Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to down-regulate the epithelial cell adhesion molecule

Bernardina T.F. Van Der Gun, Maria Maluszynska-Hoffman, Antal Kiss, Alice J. Arendzen, Marcel H.J. Ruiters, Pamela M.J. McLaughlin, Elmar Weinhold, Marianne G. Rots

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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.

Original languageEnglish
Pages (from-to)1239-1245
Number of pages7
JournalBioconjugate Chemistry
Volume21
Issue number7
DOIs
Publication statusPublished - Jul 21 2010

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ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Van Der Gun, B. T. F., Maluszynska-Hoffman, M., Kiss, A., Arendzen, A. J., Ruiters, M. H. J., McLaughlin, P. M. J., Weinhold, E., & Rots, M. G. (2010). Targeted DNA methylation by a DNA methyltransferase coupled to a triple helix forming oligonucleotide to down-regulate the epithelial cell adhesion molecule. Bioconjugate Chemistry, 21(7), 1239-1245. https://doi.org/10.1021/bc1000388