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*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

26 Citations (Scopus)

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 (he EpCAM gene in a persistent manner via targeted DNA methylation, a low activity mutant (C141S) of the CpG-specific DNA methyltransferase M.Sssl 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-2010

Keywords

  • CANCER GENE-THERAPY
  • EP-CAM PROMOTER
  • BREAST-CANCER
  • IN-VIVO
  • CYTOSINE METHYLATION
  • POTENTIAL TARGET
  • ANTIGEN EPCAM
  • STEM-CELLS
  • EXPRESSION
  • SEQUENCES

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