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. 2013 Feb 15;27(4):400-12.
doi: 10.1101/gad.209767.112. Epub 2013 Feb 7.

Multiple roles for Piwi in silencing Drosophila transposons

Nikolay V Rozhkov  1 , Molly Hammell, Gregory J Hannon
Affiliations

Multiple roles for Piwi in silencing Drosophila transposons

Nikolay V Rozhkov et al. Genes Dev. .

Abstract

Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins--Piwi, Aubergine (Aub), and Ago3--acting in concert with their small RNA guides, the Piwi-interacting RNAs (piRNAs). Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell type-specific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis.

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Figures

Figure 1.
Figure 1.
Integrated analysis of nascent RNA synthesis and steady-state RNA levels upon piwi knockdown in the soma. (A–C) Comparisons are shown of RPM mapping to each transposon in the control knockdown (Y-axis) versus the Tj-driven piwi knockdown (X-axis). Red dots indicate germline-biased element, green dots indicate soma-biased elements, yellow dots indicate intermediate elements targeted in both compartments, and black dots indicate elements with “on” designation. Analysis of the total library is displayed in A, with specific analysis of reads mapping to sense and antisense strands shown in B and C, respectively. (D–F) Data are presented for steady-state RNA levels measured by RNA-seq and organized exactly as described for A–C. (G–I) Comparisons of the change in steady-state RNA levels (Y-axis) as compared with changes in nascent RNA levels (X-axis) for total reads (G), reads antisense to transposons (H), and reads sense to transposons (I). Elements falling along the diagonal show correlated changes; those left of the diagonal are dominated by effects on steady state, presumably representing changes in RNA stability, and those right of the diagonal are dominated by transcriptional effects. Selected transposons are indicated.
Figure 2.
Figure 2.
Effects of somatic piwi knockdown on piRNAs. (A) A heat map represents fold changes (key below) in presumed piRNAs (23–29 nt in length) that are sense or antisense to transposons. Elements are ranked according to the fold change (decrease) in antisense piRNAs. Color coding for names represents the cell type bias of the element, with the colors as defined in Figure 1. (B, top) As an example, a soma-biased transposon, gtwin, is shown. piRNA density in library from the control animals is displayed along a line representing the extent of the transposon consensus. Below is shown piRNA density observed in libraries from the knockdown animals. To the right, small RNAs are shown according to their size distributions, divided into sense (above the axis) and antisense (below the axis) species. Control and piwi knockdown libraries are as indicated. (C) An analysis of the ZAM transposon, similar to what is described in B, is shown. Here, both piRNA (23–29 nt, top) and siRNA (20–21 nt, bottom) fractions are plotted along the transposon consensus.
Figure 3.
Figure 3.
Integrated analysis of nascent RNA synthesis and steady-state RNA levels upon piwi knockdown in the germline. (A–C) Comparisons are shown of RPM mapping to each transposon in the control knockdown (Y-axis) versus the nos-driven piwi knockdown (X-axis). Coloration of the dots and organization of the panels are as described in Figure 1. (D–F) Data are presented for steady-state RNA levels measured by RNA-seq and organized exactly as described for A–C. (G–I) Comparisons of the change in steady-state RNA levels (Y-axis) as compared with changes in nascent RNA levels (X-axis) as described in Figure 1. Selected transposons are indicated.
Figure 4.
Figure 4.
Impact of germline piwi knockdown on piRNAs mapping to transposons. (A) A bar graph represents the density of piRNA (RPM, strand as indicated) mapping to the 70 most highly targeted elements. These are sorted according to their cell type bias, with the coloration of the names as described in Figure 1. (Red) Germline-biased; (yellow) intermediate; (green) soma-biased; (black) undetermined. (B) A heat map represents changes in small RNAs (scale below) in comparisons of germline piwi knockdown versus control animals. To the left, densities of piRNAs along lines representing the extent of the transposon consensus are shown for the control (top) and piwi knockdown libraries (bottom) for selected elements (indicated). Adjacent to that are size distributions for small RNAs corresponding to those elements, with sense species displayed above the axis and antisense displayed below the axis (piwi knockdown and control as indicated). At the extreme right are similar plots of only that subset of piRNAs (23–29 nt) that is most probably bound to Ago3; namely; those sense to elements that lack a 5′ U and have an A at position 10.
Figure 5.
Figure 5.
Effects of germline piwi knockdown on primary piRNA levels. (A) The relative change in piRNAs that can be uniquely mapped to any of the nine most productive piRNAs is shown, comparing levels in libraries derived from nos-driven piwi knockdown with the control animals (scale below). (B) For selected clusters, piRNA densities are displayed along a line representing the extent of the cluster for the control (left) or piwi knockdown (right) libraries. To the right are shown small RNA distributions corresponding to those clusters with species derived from the top strand shown above the axis and species from the bottom strand shown below the axis. Bars corresponding to control or piwi knockdown are as indicated.
Figure 6.
Figure 6.
Impact of piwi knockdown in the germline on H3K9me3 marks at transposons. Normalized densities of the repressive mark H3K9me3 (top plots) are compared with piRNA distributions (bottom plots) in control animals for HeT-A, TART, TAHRE, and jockey. Black and red are H3K9me3 enrichments over input in control (nos-white) and nos-piwi samples, respectively.
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