Specific transcriptional enhancement of inducible nitric oxide synthase by targeted promoter demethylation

The ability to specifically reactivate epigenetically silenced genes would have great utility in experimental studies and potential therapeutic value. Here, we describe the specific targeting of thymidine DNA glycosylase (TDG), an enzyme involved in the mechanism of methylcytosine demethylation, to the promoter of Nos2, a gene silenced by methylation in fibroblasts, using artificial zinc finger DNA binding domains. Individual targeted TDG constructs had a small effect on Nos2 expression and methylation, but simultaneous targeting of a quartet of TDG constructs significantly restored responsiveness to LPS and IFN stimuli in association with marked cytosine demethylation at the promoter and CpG island; catalytically inactive TDG complexes had no effect. Whole-genome expression microarray and pathway analysis found only 42 genes that were affected by targeted TDG constructs; the majority are likely downstream of the effect on Nos2. This study therefore shows highly specific, directed reactivation of a single, silenced gene by targeting of a demethylase to the promoter.