Selective DNA demethylation by fusion of TDG with a sequence-specific DNA-binding domain

Abstract

Our ability to selectively manipulate gene expression by epigenetic means is limited, as there is no approach for targeted reactivation of epigenetically silenced genes, in contrast to what is available for selective gene silencing. We aimed to develop a tool for selective transcriptional activation by DNA demethylation. Here we present evidence that direct targeting of thymine-DNA-glycosylase (TDG) to specific sequences in the DNA can result in local DNA demethylation at potential regulatory sequences and lead to enhanced gene induction. When TDG was fused to a well-characterized DNA-binding domain [the Rel-homology domain (RHD) of NFκB], we observed decreased DNA methylation and increased transcriptional response to unrelated stimulus of inducible nitric oxide synthase (NOS2). The effect was not seen for control genes lacking either RHD-binding sites or high levels of methylation, nor in control mock-transduced cells. Specific reactivation of epigenetically silenced genes may thus be achievable by this approach, which provides a broadly useful strategy to further our exploration of biological mechanisms and to improve control over the epigenome.

Figure 1. Schematic of the hypothesis: demethylation of CpGs in regulatory sequences in the vicinity of NFκB binding sites by RHD-TDG construct but not by control constructs (e.g., RHD alone) would lead to increased transcription of the target gene. An unrelated stimulus, e.g., STAT1 induced by interferon, would produce stronger upregulation of Nos2 over background, if the promoter is demethylated.

Figure 2. Choice of target genes. Effect was expected in primary target Nos2, secondary target Cxcl11 and auxiliary target LINE-1, but not in control genes. Orange blocks denote NFκB binding sequences, blue blocks denote STAT1 binding sequences; yellow underscores indicate sites interrogated by pyrosequencing. N values represent number of PCR sample measurements.

Figure 3. RHD-TDG increases transcriptional response to IFNγ for Nos2, but not for control genes. *p < 0.01 against RHD control.

Figure 4. RHD-TDG decreases DNA methylation in vicinity of NF-kB binding sites in Nos2 promoter, Nos2 CpG island and in 4 out of 5 tested CpG sites in LINE-1. *p < 0.05 against RHD control.