piRNAs and epigenetic conversion in Drosophila

Abstract

Transposable element (TE) activity is repressed in the Drosophila germline by Piwi-Interacting RNAs (piRNAs), a class of small non-coding RNAs. These piRNAs are produced by discrete genomic loci containing TE fragments. In a recent publication, we tested for the existence of a strict epigenetic induction of piRNA production capacity by a locus in the D. melanogaster genome. We used 2 lines carrying a transgenic 7-copy tandem cluster (P-lacZ-white) at the same genomic site. This cluster generates in both lines a local heterochromatic sector. One line (T-1) produces high levels of ovarian piRNAs homologous to the P-lacZ-white transgenes and shows a strong capacity to repress homologous sequences in trans, whereas the other line (BX2) is devoid of both of these capacities. The properties of these 2 lines are perfectly stable over generations. We have shown that the maternal transmission of a cytoplasm carrying piRNAs from the first line can confer to the inert transgenic locus of the second, a totally de novo capacity to produce high levels of piRNAs as well as the ability to induce homology-dependent silencing in trans. These new properties are stably inherited over generations (n>50). Furthermore, the converted locus has itself become able to convert an inert transgenic locus via cytoplasmic maternal inheritance. This results in a stable epigenetic conversion process, which can be performed recurrently--a phenomenon termed paramutation and discovered in Maize 60 y ago. Paramutation in Drosophila corresponds to the first stable paramutation in animals and provides a model system to investigate the epigenetically induced emergence of a piRNA-producing locus, a crucial step in epigenome shaping. In this Extra View, we discuss some additional functional aspects and the possible molecular mechanism of this piRNA-linked paramutation.

Keywords: cellular memory; epigenetics; heterochromatin; piRNAs; transposable elements.

Figure 1. Paramutation linked to maternal transmission of piRNAs. (A, C, E, G, and I) The trans-silencing assay measures the capacity of a tested P-lacW transgene cluster to repress a homologous P-lacZ transgene target in the female germline. Females carrying the cluster were crossed with males carrying the P-1039 transgene (not indicated in the mating scheme), which is expressed in both nurse cells and follicle cells. Female progeny of this cross were stained for lacZ activity. Trans-silencing is germline specific and occurs only in the nurse cells and the oocytes, but not in the somatic follicle cells surrounding the egg chamber. The percentage of repressed egg chambers is given as TSE (Trans-Silencing Effect) percentage. In each case, more than 1500 egg chambers were observed. (B, D, F, H, and J) Deep sequencing of small RNAs (19–30nt) from ovaries: histograms show the length-distribution of small RNAs matching the P-lacW sequence. Positive and negative values correspond to sense and antisense reads, respectively. The T-1 and BX2 lines carry a 7-copy P-lacZ transgene cluster, generating a heterochromatin island in the middle of the 2R chromosomal arm (50C). The transgenic locus of the T-1 line does not show detectable lacZ staining in the germline (not shown) and can silence in trans the expression of a homologous transgene in the germline tissue (A). Moreover, T-1 produces abundant ovarian piRNAs homologous to the transgenes (B). The transgenic locus of the BX2 line does neither show lacZ staining in the germline (not shown), nor induce trans-silencing (C), nor produce abundant transgene matching piRNAs (D). These properties are stable over generations. G₂ females, which inherit both a cytoplasm deriving from T-1 grandmothers (but not the T-1 cluster) and a BX2 cluster can induce complete trans-silencing in the germline (E) and produce abundant P-lacW homologous piRNAs (F). These properties are stably maintained over generations in derived lines (G and H), called BX2*/CyRoi. * symbolizes the epigenetic conversion process and the green background the presence of maternally inherited P-lacW homologous small RNAs. The T-1 cluster thus has paramutagenic properties and the BX2 cluster is paramutable. Maternal transmission of the cytoplasm from paramutated BX2*/CyRoi females can again induce a conversion process similar to that induced by the T-1cytoplasm, generating thus a second order paramutation (symbolized by ^*2). BX2^*females induce trans-silencing (I) and produce abundant ovarian piRNAs (J). We performed the same experimental scheme starting with the reciprocal G₀ cross (Females BX2 x Males T-1). Progeny carrying the BX2 locus never showed a repression capacity at any generation (data not shown).

Figure 2. Assay for self-maintaining cytoplasmic components. We generated G₁ females having inherited the cytoplasm of a T-1 mother but no transgene cluster from any parent. The capacity of these G₁ females to induce paramutation was tested by crossing them with BX2 males and analyzing silencing capacities of G₂ females carrying the BX2 cluster. When tested using the TSE assay (cross with P-1039 males), these G₂ females conferred no silencing capacity to daughter ovaries (A). Therefore, G₁ females, which maternally inherit piRNAs but do not carry a homologous locus allowing zygotic piRNA biogenesis, cannot induce epigenetic conversion of their progeny. TSE was tested as in Figure 1. M lines are devoid of any P-transgene. Negative and positive TSE controls were performed by crossing w¹¹¹⁸ (M line) and T-1 females with P-1039 males, respectively, and staining daughter ovaries.

Figure 3. Models for piRNA induced paramutation. Maternal deposition of piRNAs in the embryo activates abundant piRNA production by the BX2 locus. A first model could be that maternal piRNAs associated to Piwi enter into the nuclei of primordial germ cells (PGC) and interact with BX2 nascent transcripts. This targeting could modify the chromatin state of the locus (HP1 to Rhino switch), leading to increased production of primary piRNAs and, consequently, of secondary piRNAs in the nuage. Alternatively, maternally transmitted piRNAs associated with Aub or Ago3 could directly activate ping-pong amplification of piRNAs in the nuage. AGO3 maternal transmission is shown with a dashed arrow since some observations suggest that it is less efficient than that of Piwi or Aub.