Weinberg Marc S, Villeneuve Louisa M, Ehsani Ali, Amarzguioui Mohammed, Aagaard Lars, Chen Zhao-Xia, Riggs Arthur D, Rossi John J, Morris Kevin V
Department of Molecular Medicine and Hematology, University of the Witwatersrand Medical School, Parktown, South Africa.
RNA. 2006 Feb;12(2):256-62. doi: 10.1261/rna.2235106. Epub 2005 Dec 22.
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.
为了从机制上确定小干扰RNA（siRNA）如何在人类细胞中介导转录基因沉默（TGS），我们检测了靶向启动子处的组蛋白甲基化、对活性转录的依赖性以及siRNA介导的TGS是否需要siRNA的两条链。我们在此报告，siRNA处理增加了靶向的EF1A启动子的H3K9和H3K27甲基化，且这种增加依赖于siRNA的核特异性递送。我们还发现，TGS可仅由反义链引导，并且在人类细胞中需要RNA聚合酶II的活性转录，这一点通过对α-鹅膏蕈碱的敏感性得到证明。通过靶向HIV-1长末端重复序列/启动子的U3区域，证实了EF1A的反义链特异性siRNA介导的TGS。此外，我们表明，siRNA EF52的反义链与瞬时表达的Flag标签的DNMT3A、靶向的EF1A启动子和三甲基化的H3K27相关。本文报道的观察结果表明，siRNA介导的基因组区域（启动子）靶向、RNA聚合酶II功能、组蛋白甲基化和DNMT3A之间存在功能联系，并支持一种模式，即仅siRNA的反义链就可以在人类细胞中指导序列特异性转录基因沉默。
