The capacity of target silencing by Drosophila PIWI and piRNAs

Abstract

Although Piwi proteins and Piwi-interacting RNAs (piRNAs) genetically repress transposable elements (TEs), it is unclear how the highly diverse piRNA populations direct Piwi proteins to silence TE targets without silencing the entire transcriptome. To determine the capacity of piRNA-mediated silencing, we introduced reporter genes into Drosophila OSS cells, which express microRNAs (miRNAs) and piRNAs, and compared the Piwi pathway to the Argonaute pathway in gene regulation. Reporter constructs containing several target sites that were robustly silenced by miRNAs were not silenced to the same degrees by piRNAs. However, another set of reporters we designed to enable a large number of both TE-directed and genic piRNAs to bind were robustly silenced by the PIWI/piRNA complex in OSS cells. These reporters show that a bulk of piRNAs are required to pair to the reporter's transcripts and not the reporter's DNA sequence to engage PIWI-mediated silencing. Following our genome-wide study of PIWI-regulated targets in OSS cells, we assessed candidate gene elements with our reporter platform. These results suggest TE sequences are the most direct of PIWI regulatory targets while coding genes are less directly affected by PIWI targeting. Finally, our study suggests that the PIWI transcriptional silencing mechanism triggers robust chromatin changes on targets with sufficient piRNA binding, and preferentially regulates TE transcripts because protein-coding transcripts lack a threshold of targeting by piRNA populations. This reporter platform will facilitate future dissections of the PIWI-targeting mechanism.

Keywords: Piwi; gene silencing; piRNAs.

FIGURE 1.

AGO1/miRNA and PIWI/piRNA complexes act differently on gene silencing reporter constructs. (A) Diagram of the reporter construct design, (RBS) RNA binding site, repeated in four tandem copies (4×). (B) Relative abundance of the small RNAs that were determined from early cultures of OSS cells used in these assays (Lau et al. 2009b). Relative abundance from deep-sequencing was calculated from reads per million, while similar relative abundance levels were confirmed by quantitative Northern blots (below graphs, standards are 10, 50, 100, 500, and 1000 pg). Only two miRNA and piRNA Northern blots are shown as representatives. (C) Reporter gene expression levels normalized to the respective mutated site reporter. Error bars are the standard error of biological triplicates. Asterisk indicates statistical significance relative to the mutated sites control, at P < 0.05, Wilcoxon rank-sum test. (D) The tethered construct reporter system (Pillai et al. 2004; Djuranovic et al. 2010). (E) This reporter design is sufficient to induce AGO1-mediated repression, but in a cell line with an endogenous Piwi pathway, it is unable to trigger silencing by tethered PIWI (which behaves similar to the LacZ negative control). The asterisk marks statistically significant λN-HA-AGO-mediated repression (P < 0.05, Wilcoxon rank-sum test). (F) The upper Western blot shows each of the λN-HA- and HA-fusion transgene constructs were similarly expressed. The lower Western blot shows the λN-HA-PIWI is nuclear localized like endogenous PIWI in OSS cells. Sub-cellular fractions from OSS cells transfected plasmid expressing λN-HA-PIWI, probed with anti-HA antibody.

FIGURE 2.

Reporter gene silencing requires a bulk of PIWI/piRNA complexes. (A) Design of the piRNA reporter assay in OSS cells. (B) A representative assay result from C of a piRNA reporter showing strong silencing of the “Antisense” reporter compared with the “Sense” reporter. Although it appears there is less expression from the Antisense reporter which could be targeted by piRNAs, this Antisense reporter cannot be meaningfully compared with the different sequence composition of the Sense reporter. The siPIWI/siGFP normalization is critical because it shows only the Antisense reporter is derepressed upon PIWI knockdown. (C) (Left) Reporter genes bearing segments of flamenco elements inserted into the 3′ UTR of luciferase. (Middle) Graph of the amounts of endogenous piRNAs in OSS cells that target the Antisense reporters. Sense reporters have the same sequence as these piRNAs. Relative numbers of piRNAs were determined from early cultures of OSS cells used in these assays, based on reads per million (RPM) (Lau et al. 2009b). (Right) Graph of normalized ratios of luciferase expression between PIWI knockdown with siPIWI and the control siGFP siRNA. (D) Graphics and graphs analogous to C showing the effect on reporters bearing a segment of tj 3′ UTR. Reporter assay values depict ratio of each reporter transgene tested by transfecting OSS cells with PIWI knockdown (siPIWI) and normalized over control OSS cell (siGFP). Error bars represent standard error of mean of biological triplicates. Asterisks indicate statistical significance in the difference between the Antisense reporters compared with the Sense reporters (P < 0.05, Wilcoxon rank-sum test).

FIGURE 3.

PIWI/piRNA complexes robustly mediate TGS on transgenes. (A) Reporter constructs with piRNA-binding sequences placed into the intron within the 5′ UTR of the Renilla luciferase gene. (B) RT-PCR analysis of the reporter mRNAs from transfected OSS cells. Specificity of spliced transcripts was confirmed next to negative amplification controls and the plasmid DNA which contained the intron and piRNA-binding element sequence. (C) Reporter assay values depict ratio of each reporter tested by transfecting OSS cells with PIWI knockdown (siPIWI) and normalized over control OSS cell (siGFP). Error bars represent standard error of mean of biological triplicates. Asterisks indicate statistical significance in the difference between the Antisense reporters compared with the Sense reporters (P < 0.05, Wilcoxon rank-sum test).

FIGURE 4.

PIWI-mediated TGS operates through chromatin changes. (A) Chromatin immunoprecipitation (ChIP) assays measuring RNA Polymerase II (RNA Pol II) and Histone H3 lysine 9 tri-methylation (H3K9me3) enrichment at OSS cell genomic loci and the flamenco piRNA reporter. Blood is an endogenous TE that is epigenetically silenced, Actin5C is a constitutively expressed housekeeping gene locus, Mec2 is a locus with a de novo 297 TE and under PIWI-mediated silencing in OSS cells (Sytnikova et al. 2014), and the flam element 3′-UTR reporter constructs were transiently transfected plasmids. Error bars represent standard error of mean of biological triplicates. Wilcoxon rank-sum tests for certain comparisons of siPIWI versus siGFP conditions are represented by (***) P < 0.05, (**) P < 0.1, and (*) P < 0.2. The inset for the Blood ChIP is a different scale of the main figure to show the RNA Pol II levels. (B) A model that suggests the dominant mechanism for PIWI directed gene silencing is TGS and this mechanism requires a bulk of piRNAs interacting with the nascent transcript. Most protein-coding transcripts are unaffected by less-frequent PIWI/piRNA interactions.