Epigenetic inhibition of lysyl oxidase transcription after transformation by ras oncogene

Lysyl oxidase is an extracellular enzyme involved in connective tissue matu ration that also acts as a phenotypic suppressor of the ras oncogene. To un derstand how this suppressor is controlled, gene transcription was studied and the promoter was characterized. Nuclear runoff transcription assays ind icated that the markedly reduced amounts of lysyl oxidase message detected after ras transformation resulted from inhibition of lysyl oxidase transcri ption. Interferon-mediated phenotypic reversion of ras transformed cells, i n which the ras oncogene continued to be expressed, was accompanied by the restoration of lysyl oxidase transcription. Reporter gene assay of a transf ected mouse lysyl oxidase promoter indicated that it was active in the tran sformed background, despite the silencing of the endogenous lysyl oxidase p romoter. The detection of comparable amounts of mRNA for transcription fact ors 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 o f IRFs. Lysyl oxidase promoter activity was localized to a 126 bp region th at includes two consensus TATA boxes with associated confirmed cap signals. Analysis of a human lysyl oxidase promoter sequence indicated similar prom oter elements and extensive sequence identity with the mouse promoter. The binding of transcription factor AP2 to sites predicted in the control regio n was confirmed by DNase footprinting. Lysyl oxidase transcription was stim ulated by dexamethasone treatment of cells, but this effect could not be as signed within the similar to 3 kb region tested in reporter gene constructs . The promoter activity of the lysyl oxidase reporter gene construct was co mpletely abolished by in vitro DNA methylation, suggesting that the transcr iptional suppression after transformation by the ras oncogene may involve D NA methylation.