Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters.

International audienceExpression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a heterochromatin protein 1 (HP1) homolog. Previously, we have shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. We noted that identical transgenes located in different genomic sites vary considerably in piRNA production and classified them as "strong" and "weak" piRNA clusters. Here, we investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. We found that the formation of a transgenic piRNA cluster is accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the "empty" sites before transgene insertion. Finally, we have shown that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an "all-or-nothing" model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production

Subcellular localization and Egl-mediated transport of telomeric retrotransposon HeT-A ribonucleoprotein particles in the Drosophila germline and early embryogenesis.

The study of the telomeric complex in oogenesis and early development is important for understanding the mechanisms which maintain genome integrity. Telomeric transcripts are the key components of the telomeric complex and are essential for regulation of telomere function. We study the biogenesis of transcripts generated by the major Drosophila telomere repeat HeT-A in oogenesis and early development with disrupted telomeric repeat silencing. In wild type ovaries, HeT-A expression is downregulated by the Piwi-interacting RNAs (piRNAs). By repressing piRNA pathway, we show that overexpressed HeT-A transcripts interact with their product, RNA-binding protein Gag-HeT-A, forming ribonucleoprotein particles (RNPs) during oogenesis and early embryonic development. Moreover, during early stages of oogenesis, in the nuclei of dividing cystoblasts, HeT-A RNP form spherical structures, which supposedly represent the retrotransposition complexes participating in telomere elongation. During the later stages of oogenesis, abundant HeT-A RNP are detected in the cytoplasm and nuclei of the nurse cells, as well as in the cytoplasm of the oocyte. Further on, we demonstrate that HeT-A products co-localize with the transporter protein Egalitarian (Egl) both in wild type ovaries and upon piRNA loss. This finding suggests a role of Egl in the transportation of the HeT-A RNP to the oocyte using a dynein motor. Following germline piRNA depletion, abundant maternal HeT-A RNP interacts with Egl resulting in ectopic accumulation of Egl close to the centrosomes during the syncytial stage of embryogenesis. Given the essential role of Egl in the proper localization of numerous patterning mRNAs, we suggest that its abnormal localization likely leads to impaired embryonic axis specification typical for piRNA pathway mutants

Key role of piRNAs in telomeric chromatin maintenance and telomere nuclear positioning in Drosophila germline

Abstract Background Telomeric small RNAs related to PIWI-interacting RNAs (piRNAs) have been described in various eukaryotes; however, their role in germline-specific telomere function remains poorly understood. Using a Drosophila model, we performed an in-depth study of the biogenesis of telomeric piRNAs and their function in telomere homeostasis in the germline. Results To fully characterize telomeric piRNA clusters, we integrated the data obtained from analysis of endogenous telomeric repeats, as well as transgenes inserted into different telomeric and subtelomeric regions. The small RNA-seq data from strains carrying telomeric transgenes demonstrated that all transgenes belong to a class of dual-strand piRNA clusters; however, their capacity to produce piRNAs varies significantly. Rhino, a paralog of heterochromatic protein 1 (HP1) expressed exclusively in the germline, is associated with all telomeric transgenes, but its enrichment correlates with the abundance of transgenic piRNAs. It is likely that this heterogeneity is determined by the sequence peculiarities of telomeric retrotransposons. In contrast to the heterochromatic non-telomeric germline piRNA clusters, piRNA loss leads to a dramatic decrease in HP1, Rhino, and trimethylated histone H3 lysine 9 in telomeric regions. Therefore, the presence of piRNAs is required for the maintenance of telomere chromatin in the germline. Moreover, piRNA loss causes telomere translocation from the nuclear periphery toward the nuclear interior but does not affect telomere end capping. Analysis of the telomere-associated sequences (TASs) chromatin revealed strong tissue specificity. In the germline, TASs are enriched with HP1 and Rhino, in contrast to somatic tissues, where they are repressed by Polycomb group proteins. Conclusions piRNAs play an essential role in the assembly of telomeric chromatin, as well as in nuclear telomere positioning in the germline. Telomeric arrays and TASs belong to a unique type of Rhino-dependent piRNA clusters with transcripts that serve simultaneously as piRNA precursors and as their only targets. Telomeric chromatin is highly sensitive to piRNA loss, implying the existence of a novel developmental checkpoint that depends on telomere integrity in the germline

Natural variation of piRNA expression affects immunity to transposable elements

<div><p>In the <i>Drosophila</i> germline, transposable elements (TEs) are silenced by PIWI-interacting RNA (piRNA) that originate from distinct genomic regions termed piRNA clusters and are processed by PIWI-subfamily Argonaute proteins. Here, we explore the variation in the ability to restrain an alien TE in different <i>Drosophila</i> strains. The <i>I</i>-element is a retrotransposon involved in the phenomenon of I-R hybrid dysgenesis in <i>Drosophila melanogaster</i>. Genomes of R strains do not contain active <i>I</i>-elements, but harbour remnants of ancestral <i>I</i>-related elements. The permissivity to <i>I</i>-element activity of R females, called reactivity, varies considerably in natural R populations, indicating the existence of a strong natural polymorphism in defense systems targeting transposons. To reveal the nature of such polymorphisms, we compared ovarian small RNAs between R strains with low and high reactivity and show that reactivity negatively correlates with the ancestral <i>I</i>-element-specific piRNA content. Analysis of piRNA clusters containing remnants of <i>I</i>-elements shows increased expression of the piRNA precursors and enrichment by the Heterochromatin Protein 1 homolog, Rhino, in weak R strains, which is in accordance with stronger piRNA expression by these regions. To explore the nature of the differences in piRNA production, we focused on two R strains, weak and strong, and showed that the efficiency of maternal inheritance of piRNAs as well as the <i>I</i>-element copy number are very similar in both strains. At the same time, germline and somatic uni-strand piRNA clusters generate more piRNAs in strains with low reactivity, suggesting the relationship between the efficiency of primary piRNA production and variable response to TE invasions. The strength of adaptive genome defense is likely driven by naturally occurring polymorphisms in the rapidly evolving piRNA pathway proteins. We hypothesize that hyper-efficient piRNA production is contributing to elimination of a telomeric retrotransposon <i>HeT-A</i>, which we have observed in one particular transposon-resistant R strain.</p></div

