title: The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells.
abstract: To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.
authors.affiliation: Department of Molecular Medicine and Hematology, University of the Witwatersrand Medical School, Parktown, South Africa.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: Louisa M
authors.initials: LM
authors.lastname: Villeneuve
authors.forename: Ali
authors.initials: A
authors.lastname: Ehsani
authors.forename: Mohammed
authors.initials: M
authors.lastname: Amarzguioui
authors.forename: Lars
authors.initials: L
authors.lastname: Aagaard
authors.forename: Zhao-Xia
authors.initials: ZX
authors.lastname: Chen
authors.forename: Arthur D
authors.initials: AD
authors.lastname: Riggs
authors.forename: John J
authors.initials: JJ
authors.lastname: Rossi
authors.forename: Kevin V
authors.initials: KV
authors.lastname: Morris
mesh_terms: D000546:Amanitins / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism; D000090205:DNA Methyltransferase 3A; D016325:HIV Long Terminal Repeat; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D034622:RNA Interference / Q000187:drug effects; D012319:RNA Polymerase II / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D000546:Amanitins; C000717967:DNMT3A protein, human; D006657:Histones; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; D016372:RNA, Antisense; D034741:RNA, Small Interfering; D014157:Transcription Factors; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D012319:RNA Polymerase II