Fusion of TDG and RHD for selective DNA demethylation and transcriptional enhancement of NOS2 (58.2)

Abstract DNA methylation is a key mechanism in epigenetic silencing of transcription. Removal of methylation marks in promoters and other key regulatory sequences is associated with increased gene transcription, which could be beneficial for immunomodulatory and other purposes. However there is no method to selectively demethylate DNA in living cells. We designed a molecular construct in which a putative demethylase thymine-DNA-glycosylase (TDG) is targeted to a specific sequence by fusion with a DNA binding domain of NF-κB, the Rel-homology domain (RHD). 3T3 murine fibroblasts were transduced with fusion TDG-RHD or control constructs. In these cells the promoter of inducible nitric oxide synthase (NOS2) gene is highly methylated and contains RHD-binding sequences. We observed decreased DNA methylation (by 5-10 %, p<0.05) in several C’s in the NOS2 promoter and nearest CpG island. We also observed decreased methylation in the vicinity of RHD-binding sites in LINE-1, suggesting that the effect was available across the genome. Stimulation with IFNγ (250 ug/ml, 6 hrs) led to increased NOS2 message (fold over unstimulated 97.7±27.3 vs. 18.9±6.3 in control, p<0.01) and protein, indicating increased gene function. The effect was not seen for control genes lacking either RHD-binding sites or high levels of methylation, nor in mock-transduced cells. Results suggest that direct targeting of TDG to specific DNA loci may be practical for selective re-activation of epigenetically silenced genes.