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.

Figure 1.

U6-antisense RNA cassettes generated to target regions flanking the LTR 247 site. (A) A set of nine asRNAs were selected that targeted either site 247, or target regions which span upstream (+) and/or downstream (−) of 247. The respective asRNAs are shown aligned with the HIV-1 subtype B LTR and site 247 (targeted by LTR-247as). (B) Selected U6-expressing asRNAs were cloned into the pTopoTA-based vector system as described (18).

Figure 2.

Effects of various U6 expressed antisense RNAs on luciferase expression. 1G5 cells containing an integrated HIV-1 subtype B LTR-Luciferase-SV40 poly A cassette were co-electroporated with pTat-dsRed and the respective LTR-247as vectors (2.5 µg of each plasmid respectively) and assessed for luciferase expression. (A) Relative luciferase expression standardized to GAPDH per cell presented relative to the control treated cultures (pU6 GFPas) as determined by real-time RT-PCR 24 h post-transfection. Data represent three independent experiments with the standard deviations shown. LTR-247as+7-mediated increased transcription (P = 0.06093) relative to the control GFPas based on a single-sided f-test. (B) Non-specific LTR-247as+7-mediated enhancement of luciferase expression. 1G5 cells (3 × 10⁶) were co-transfected with either pBSK+ or pTat-dsRed and either pU6 LTR-247as+7, pU6 LTR-247as or the control pU6 GFPas. Twenty-four hours later cultures were collected, qRT-PCR was performed and luciferase expression determined and normalized to GAPDH. Results represent the mean ± standard deviations of three independent experiments.

Figure 3.

H3K4me2 in various asRNA-treated 1G5 cells. A total of 4 × 10⁶ 1G5 cells were co-transfected with pTatDSRed or one of the various U6 expressed asRNA constructs [pU6 GFPas (control), pU6 LTR-247as, pU6 LTR-247as+7, pU6 LTR-247as+13 and pU6 LTR-247as-7]. Twenty-four hours later the cultures were collected and ChIP analysis performed specifically for H3K4me2 as described (18). The relative enrichment following ChIP was determined by qPCR (SYBR green, BioRad™) with LTR-specific oligonucleotides (18). Data represent four independent experiments standardized to no antibody controls with the standard deviation shown.

Figure 4.

Characterization of LTR-247as+7 asRNA mutants. A single mutation in LTR-247as+7 at nucleotide 7 abrogates off-target gene activation in 1G5 cells. (A) Cultures were co-transfected with the various U6 expressed LTR-247as+7 RNAs containing different point mutations, along with HIV-1 Tat. luciferase/GAPDH expression was measured by qRT PCR 24 h later. (B) Triplicate transfected 1G5 cultures are shown with the respective ranges. A Student's t-test was performed using the web-based tool http://home.clara.net/sisa/t-test.htm and statistical significance was determined for P < 0.05.

Figure 5.

Microarray analysis of 23 deregulated genes. (A) A heatmap was produced depicting the variation in gene expression in triplicate treated LTR-247as+7 relative to the corresponding expression profile for the control GFPas treated 1G5 cells. (B) The microarray analysis was utilized to validate the increase in expression from 3 of the 23 candidate genes by real-time RT PCR analysis. Results represent the mean ± standard deviations of three independent transfection experiments.

Figure 6.

The effect of C10orf76 on 247as+7-mediated gene activation. (A) Suppression of C10orf76 RNA by siRNAs. 293-CCR5-GFP cells were transfected with C10orf76-specific siRNAs (50 nM) in triplicate and the standard deviations are shown. (B) Suppression of C10orf76 is inversely proportional to gene activation of GFP, NSBP1 and NFYB. 293-R5-GFP cells. Results represent the mean ± standard deviations of three independent transfection experiments. (C) The complete LTR-247as+7 target site and putative LTR-247as+7 target sites within C10orf76 were inserted downstream of a Renilla luciferase 3′ UTR within the psiCheck vector. (D) Knockdown efficiency of LTR-247as+7 was determined against each of the C10orf76 targets. Values are normalized to the ratio of Renilla to firefly luciferase activity in the Mock transfected cells and are presented as the mean ± SEM of three independent experiments. A single-tailed Student's t-test was used to determine statistical significance (where shown, for P < 0.05). (E) The LTR-247as+7 siRNA is capable of reverse-priming C10orf76 mRNA. Cellular RNA from transfected HEK293T cells was converted to cDNA using siRNAs as the template primer for cDNA conversion (44). The siRNAs targeted to C10orf76 (C10-siRNA-1), LTR-247as+7 (247as+7) and targeted to Akt-19 (AKT-19) as well as control RNA in the absence of RT (RNA-RT) or RNA in the presence of RT (RNA+RT) were converted to cDNA and PCR was performed using C10orf76-specific oligos for an expected band ∼136 bp.