Fig. 5: MEP-1, Mi-2, and Rpd3 involvement in piRNA-dependent repression.

a Schematic representation of the krimper (krimp) piRNA-mediated repression assay after siRNA-mediated depletion of MEP-1, Mi-2, Rpd3, or Piwi in OSCs. Transfection of the apiRNA vector allows the production of antisense artificial piRNAs against krimp. Transfection of the GFPctrl vector is used as a piRNA-less negative control. b Fold change (relative to GFPctrl) in krimp RNA levels caused by apiRNA in OSCs previously transfected with the indicated siRNAs. RNA levels were quantified relative to RpL32. Box plots display median (line), first and third quartiles (box), and highest/lowest value within 1.5× interquartile range (whiskers) for n = 6 values calculated over three independent samples. The siRNA effect was tested using the ANOVA test and differences using the pairwise t-test. P-values were calculated using sample data that displayed normal distribution (tested with the Shapiro–Wilkinson test). Variance homogeneity was tested with the Levene’s test and then the two-tailed Student’s t-test was used. ***P-value < 0.05 when each siRNA experiment is compared to the sictrl experiment. c Quantification of krimp RNA levels relative to RpL32 in OSCs transfected by the indicated siRNAs with (+) or without (−) production of apiRNAs against krimp. Note that the cells that were not transfected with the apiRNA vector were transfected with GFPctrl vector, instead. Means ± SD from n = 3 biologically independent samples are represented. d Fold change (relative to GFPctrl) of H3K9me3 marks on krimp quantified by ChIP-qPCR. OSCs were transfected with the indicated siRNAs and either the apiRNA or the GFPctrl vector. H3K9me3 quantification was normalized to a positive control (1360-element) and to input. Means ± SD from n = 3 biologically independent samples are represented. Statistical test was performed as in b. ***P-value < 0.05 compared with sictrl. Source data for b–d: Supplementary Data set 5 and Supplementary Table 2 for b, c.