De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment

Abstract

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Paramutation inDrosophilaRequires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and InducesCis-spreading of piRNA Production

“…A spreading process for piRNA production was also observed for the flamenco piRNA-producing locus, which functions in the somatic follicle cells that surround germline cysts (Muerdter et al 2011). Further, analysis of previously established transgenic lines that carry an I-fragment within a P-transgene have shown that piRNA production capacity can spread within transgenes (Olovnikov et al 2013). The capacity to produce piRNAs invaded the whole I-derived transgene and reached in some cases flanking sequences.…”

“…Figure 3C shows that, as early as generation 3, the three types of G 0 maternal inheritance resulted in strong production of small RNAs covering the entire BX2 P{lacW} transgene length. Thus, partially homologous paramutations are associated with rapid and stable spreading of piRNA production by nonhomologous sequences of the targets, similarly to what was found for target transgenes (Muerdter et al 2011;Olovnikov et al 2013;Han et al 2015;Mohn et al 2015) or transposable elements (Shpiz et al 2014).Further, the paramutagenicity of P-1152 BX2*, RS3 BX2*, and P-1152+RS3 BX2* females was tested. Figure 4 shows that maternal transmission of cytoplasm produced by these females, combined to paternal transmission of a BX2 naive locus, resulted in female progeny showing strong trans-silencing properties.…”

“…Further, these G 1 females were crossed with Dcr-2 +/+ males and increased ovarian P{lacW} siRNA levels were found in BX2* G 2 females having recovered a Dcr-2 +/+ genotype. Therefore, production of ovarian P{lacW} siRNAs by females having a functional siRNA pathway does not require homologous siRNA inheritance and can be induced by inheritance of solely homologous piRNAs.Paramutation of a locus only partially covered by maternally inherited piRNAs can be stable and is accompanied by cis-spreading of piRNA production capacityTargeting of transgenes or natural transposable elements by piRNAs can lead to spreading of piRNA production to sequences adjacent to the targeted elements (Muerdter et al 2011;Olovnikov et al 2013;Shpiz et al 2014). We tested if spreading could be observed for a silencer locus induced by paramutation.…”

“…Targeting of transgenes or natural transposable elements by piRNAs can lead to spreading of piRNA production to sequences adjacent to the targeted elements (Muerdter et al 2011;Olovnikov et al 2013;Shpiz et al 2014). We tested if spreading could be observed for a silencer locus induced by paramutation.…”

Reexamining the P-Element Invasion of Drosophila melanogaster Through the Lens of piRNA Silencing

“…For a genomic region of interest, the presence of homologous piRNAs in the ooplasm is associated with the deposition of H3K9me3 and the onset of piRNA production (Olovnikov et al 2013;Le Thomas et al 2014a,b;Shpiz et al 2014). Experiments in which tandemly-duplicated arrays of transgenes were epimutated into piRNA clusters suggest that higher-copy arrays are both more likely to undergo epimutation, and more likely to stably maintain piRNA production over successive generations after they have been epimutated, rather than spontaneously reverting to the quiescent state (de Vanssay et al 2012).…”