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. 2011 Dec 27;108(52):21164-9.
doi: 10.1073/pnas.1107892109. Epub 2011 Dec 12.

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Sidney H Wang  1 , Sarah C R Elgin
Affiliations

Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line

Sidney H Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Transposon control is a critical process during reproduction. The PIWI family proteins can play a key role, using a piRNA-mediated slicing mechanism to suppress transposon activity posttranscriptionally. In Drosophila melanogaster, Piwi is predominantly localized in the nucleus and has been implicated in heterochromatin formation. Here, we use female germ-line-specific depletion to study Piwi function. This depletion of Piwi leads to infertility and to axis specification defects in the developing egg chambers; correspondingly, widespread loss of transposon silencing is observed. Germ-line Piwi does not appear to be required for piRNA production. Instead, Piwi requires Aubergine (and presumably secondary piRNA) for proper localization. A subset of transposons that show significant overexpression in germ-line Piwi-depleted ovaries exhibit a corresponding loss of HP1a and H3K9me2. Germ-line HP1a depletion also leads to a loss of transposon silencing, demonstrating the functional requirement for HP1a enrichment at these loci. Considering our results and those of others, we infer that germ-line Piwi functions downstream of piRNA production to promote silencing of some transposons via recruitment of HP1a. Thus, in addition to its better-known function in posttranscriptional silencing, piRNA also appears to function in a targeting mechanism for heterochromatin formation mediated by Piwi.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Germ-line–specific Piwi depletion leads to axis specification defects in developing egg chambers. (A) Quantitative RT-PCR analysis of the piwi expression level in germ-line Piwi knockdown ovaries. Expression levels are given relative to the RPL32 locus. (Bars represent the mean ± SEM) (B) Piwi antibody staining of developing egg chambers. Piwi depletion specifically in the germ line (red arrows) is achieved with either of two independent knockdown constructs without affecting the surrounding somatic follicle cells. (Full genotypes of piwiKD1 and piwiKD2 are given in SI Appendix, Table S5.) (C) The cumulative percentage of dorsal appendage phenotype of embryos produced by germ-line Piwi knockdown females. (N represents the total number of embryos scored for each genotype.) (D) Gurken (green) immunofluorescent staining of stage 9 developing egg chambers. The oocyte nucleus is indicated (asterisk). DAPI staining (blue) marks the nuclei and the actin filament (red) marks the cell boundaries. Gurken localization is diminished in the Piwi knockdown lines.
Fig. 2.
Fig. 2.
Depletion of germ-line Piwi does not disrupt Aub function. (A) Aub immunofluorescent staining of stage 4/5 egg chambers bearing piwi1/+ or piwi1/piwi1 germ line. The peri-nuclear structure nuage (black arrow) and Aub localization are not perturbed in the piwi1/piwi1 germ line. (B) Small RNA Northern blot analysis using three different piRNA probes, HeT-A-2801, AT-chX-1, and Aub-bound roo, along with a microRNA probe, miR-8, as a loading control. (A and B) Genotypes indicated are germ-line genotype at the piwi locus.
Fig. 3.
Fig. 3.
Germ-line Aub knockdown perturbs proper Piwi nuclear localization and leads to overexpression of some transposons. (A) Western blot analysis of Piwi or Aub protein levels in Aub knockdown ovaries shows no significant loss of Piwi. Myosin VI is used as the loading control; the volume of lysate loaded in each lane is indicated beneath. (B) Quantitative RT-PCR analysis of transposon expression levels in germ-line Aub knockdown ovaries. Expression levels are given relative to the RPL32 locus. (Bars represent the mean ± SEM.) (C) Piwi immunofluorescent staining of ovarioles. In the Aub knockdown germ line, Piwi is barely visible in the nuclei of early stage egg chambers (arrow) in contrast to wild type. (D) The diffuse pattern of Piwi staining in an Aub knockdown germ line is most apparent in stage 2/3 egg chambers. (E) Piwi immunofluorescent staining of stage 6/7 egg chambers. DNA staining is shown in red to delineate the nucleus. The overall Piwi signal in the Aub knockdown egg chambers is adjusted so that the signal strength in the germline nuclei matches the corresponding region in the wild-type egg chamber. (Scale bars: 5 μm.)
Fig. 4.
Fig. 4.
Germ-line Piwi functions in silencing some transposons through an HP1a-dependent chromatin-based mechanism. (A) ChIP-quantitative PCR analysis at 5′ ends or promoter regions (as indicated in the label) of a set of transposons using antibodies against HP1a in germ-line Piwi knockdown ovaries. The enrichment levels are relative to the α-actinin locus. (B) Quantitative RT-PCR analysis of expression levels for the same set of transposons in germ-line HP1a knockdown ovaries. Fold expression levels are relative to RPL32 expression. (C) ChIP-quantitative PCR analysis at 5′/promoter regions of a set of transposons using antibodies against H3K9me2 in germ-line Piwi knockdown ovaries. The enrichment levels are relative to the 18S ribosomal DNA locus. Bars represent mean ± SEM of three biological replicate experiments.
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