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.
van der Gun
Bernardina T F
BT
Epigenetic Editing, Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9713 GZ Groningen, The Netherlands.
Maluszynska-Hoffman
Maria
M
Kiss
Antal
A
Arendzen
Alice J
AJ
Ruiters
Marcel H J
MH
McLaughlin
Pamela M J
PM
Weinhold
Elmar
E
Rots
Marianne G
MG
D015815:Cell Adhesion Molecules / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004247:DNA / Q000187:drug effects* / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects* / Q000235:genetics; D015254:DNA Modification Methylases / Q000737:chemistry / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics
D015815:Cell Adhesion Molecules; D009841:Oligonucleotides; D049452:Green Fluorescent Proteins; D004247:DNA; D015254:DNA Modification Methylases