Trapping a somatic endogenous retrovirus into a germline piRNA cluster immunizes the germline against further invasion

Abstract

Background: For species survival, the germline must faithfully transmit genetic information to the progeny. Transposable elements (TEs) constitute a significant threat to genome stability due to their mobility. In the metazoan germline, their mobilization is limited by a class of small RNAs called PIWI-interacting RNAs (piRNAs) produced by dedicated genomic loci called piRNA clusters. Although the piRNA pathway is an adaptive genomic immunity system, it remains unclear how the germline gains protection from a new transposon invasion.

Results: To address this question, we analyze Drosophila melanogaster lines harboring a deletion within flamenco, a major piRNA cluster specifically expressed in somatic follicular cells. This deletion leads to derepression of the retrotransposon ZAM in the somatic follicular cells and subsequent germline genome invasion. In this mutant line, we identify de novo production of sense and antisense ZAM-derived piRNAs that display a germinal molecular signature. These piRNAs originated from a new ZAM insertion into a germline dual-strand piRNA cluster and silence ZAM expression specifically in germ cells. Finally, we find that ZAM trapping in a germinal piRNA cluster is a frequent event that occurs early during the isolation of the mutant line.

Conclusions: Transposons can hijack the host developmental process to propagate whenever their silencing is lost. Here, we show that the germline can protect itself by trapping invading somatic-specific TEs into germline piRNA clusters. This is the first demonstration of "auto-immunization" of a germline endangered by mobilization of a surrounding somatic TE.

Keywords: Drosophila; Genome stability; Germline; Inheritance; Transposable elements; piRNA cluster; piRNAs.

Fig. 1

The ZAM sensor transgene is not repressed in the germline of D. melanogaster ovaries. a Structure of the pGFP-ZAM sensor transgene. The UASp promoter contains the Gal4 target sequence upstream of the GFP reporter gene fused to 467 bp of the ZAM env gene (light grey box, sense orientation). The ZAM sequence is flanked by two FRT sites. The arrow indicates the transcription initiation site. b Confocal images of ovarioles after GFP (green, left) and DNA (blue, middle) staining. Ovarioles were from the progeny of a cross between w¹¹¹⁸ females and males harboring the pGFP-ZAM transgene driven by the actin-Gal4 driver. Merged images for GFP and DNA labeling are displayed on the right

Fig. 2

Deletion of some TE fragments in flamenco does not impair the global piRNA production from this piRNA cluster. a Pie chart showing the proportion of unique piRNAs that map to each of the 142 piRNA clusters in ovarian somatic sheath cells (no mismatch allowed, piRNA clusters defined as in [9]). b Density profile of unique piRNAs from the w^IR6 (left) and RevI-H2 (right) lines that map to the flamenco piRNA cluster. Sense and antisense reads are presented in black and grey, respectively. Almost no antisense reads map to the flamenco piRNA cluster. ZAM location in flamenco is displayed by a purple box. The flamenco deletion distal break-point in RevI-H2 [26] (Additional file 1: Figure S1B) is indicated by a red arrow and the sense of transcription by a black arrow. The count of piRNA reads per million (RPM) mapping the non-deleted region of flamenco, indicated below, does not differ between w^IR6 and RevI-H2

