title: An RNA targeted to the HIV-1 LTR promoter modulates indiscriminate off-target gene activation.
abstract: Transcriptional gene silencing (TGS) can be achieved by small RNAs targeted to upstream promoter regions. Previously we characterized siRNAs targeted to the HIV-1 long terminal repeat (LTR) promoter at site 247, and found that a 21-base antisense strand of siRNA-247 (LTR-247as) suppressed LTR-mediated expression. To characterize the specificity of LTR-247as, vectors expressing antisense RNAs targeted to a region spanning 50 bases up- and downstream of the 247 target site were generated. LTR-247as+7, a approximately 22 base antisense RNA that is shifted by only seven bases upstream of LTR-247as, showed a significant increase in LTR-driven reporter gene expression that was independent of cell type and active chromatin methyl-marks. Promoter-targeting siRNAs have been recently shown to induce gene activation. However, here we demonstrate gene activation via a sequence-specific off-target effect. Microarray analysis of LTR-247as+7-treated cultures resulted in the deregulation of approximately 185 genes. A gene of unknown function, C10orf76, was responsive to inhibition by LTR-247as+7 and the loss of C10orf76 resulted in the upregulation of several genes that were activated by LTR-247as+7. These data suggest caution when using short antisense RNAs or siRNAs designed to target promoter sequences, since promoter-targeted RNAs may have unintended inhibitory effects against factors with suppressive gene activity.
authors.affiliation: Antiviral Gene Therapy Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, 7 York Rd Parktown 2193, South Africa.
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: Samantha
authors.initials: S
authors.lastname: Barichievy
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.forename: Jiang
authors.initials: J
authors.lastname: Han
authors.forename: Kevin V
authors.initials: KV
authors.lastname: Morris
mesh_terms: D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006657:Histones / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000037:antagonists & inhibitors; D015533:Transcriptional Activation
chemical_list: D006657:Histones; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; D008156:Luciferases