Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons

Abstract

In the Drosophila germline, retrotransposons are silenced by the PIWI-interacting RNA (piRNA) pathway. Telomeric retroelements HeT-A, TART and TAHRE, which are involved in telomere maintenance in Drosophila, are also the targets of piRNA-mediated silencing. We have demonstrated that expression of reporter genes driven by the HeT-A promoter is under the control of the piRNA silencing pathway independent of the transgene location. In order to test directly whether piRNAs affect the transcriptional state of retrotransposons we performed a nuclear run-on (NRO) assay and revealed increased density of the active RNA polymerase complexes at the sequences of endogenous HeT-A and TART telomeric retroelements as well as HeT-A-containing constructs in the ovaries of spn-E mutants and in flies with piwi knockdown. This strongly correlates with enrichment of two histone H3 modifications (dimethylation of lysine 79 and dimethylation of lysine 4), which mark transcriptionally active chromatin, on the same sequences in the piRNA pathway mutants. spn-E mutation and piwi knockdown results in transcriptional activation of some other non-telomeric retrotransposons in the ovaries, such as I-element and HMS Beagle. Therefore piRNA-mediated transcriptional mode of silencing is involved in the control of retrotransposon expression in the Drosophila germline.

Multiple roles for Piwi in silencing Drosophila transposons

“…Patterns of HP1 association with the promoter regions of some transposons were altered in knockdown cells, with coincident changes in the presence of H3K9me3 marks. This joins another recent study implicating transcriptional gene silencing (TGS) in the regulation of telomeric transposons (Shpiz et al 2011). spn-E mutation or germline-specific piwi knockdown resulted in transcription activation of HeT-A and TART.…”

“…However, these have been based on genetic mutants, which lose Piwi function in both the germline and somatic compartments with consequent severe effects on ovary morphology. Only a few studies have addressed this problem with selective loss of Piwi in clonal cell populations within the ovary (Rangan et al 2011) or using cell type-specific knockdown (Shpiz et al 2011;Wang and Elgin 2011). We sought to use the latter approach as a basis for our studies of the mechanisms by which Piwi contributes to transposon silencing.…”

“…The overall procedure was as described (Core et al 2008) with modifications as described (Shpiz et al 2011;Core et al 2012). Briefly, ;100 freshly dissected ovaries were homogenized on ice in 350 mL of HB-0.35 buffer (15 mM HEPES at pH 7.5, 10 mM KCl, 2.5 mM MgCl 2 , 0.1 mM EDTA, 0.5 mM EGTA, 0.05% NP40, 0.35 M sucrose).…”

Control of Telomere Length in Drosophila

“…No detailed information about the cap structure of the germ cell chromosomes has been obtained. HP1 has been found at the ends of normal chromosomes as well as of terminally deleted chromosomes in the nuclei of nurse cells in Drosophila ovaries by immunohistochemistry (Shpiz et al, 2011) . Chromatin immunoprecipitation (ChIP) analysis revealed binding of both HP1 and HOAP with HeT-A in ovaries (Khurana et al, 2010).…”

“…The abundance of telomeric retroelement transcripts, both sense and antisense, as well as the frequency of their transpositions onto chromosome ends, are controlled by a piRNA-mediated mechanism (Savitsky et al, 2006;Shpiz et al, 2007). piRNAs induce transcriptional silencing of the telomeric retrotransposons (Shpiz et al, 2011), suggesting a putative role for the piRNA pathway in the formation of the telomeric chromatin that protects chromosome ends from fusion and is involved in meiotic and mitotic telomere behavior. Fig.…”