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

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

Super-specific DNA methylation by a DNA methyltransferase coupled with a triple helix-forming oligonucleotide

In this work, an engineered variant of the DNA cytosine-C5 methyltransferase (MTase) M.SssI has been conjugated with a triple helix-forming oligonucleotide (TFO) and the properties of the enzyme-TFO conjugate were evaluated using a variety of biochemical and biophysical methods. M.SssI is the only known prokaryotic DNA MTase which shares the 5’-CG-3’ recognition sequence (CpG) with mammalian DNA MTases. It has been rendered a super-specific MTase by conjugation with a TFO designed to bind a triple helix-forming site (TFS) in the promoter of the epithelial cell adhesion molecule (EpCAM) with high specificity and affinity. This transmembrane glycoprotein is strongly overexpressed in several carcinomas and its expression levels have been shown to correlate with the EpCAM promoter methylation status (van der Gun et al., 2008). EpCAM down-regulation via sequence-specific DNA methylation could find potential application in gene therapy. Three M.SssI variants have been selectively conjugated with DNA single strand, either by bifunctional crosslinking or expressed protein ligation (EPL). Only one conjugate has been further investigated, as a tool for super-specific TFO-directed DNA methylation. M.SssI(C141S) has been conjugated with the chosen TFO using a well known maleimide based bifunctional crosslinking method. In M.SssI(C141S), the catalytic cysteine has been replaced with serine and thus it possesses only one C-terminal cysteine residue, which can be crosslinked with the TFO via a long polyethylaene linker (Mal-PEG27-TFO). Wild type M.SssI, which possesses more than one cysteine residue, has been coupled with an oligodeoxynucleotide (ODN) also by heterobifunctional crosslinking, but in a longer synthetic route featuring the temporary protection of the active site cysteine (C141). Coupling with catalytic cysteine could lead to a loss of DNA MTase activity. Therefore, trapping and subsequent conjugation reaction was analyzed and the desired C-terminally ODN-linked conjugate could be purified by anion exchange HPLC. However, the obtained overall yield was 2%, which excluded this approach to obtain larger amounts of M.SssI-ODN conjugate from further investigations. Trapping M.SssI is a good way to temporally block the catalytic cysteine residue, but can be applied only to variants displaying good binding properties. Isolating the formed trapping complex can be also seen as a selection of the catalytically active M.SssI molecules and way to determine the amount of active enzyme in the preparation. This approach, despite a very low yield, enabled conjugation and purification of an ODN-conjugate with catalytically active M.SssI. Such discrimination is infrequent in DNA-protein coupling methods. Finally, coupling of the low binding variant M.SssI(Q147L) with the TFO was carried out by EPL. Coupling of M.SssI(Q147L)-thioester with the TFO was analyzed by SDS-PAGE and resulted in 10% conversion. Applying EPL for producing the M.SssI(Q147L)-TFO conjugate was not successful in terms of high conversion. This and high precipitation levels reduced the usefulness of this method for producing M.SssI(Q147L)-TFO conjugate in an amount needed for testing TFO-directed super-specific DNA methylation in vitro. The specificity of M.SssI(C141S)-TFO conjugate obtained by heterobifunctional crosslinking has been tested in an in vitro methylation assay on Litcon54. The results showed super-specific DNA methylation of the targeted site and that background DNA methylation levels were only slightly elevated even in the sample with the highest conjugate excess. In parallel the thermodynamic and kinetic properties of triple helix formation were studied. The chosen TFO is a polypyrimidine T-rich strand, which binds in a parallel orientation to the polypurine strand of the duplex DNA. The influence of magnesium on triple helix formation was analyzed by thermal denaturation. In addition, the FRET assay was used to confirm the binding orientation of the TFO to the TFS. The kinetics of triple helix formation were analyzed in the UV assay. DNA binding affinity of M.SssI(C141S) and M.SssI(C141S)-TFO was investigated in the 2-aminopurine (2Ap) fluorescence assay, where the M.SssI(C141S)-TFO conjugate displayed a much reduced DNA binding. Yet, when a DNA substrate with the TFS sequence next to the target site was investigated, the DNA binding of the enzyme within the M.SssI(C141S)-TFO conjugate could be significantly restored. The imposing effect of a single methylated 5’-CG-3’ sequence by M.SssI(C141S)-TFO conjugate on gene expression was shown in transfection experiments. SKOV3 cell were transfected with a conjugate-treated reporter plasmid p39E, possessing the EpCAM promoter sequence upstream of the green fluorescent protein (GFP) gene. Selective methylation resulted in GFP expression down-regulation of 35% compared with the fully methylated plasmid