Chimeric DNA methyltransferases target DNA methylation to specific DNA sequences and repress expression of target genes

Abstract

Gene silencing by targeted DNA methylation has potential applications in basic research and therapy. To establish targeted methylation in human cell lines, the catalytic domains (CDs) of mouse Dnmt3a and Dnmt3b DNA methyltransferases (MTases) were fused to different DNA binding domains (DBD) of GAL4 and an engineered Cys2His2 zinc finger domain. We demonstrated that (i) Dense DNA methylation can be targeted to specific regions in gene promoters using chimeric DNA MTases. (ii) Site-specific methylation leads to repression of genes controlled by various cellular or viral promoters. (iii) Mutations affecting any of the DBD, MTase or target DNA sequences reduce targeted methylation and gene silencing. (iv) Targeted DNA methylation is effective in repressing Herpes Simplex Virus type 1 (HSV-1) infection in cell culture with the viral titer reduced by at least 18-fold in the presence of an MTase fused to an engineered zinc finger DBD, which binds a single site in the promoter of HSV-1 gene IE175k. In short, we show here that it is possible to direct DNA MTase activity to predetermined sites in DNA, achieve targeted gene silencing in mammalian cell lines and interfere with HSV-1 propagation.

Persistent downregulation of the pancarcinoma‐associated epithelial cell adhesion molecule via active intranuclear methylation

“…21 Methyltransferases fused to zinc finger proteins targeting predetermined sites in the DNA to repress gene expression have been reported. 12,22,23 Engineering ZFPs targeted to the EpCAM promoter 21 fused to M.SssI as an effector domain, provides a powerful tool to achieve targeted methylation. The ZFP binds specific to the promoter where after the enzyme will methylate only the CpGs close to the ZFP target sequence.…”

Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain

“…These results confirm that the TK promoter is highly sensitive to DNA methylation, as previously reported. 24 We determined the effect exerted by the transient expression of different GBD-ROS1_CD versions on this silenced luciferase activity (Fig. 3).…”

“…A fragment containing the GAL4 DNA Binding Domain (GBD, 1-147 amino acids), a c-myc tag and a nuclear localization signal (NLS, PKKKKRK), was amplified by PCR from pcDNA3.1-GBD, 24 using primers with NheI and EcoRI restriction sites (Table S1). The PCR product was digested, subcloned between the His 6 -tag and ROS1_CD into pET28c-ROS1ND294, and verified by DNA sequencing.…”

“…23 GBD has been previously used for targeting the catalytic domain of mouse DNA methyltransferases DNMT3a and DNMT3b in human cells. 24 The targeted binding capacity of GBD-ROS1_CD was first analyzed in vitro and then the fusion protein was transiently expressed in a human cell line. We have found that transient expression of GBD-ROS1_CD in human cells promotes targeted DNA demethylation and reactivation of a methylationsilenced reporter gene.…”

Manipulation of prostate cancer metastasis by locus-specific modification of the CRMP4 promoter region using chimeric TALE DNA methyltransferase and demethylase

“…Although site-selective methylation with chimeric DNA methyltransferases has been achieved using DNA-binding domains derived from various native DNA-binding proteins [21] and artificial zinc finger DNAbinding domains [22], these modes of methylation suffer from poor specificity.…”