title: Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation.
abstract: Small interfering RNAs (siRNAs) are newly identified molecules shown to silence genes via targeted mRNA degradation. In this study, we used specific siRNAs as a tool to probe the relationship between two DNA methyltransferase genes, DNMT3b and DNMT1, in the maintenance of DNA methylation patterns in the genome. Levels of DNMT3b or DNMT1 mRNAs and proteins were markedly decreased (up to 80%) on transfecting these siRNAs into the ovarian cancer cell line CP70. The resulting RNA interference showed differential effects on DNA demethylation and gene reactivation in the treated cells. The DNMT1 siRNA treatment led to a partial removal of DNA methylation from three inactive promoter CpG islands, TWIST, RASSF1A, and HIN-1, and restored the expression of these genes. This epigenetic alteration appeared less effective in cells transfected with DNMT3b siRNA. However, the combined treatment of DNMT3b and DNMT1 siRNAs greatly enhanced this demethylation effect, producing 7-15-fold increases in their expression. We also used a microarray approach to examine this RNA interference on 8640 CpG island loci in CP70 cells. The combined siRNA treatment had a greater demethylation effect on 241 methylated loci and selected repetitive sequences than that of the single treatment. Our data thus suggest that whereas DNMT1 plays a key role in methylation maintenance, DNMT3b may act as an accessory to support the function in CP70 cells. This study also shows that siRNA is a powerful tool for interrogating the mechanisms of DNA methylation in normal and pathological genomes.
authors.affiliation: Division of Human Cancer Genetics, Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, The Ohio State University, 420 West 12th Avenue, Columbus, OH 43210, USA.
authors.forename: Yu-Wei
authors.initials: YW
authors.lastname: Leu
authors.forename: Farahnaz
authors.initials: F
authors.lastname: Rahmatpanah
authors.forename: Huidong
authors.initials: H
authors.lastname: Shi
authors.forename: Susan H
authors.initials: SH
authors.lastname: Wei
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Liu
authors.forename: Pearlly S
authors.initials: PS
authors.lastname: Yan
authors.forename: Tim Hui-Ming
authors.initials: TH
authors.lastname: Huang
mesh_terms: D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics*; D019175:DNA Methylation; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D015894:Genome, Human; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000473:pathology / Q000628:therapy; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection; D000097584:DNA Methyltransferase 3B
chemical_list: D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D034741:RNA, Small Interfering; D000097584:DNA Methyltransferase 3B; C000619380:DNMT1 protein, human