Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

Abstract

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that has been identified as a marker of cancer-initiating cells. EpCAM is highly expressed on most carcinomas, and transient silencing of EpCAM expression leads to reduced oncogenic potential. To silence the EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.SssI was coupled to a triple-helix-forming oligonucleotide (TFO−C141S) specifically designed for the EpCAM gene. Reporter plasmids encoding the green fluorescent protein under control of different EpCAM promoter fragments were treated with the TFO−C141S conjugate to determine the specificity of targeted DNA methylation in the context of a functional EpCAM promoter. Treatment of the plasmids with TFO−C141S resulted in efficient and specific methylation of the targeted CpG located directly downstream of the triple helix forming site (TFS). No background DNA methylation was observed neither in a 700 bp region of the EpCAM promoter nor in a 400 bp region of the reporter gene downstream of the TFS. Methylation of the target CpG did not have a detectable effect on promoter activity. This study shows that the combination of a specific TFO and a reduced activity methyltransferase variant can be used to target DNA methylation to predetermined sites with high specificity, allowing determination of crucial CpGs for promoter activity.

Transcription factors and molecular epigenetic marks underlying EpCAM overexpression in ovarian cancer

“…DNA methylation analysis was performed as described (van der Gun et al , 2010a). Bisulphite primer sequences for regions A and B are depicted in Figure 1A.…”

Small-molecule regulators that mimic transcription factors

“…Snowden et al fused a ZF DNA binding domain to histone modifying enzymes to (i.e., a histone deacetylase and a histone methyltransferae) [59, 60]. More recently a DNA methylase enzyme was fused to TFO DNA binding domain and shown to specifically methylate the targeted promoter in a reporter plasmid [61]. …”

Cytosine-to-Uracil Deamination by SssI DNA Methyltransferase

“…It is worth mentioning that G19D has two features that can be beneficial for the planned application. The relatively low cytosine deaminase activity can be an advantage for achieving high targeting specificity as has been shown for targeted DNA methylation [41,42]. The high level of insensitivity to uracil excision repair observed in E. coli Ung + hosts can be important for preventing repair of the pre-mutagenic U:G mismatch generated by the enzyme before conversion into a stable T:A mutant base pair.…”