title: Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells.
abstract: 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.
authors.affiliation: Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Castanotto
authors.forename: Stella
authors.initials: S
authors.lastname: Tommasi
authors.forename: Mingjie
authors.initials: M
authors.lastname: Li
authors.forename: Haitang
authors.initials: H
authors.lastname: Li
authors.forename: Stephanie
authors.initials: S
authors.lastname: Yanow
authors.forename: Gerd P
authors.initials: GP
authors.lastname: Pfeifer
authors.forename: John J
authors.initials: JJ
authors.lastname: Rossi
mesh_terms: D001483:Base Sequence; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D012313:RNA; D003596:Cytosine