title: 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.
authors.affiliation: Department of Environmental Health, Harvard School of Public Health, Boston, MA, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Gregory
authors.forename: Lyudmila
authors.initials: L
authors.lastname: Mikhaylova
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Fedulov
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D018899:CpG Islands; D019175:DNA Methylation; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
chemical_list: D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D007371:Interferon-gamma; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D044502:Thymine DNA Glycosylase