Fig. 3

De novo production of functional ZAM-derived piRNAs in the germline of the RevI-H2 line. a Pie chart summarizing the proportion of ZAM-derived piRNAs (allowing up to 3 mismatches) that map to the 142 piRNA clusters in w^IR6 (no mismatch allowed, piRNA clusters defined as in [9]). b Density profile of ZAM-derived piRNAs along the 8.4 kb ZAM sequence in w^IR6 ovaries (allowing up to 3 mismatches). Sense and antisense reads are represented in black and grey, respectively. ZAM organization is displayed above the profile. LTR, long terminal repeats. c Logo of nucleotide bias for the first ten positions in ZAM-derived piRNAs produced in w^IR6 ovaries. The nucleotide height represents its relative frequency at that position. d Density profile of ZAM-derived piRNAs along the ZAM sequence produced in RevI-H2 ovaries (allowing up to 3 mismatches). Sense and antisense reads are represented in black and grey, respectively. e Bar diagram showing the total amount of ZAM-derived piRNAs produced in w^IR6 and RevI-H2 ovaries, quantified from the profiles shown in b and c, respectively. f Logo of nucleotide bias for the first ten positions of ZAM-derived piRNAs produced in RevI-H2 ovaries. g Histogram showing the percentage of 5′-overlap between sense and antisense ZAM-derived piRNAs (23–29 nt) in w^IR6 (top) and RevI-H2 (bottom) ovaries. The proportion of 10-nt overlapping pairs is in red, and the Z-score is indicated. h Bar diagram indicating the percentage of ZAM-derived piRNAs with ping-pong partners (PPP) in the w^IR6 and RevI-H2 lines. i Analysis of nucleotide bias for sense (+) and antisense (−) ZAM-derived piRNAs with PPP in RevI-H2 ovaries. The percentage of PPP with a uridine at position 1 (1 U) and with an adenosine at position 10 (10A) is shown. j Confocal images of ovarioles after GFP (green, left panels) and DNA (blue, middle panels) staining. Ovarioles were from the progeny of a cross between w^IR6 or RevI-H2 females and males carrying the pGFP-ZAM sensor transgene driven by actin-Gal4. Right panels, merged images of GFP and DNA labeling

Fig. 4

ZAM-derived piRNAs are produced from a pre-existing germline piRNA cluster in RevI-H2 ovaries. a–b Confocal images of ovarioles after GFP (green, left panels) and DNA (blue, middle panels) staining. Merged images of the GFP and DNA signals are displayed on the right. Ovarioles were from the progeny of a cross between RevI-H2 females and control males (ZAM maternal deposition, ZMD) in a and from a cross between RevI-H2 males and control females (No ZAM maternal deposition, NZMD) in b. In both crosses, the pGFP-ZAM line in which ZAM expression is driven in germline cells by a nanos-Gal4 driver was the control line. c Western blotting of proteins extracted from ovaries of progenies of crosses between w^IR6 or RevI-H2 and the same control line as in a and b. The lines used for the crosses are indicated above. Proteins were from two biological replicates (1 and 2) prepared from 5 pairs of ovaries; α-tubulin was used as loading control. d Density profile of piRNAs mapping along the GFP-ZAM transgene sequence (allowing up to 3 mismatches). Sense and antisense reads are in black and grey, respectively. The profiles are for crosses between w^IR6 (left, control) or RevI-H2 (right, ZMD) females and control males harboring the pGFP-ZAM transgene

Fig. 5

Production of Phidippo- and Pifo-derived piRNAs is lost in RevI-H2. a Histogram for the percentage of 5′-overlaps between sense and antisense Adoxo-, Gedeo-, Idefix-, Phidippo-, Pifo-, and Vatovio-derived piRNAs (23–29 nt) in w^IR6 ovaries. The peak in red defines the 10-nt-overlapping pairs, and the Z-score is indicated. b, c Density profile of Phidippo- (b) and Pifo- (c) derived piRNAs along the 7.3 kb Philippo sequence and 7.7 kb Pifo sequence, respectively, in w^IR6 (left) and RevI-H2 (right) ovaries (using all piRNAs mapped to the corresponding TE allowing up to 3 mismatches). Sense and antisense reads are represented in black and grey, respectively. The organization of the two TEs is displayed above their respective profile

Fig. 6

ZAM is trapped in a germline piRNA cluster in all analyzed Rev lines. a, d Density profile of ZAM-derived piRNAs along the 8.4Kb ZAM sequence in the RevII-7 (a) and RevI-H3 (d) lines (allowing up to 3 mismatches). Sense and antisense reads are represented in black and grey, respectively. The organization of ZAM is displayed above the profiles. b, e Logo of nucleotide bias for the first ten positions of ZAM-derived piRNAs produced in RevII-7 (b) and RevI-H3 (e) ovaries. The nucleotide height represents its relative frequency at that position. c, f Histogram showing the percentage of 5′-overlaps between sense and antisense ZAM-derived piRNAs (23–29 nt) in RevII-7 (c) and RevI-H3 (f) ovaries. The peak in red defines the 10-nt-overlapping pairs, and the Z-score is indicated