Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells

Daniela Castanotto, Stella Tommasi, Mingjie Li, Haitang Li, Stephanie Yanow, Gerd P. Pfeifer, John J. Rossi

Research output: Contribution to journalArticlepeer-review

126 Scopus citations


Methylation of cytosines in CpG motifs is an important mechanism for epigenetic regulation of gene expression in mammalian cells. The initiating event(s) for de novo methylation in mammalian cells, particularly in cancer, is unknown. In plants, short RNAs homologous to DNA sequences are known to initiate de novo methylation. To investigate whether short hairpin RNAs (shRNAs) may also serve as initiators for de novo methylation in human cells we have expressed short hairpin RNAs complementary to the CpG island including the promoter and early transcribed regions of the human RASSF1A gene. RASSF1A encodes a putative tumor suppressor that is hypermethylated in a variety of human cancers, whereas in some human cell lines, such as HeLa, RASSF1A is unmethylated and transcriptionally active. We demonstrate that shRNAs complementary to the RASSF1A promoter or early transcribed regions can direct low levels of de novo DNA methylation and partial gene silencing in HeLa cells. In contrast, an shRNA harboring four central mismatches with the target cannot direct such methylation. The results presented suggest provocative potential mechanisms for transcriptional gene silencing via DNA methylation in cancer cells.

Original languageEnglish
Pages (from-to)179-183
Number of pages5
JournalMolecular Therapy
Issue number1
StatePublished - Jul 2005


  • Gene silencing
  • Methylation
  • RNA interference


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