Figure 1. Nxf2 is a piRNA pathway factor that functions in transcriptional gene silencing.

(A) Volcano plot showing enrichment values and corresponding significance levels for proteins co-purified with GFP-Panx from ovary lysates (n = 4 for GFP-Panx and n = 3 for control ovaries). Proteins with fold change >2 and adjusted P value < 0.05 are highlighted in blue. The bait protein is labeled in red and Piwi is shown in purple. (B) Cartoon displaying the Nxf2 domain structure and generated mutants. NTR, amino-terminal region; LRR, leucine rich repeats; RBD, RNA-binding domain; NTF2, NTF2-like domain; UBA, Ubiquitin associated domain. (C) Expression and localization of GFP-Nxf2 in an egg chamber is shown by immunofluorescence (see also Figure 1—figure supplement 1C). Green, GFP-Nxf2; magenta, Aubergine; blue, DNA. Scale bar, 10 µm. (D) as (A) but co-purification from GFP-Nxf2 ovary lysates (GFP-Nxf2, n = 4; control, n = 3). (E) Bar graphs showing fold changes in steady-state RNA levels of soma- (mdg1, gypsy) and germline- (HeT-A, burdock) specific transposons in total ovarian RNA from the indicated genotypes (relative to wild-type and normalized to rp49). * denotes P value < 0.05; ** denotes p<0.001 (unpaired t-test). Error bars indicate standard deviation (n = 3). (F) Scatter plots showing expression levels (reads per million sequenced reads) of genes (in grey) and transposons (in purple) from total RNA from ovaries of the indicated genotypes (left, nxf2; right, panx; n = 3; r² values represent expression of genes only). Transposons whose abundance change more than four-fold compared to heterozygotes are highlighted in blue. (G) Heat maps showing RNA-seq (left) and H3K9me3 ChIP-seq (right) of the 30 most expressed transposons in OSCs (compared with siGFP) upon the indicated knockdowns. Density profiles of normalized reads from RNA-seq (left) and H3K9me3 ChIP-seq (right) experiments mapping to the indicated transposons.

Figure 1—figure supplement 1. Nxf2 is a piRNA pathway factor acting in TGS.

(A) Western blot analyses of GFP-trap co-immunoprecipitation from lysates of ovaries from the indicated genotypes (representative replicate subjected to mass spectrometry analysis; IN, input; UB, unbound; IP, immunoprecipitate). (B) Cartoon displaying the domain structure of the Drosophila nuclear export factor (NXF) family. NTR, amino-terminal region; LRR, leucine rich repeats; RBD, RNA-binding domain; NTF2, NTF2-like domain; UBA, Ubiquitin associated domain. (C) Expression and localization of GFP-Nxf2 and GFP-Panx alongside Aubergine (Aub) and DNA (visualized by DAPI) in an egg chamber is shown by immunofluorescence. Scale bar, 10 µm. (D) Western blot analyses of FLAG-tag co-immunoprecipitation from lysates of OSCs transfected with the indicated expression constructs (IN, input; UB, unbound; IP, immunoprecipitate). The bottom panel shows Piwi signal with enhanced contrast. (E) Cartoon displaying the domain structure of wild-type Nxf2 and the generated mutants as well as their underlying sequence alterations. (F) Bar graphs showing the egg hatching rate of female flies of the indicated genotypes. (G) Bar graphs showing the size distribution of transposon-mapping small RNAs from ovaries of the indicated genotypes (sense in blue, antisense in red). (H) Expression and localization of Piwi in egg chambers from nxf2 and panx heterozygote and mutant ovaries is shown by immunofluorescence. Scale bar, 10 µm.

Figure 1—figure supplement 2. Loss of Nxf2 impairs TGS at levels comparable to Panx.

(A) Box plot showing changes in H3K9me3 levels between nxf2 heterozygotes and mutants in ovarian ChIP-seq for the indicated categories (coloring of transposons is based on their fold change in RNA-seq in Figure 1F; 100 random genomic intervals of 5 kb bin size are shown in grey). ** denotes P value < 0.001; *** denotes P value < 0.0001 (Welch two sample t-test). (B) Density plots representing ChIP-seq signal from ovaries mutant for nxf2 and panx over the Het-A transposon consensus sequence are shown along with heterozygote flies. (C) Scatter plots showing expression levels (reads per million sequenced reads) of genes (in grey) and transposons (in purple) from total RNA from OSCs of the indicated knockdowns (n = 3; r² values represent expression of genes only). Transposons whose abundance change more than four-fold are highlighted in blue. (D) Heat maps showing H3K4me2 levels calculated from 233 Piwi-dependent transposon insertions in the indicated knockdowns in OSCs (sorted for decreasing intensity in siGFP). Genomic regions flanking the transposon insertions (5 kb upstream and downstream) are shown. (E) As in (D) but H3K9me3 levels are shown for 15 kb up- and downstream of the transposon insertions (sorted for decreasing intensity in siGFP). (F) Shown are metaprofiles of the average H3K4me2 signals 5 kb up- and downstream of 233 euchromatic transposon insertions in the gfp, piwi, panx and nxf2 knockdowns shown in (D). (G) as in (F) but showing metaprofiles displaying average H3K9me3 signals 15 kb up- and downstream of the insertions shown in (E). (H) Genome browser shot displaying profiles of RNA-seq levels and the density of H3K4me2 and H3K9me3 chromatin marks in OSCs upon the indicated knockdowns. Shown is an euchromatic gypsy insertion located within an intron of the 5' UTR of the gene ex located on chromosome 2L.