Maternally deposited germline piRNAs silence the tirant retrotransposon in somatic cells

Abstract

Transposable elements (TEs), whose propagation can result in severe damage to the host genome, are silenced in the animal gonad by Piwi-interacting RNAs (piRNAs). piRNAs produced in the ovaries are deposited in the embryonic germline and initiate TE repression in the germline progeny. Whether the maternally transmitted piRNAs play a role in the silencing of somatic TEs is however unknown. Here we show that maternally transmitted piRNAs from the tirant retrotransposon in Drosophila are required for the somatic silencing of the TE and correlate with an increase in histone H3K9 trimethylation an active tirant copy.

Figure 1

Progenies of crosses between two wild-type strains of D. simulans show different tirant expression. (A) Schematic representation of the NRT (non-regulated tirant) and RT (regulated tirant) crosses. (B) Quantitative real-time PCR of randomly reverse-transcribed total RNA from the ovaries of the parental strains (grey bars), NRT (white bar) and RT (black bar) daughters. tirant transcript levels are expressed relative to an internal rp49 mRNA control (n=3; error bars mean±s.d., two-tailed Student’s t-test, ***P<0.0001). (C) RNA FISH on whole-mount ovaries from NRT (left panel) and RT (right panel) daughters. tirant transcripts are in (red) and DNA is labelled in green (Sytox Green). White arrows indicate tirant transcripts localization. (D) Immunostaining of tirant envelope protein (green) in ovaries from NRT (left panel) and RT (right panel) daughters. DNA is stained in red (propidium iodide). (E) No disruption of sub-cellular organization of the germline transposon silencing machinery in both NRT (upper panel) and RT (lower panel) daughters. Immunostaining of Vasa (green) and Ago3 (red) proteins. DNA is labelled with 4,6-diamidino-2-phenylindole (DAPI; blue).

Figure 2

Differences in tirant Piwi-interacting RNA (piRNA) populations between regulated tirant (RT) and non-regulated tirant (NRT) embryos reflect a difference in maternal piRNA populations. (A) Transposon piRNA ratios between RT and NRT embryos are shown as a heatmap for each of the 85 most highly targeted Drosophila transposable elements (TEs) (up to four mismatches allowed between reads and RepBase sequences). (B) (Left) Pairwise comparison of the TE piRNA levels in NRT and RT embryos. A scatter plot displays correlation between normalized piRNA abundances for each of the 85 major TEs. (Right) Normalized counts of tirant piRNAs in NRT and RT embryos. (C) Both maternally deposited tirant piRNA populations displayed a strong ping-pong signature. (D) Density profile of piRNAs matching tirant sequence (up to four mismatches) split into piRNAs with ping-pong partners (orange) and other piRNAs (blue) (see supplementary information online for ping-pong partner bioinformatic identification). Tirant structure with gag, pol and env open-reading frames is reported (red rectangles represent long terminal repeats).

Figure 3

tirant expression in the somatic gonadal cells. (A) Quantitative real-time PCR of randomly reverse-transcribed total RNA from 12- to 16-h embryos of Makindu and Chicharo parental strains (grey bars) and of regulated tirant (RT; black bar) and non-regulated tirant (NRT; white bar) crosses. tirant transcript levels are expressed relative to an internal rp49 mRNA control (n=3; error bars mean±s.d., two-tailed Student’s t-test, ***P<0.0001). (B,C) RNA FISH on whole-mount 12- to 16-h embryos from Makindu strain (left panel), from RT (middle panel) and NRT (right panel) crosses. tirant transcripts were labelled in red. Anterior is to the left in all panels. DNA is labelled with 4,6-diamidino-2-phenylindole (DAPI; blue). White arrows indicate gonadal cells; go, gonad; ph, pharynx; mt, Malpighian tubules. (C) Higher magnification of gonadal cells of NRT embryos expressing tirant transcripts (scale bar, 22 μm). (D) Double immunostaining on 12- to 16-h embryos using anti-Traffic Jam (TJ) antibody (red) and anti-Vasa antibody (green) labelled the somatic and the germline gonadal cells, respectively. DNA is labelled with DAPI (blue).

Figure 4

tirant repression correlates with the accumulation of both H3K9me3 on its active sequence and PIWI-associated tirant piRNAs in follicle cells. (A) Structure of tirant with gag, pol and env domains, inserted in the tkv gene. The positions of the amplicons obtained in ChIP experiments are indicated by the bottom lines. (B) Chromatin immunoprecipitation assay using chromatin extracts from the ovaries of the offspring of regulated tirant (RT; black bars) and non-regulated tirant (NRT; grey bars) crosses. H3K9me3-relative enrichment was studied in three regions: the 5′- and 3′- flanking regions of the full-length tirant insertion in the tkv gene and in the 18S gene. Enrichment is computed relative to the input (n=3; error bars mean±s.d., two-tailed Student’s t-test, **P<0.001). (C) Quantitative real-time PCR of reverse-transcribed total RNA from ovaries (Ov) and purified follicle cells (FC) of the offspring of RT (black bars) and NRT (grey bars) crosses. piRNA levels and Ago3 mRNA are expressed relative to Traffic jam mRNA (n=3; error bars mean±s.d., two-tailed Student’s t-test, ***P<0.0001).