Characteristics of telomeres in the <i>Paris</i> strain.

<p>(A) The number of <i>HeT-A</i>-specific small RNA is dramatically reduced in strain <i>Paris</i>. The density of small RNAs along the <i>HeT-A</i> canonical sequence is shown. Length distribution of <i>HeT-A</i> small RNAs is plotted on the right. Percentages of reads having 1U bias are indicated for each strand (only 24–29 nt reads were considered). Most of the <i>HeT-A</i>-specific small RNAs are 24–29 nt in size and show the characteristic 1U bias of piRNA species. (B) Northern analysis of the RNA isolated from the ovaries of <i>iso-1</i>, <i>Misy</i>, <i>Paris</i>, RAL-852 and <i>GIII</i> strains. Hybridization was done with the <i>HeT-A</i> antisense riboprobe. Lower panel represents hybridization to the <i>mir-13b1</i> microRNA. (C) Analysis of genomic DNA libraries. Telomeric retrotransposon <i>HeT-A</i> genomic sequence coverage in <i>Paris</i>, <i>Misy</i> and <i>w</i>^<i>K</i> strains. (D) The number of <i>HeT-A</i> genomic reads is dramatically reduced in <i>Paris</i>. Normalized number of genomic reads (RPM) mapping to the canonical telomeric retrotransposons in R strains. Analysis of mate-paired (made in 2013) and paired-end (made in 2016) <i>Paris</i> and <i>Misy</i> genomic DNA libraries. Paired-end genomic sequences of <i>w</i>^<i>K</i> were described previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006731#pgen.1006731.ref005" target="_blank">5</a>]. P-values: * <1e-5, ** <1e-10, *** <1e-15, two-sided Wilcoxon rank sum test).</p

A model explaining the mechanism of higher resistance against the <i>I</i>-element in the R strain <i>Paris</i> compared to another R strain, <i>Misy</i>, in dysgenic crosses.

<p>In <i>Paris</i>, stronger primary processing of piRNA cluster transcripts in ovarian somatic cells and in female germline generates a bigger pool of primary piRNAs, including <i>I</i>-element specific piRNAs, compared to <i>Misy</i>. For most active TEs this difference is masked by abundant secondary piRNAs, which arise as a result of the ping-pong amplification loop. For the <i>I</i>-element, however, additional primary piRNAs in <i>Paris</i> are capable of inducing more efficient suppression of <i>I</i>-element in dysgenic crosses. The occurrence of ping-pong between piRNA cluster transcripts and the existence of secondary piRNAs, specific to non-active transposons including the <i>I</i>-element [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006731#pgen.1006731.ref016" target="_blank">16</a>], is omitted from the scheme for the sake of simplicity.</p

Abundance of <i>I</i>-element-specific piRNA correlates with the reactivity of R strains.

<p>(A) Reactivity of R strains measured as a percent of non-hatched embryos. Standard deviation (SD) for three replicates is shown; n designates the number of embryos analyzed. (B) Different manifestation of hybrid dysgenesis for <i>Misy</i> (strong) and <i>Paris</i> (weak) R strain. spnE/spnE is a transheterozygous <i>spindle-E</i> mutants (<i>spn-E</i>^<i>1</i><i>/spn-E</i>^{<i>hls3987</i>}<i>)</i>. RNA <i>in situ</i> hybridization of ovaries with <i>I</i>-antisense riboprobe is shown. (C) Weak R strain produces more abundant <i>I</i>-element specific piRNA. Northern analysis of the RNA isolated from ovaries of <i>Paris</i> and <i>Misy</i> strains. Hybridization was done with <i>I</i>-element riboprobes to detect sense (I sense) and antisense (I antisense) piRNAs. Lower panel represents hybridization to <i>mir-13b1</i> microRNA. P³²-labeled RNA oligonucleotides were used as size markers. (D) <i>I</i>-element specific small RNA mapping to canonical <i>I</i>-element. Analysis of ovarian small RNA libraries from strong (<i>Misy</i> and <i>w</i>^<i>K</i>) and weak (<i>Paris</i>, <i>Zola</i>, <i>cn bw;e</i>) R strains (0–3 mismatches allowed). Reads mapped to the sense strand are shown in blue, and antisense in brown. Length distribution of small RNAs mapping to <i>I</i>-element is plotted on the right. Percentages of reads having 1U bias are indicated for each strand (only 24–29 nt reads were considered) (E) Negative correlation between reactivity and <i>I</i>-element specific piRNA counts for R strains. Spearman correlation tests: r = -0.90, P-value <0.1. The line depicts the results of linear regression analysis for the level of reactivity and amount of piRNAs. R²—adjusted squared R (P-value < 0.1); the grey zone illustrates the 90% confidence interval.</p