that includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar promoter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control region was confirmed by DNase footprinting. Lysyl oxidase transcription was stimulated by dexamethasone treatment of cells, but this effect could not be assigned within the similar to 3 kb region tested in reporter gene constructs. The promoter activity of the lysyl oxidase reporter gene construct was completely abolished by in vitro DNA methylation, suggesting that the transcriptional suppression after transformation by the ras oncogene may involve DNA methylation.