Author response: piRNA-guided co-transcriptional silencing coopts nuclear export factors

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. Here we show that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. We mapped the protein domains crucial for the Nxf2/Panx complex formation and show that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing. https://doi.org/10.7554/eLife.47999.001 eLife digest For an organism to form and grow correctly, it must rely on the genetic information it has received from its parents. DNA, however, is full of elements called transposons that can disrupt this information by moving around the genome and inserting themselves into genes. Changes caused by these invading elements can lead to devastating effects, such as cancer and cell death. To shield their DNA from harm, organisms have evolved regulatory machineries to recognize and correct alterations that may be damaging. One way cells can protect their DNA is by silencing disruptive transposons using small molecules known as piRNAs. These protective molecules detect transposons as soon as they are active and recruit other proteins to switch them off. However, questions still remain about how specific proteins recruited by piRNAs are involved in this process. In flies, a protein called Panoramix (Panx) is known to trigger transposon silencing, but how it does this is still unclear. Now, Fabry, Ciabrelli, Munafò et al. set out to investigate how Panx silences active transposons in the ovaries of flies and whether other proteins are involved. Like so many other proteins, Panx was found not to work alone but to form a complex with two other proteins, called Nxf2 and Nxt1. The experiments showed that all three components of the complex, named PICTS, are critical for transposon control in flies, but Panx is the engine that drives the machine. Panx and Nxf2 were found to stabilize each other, and together with co-factor Nxt1 place a mark on the genome at the point where the transposon emerges, effectively switching it off. Notably, although Nxf2 and Nxt1 are part of a family of proteins that export molecules from the nucleus, both these factors appear to have been repurposed to silence transposable elements within the genome. This work expands our understanding of how cells employ regulatory machineries, like the PICTS complex, to guard against disruptive genetic changes. These mechanisms are often conserved throughout evolution, and the findings presented here may help identify ways to counteract harmful changes caused by transposons in other organisms, including humans. However, more work would be required to deepen our knowledge of how these processes work. https://doi.org/10.7554/eLife.47999.002 Introduction The piRNA pathway is a small RNA-based immune system that represses transposable elements in animal gonadal tissues (Czech et al., 2018; Ozata et al., 2019). At the core of this pathway are PIWI-clade Argonaute proteins that are guided by 23-30nt piRNA partners to silence transposon targets via two main mechanisms. In Drosophila, Aubergine and Argonaute 3 enforce post-transcriptional gene silencing (PTGS) via direct cleavage of transposon mRNAs in the cytoplasm (Brennecke et al., 2007; Gunawardane et al., 2007). Piwi, in contrast, operates in the nucleus where it instructs the co-transcriptional gene silencing (TGS) of transposon insertions (Brennecke et al., 2007; Klenov et al., 2011; Sienski et al., 2012). Mutations that compromise TGS result in severe loss of transposon control, despite normal piRNA levels (Dönertas et al., 2013; Le Thomas et al., 2013; Muerdter et al., 2013; Ohtani et al., 2013; Rozhkov et al., 2013; Sienski et al., 2015; Sienski et al., 2012; Yu et al., 2015). Piwi, in complex with piRNAs, detects nascent transposon RNAs arising from active insertions and directs the silencing of these loci. Target silencing is achieved via recruitment of histone modifying enzymes that deposit repressive chromatin marks, mainly trimethylation of Lysine 9 on Histone 3 (H3K9me3) (Iwasaki et al., 2016; Klenov et al., 2014; Le Thomas et al., 2013; Rozhkov et al., 2013; Sienski et al., 2012; Wang and Elgin, 2011). Panoramix (Panx) is a key TGS effector, acting downstream of Piwi at the interface between the piRNA pathway and the general chromatin silencing machinery (Sienski et al., 2015; Yu et al., 2015). Strikingly, RNA-mediated recruitment of Panx, but not Piwi, to a locus is sufficient to trigger its epigenetic silencing, thus placing Panx at a critical node of the TGS mechanism. Downstream of Panx, the concerted action of dLsd1/Su(var)3–3 and Eggless/dSETDB1 erases H3K4me2 and concomitantly deposits H3K9me3, followed by chromatin compaction via Heterochromatin Protein 1a (HP1a/Su(var)205) (Czech et al., 2013; Iwasaki et al., 2016; Rangan et al., 2011; Sienski et al., 2015; Wang and Elgin, 2011; Yu et al., 2015). Precisely how Panx recruits these histone modifying enzymes and what other factors participate in this process remains an outstanding question. Here we show that Panx coopts elements of the nuclear RNA export machinery to trigger transcriptional silencing. Panx is part of a complex that also contains Nuclear Export Factor 2 (Nxf2) and Nxt1/p15. Panx and Nxf2 are interdependent for their protein stability. nxf2 mutants show strong de-repression of Piwi-regulated transposons and severe loss of H3K9me3 at affected loci, similarly to panx mutants. We find that the amino-terminus of Panx delivers the critical silencing signal, as it is necessary and sufficient to trigger the deposition of repressive chromatin marks if tethered to a reporter construct, while its carboxyl-terminal region is involved in the interaction with Nxf2. Nxf2 is closely related to the mRNA export factor Nxf1, which also interacts and functions with Nxt1 (Fribourg et al., 2001; Herold et al., 2001; Herold et al., 2000). Thus, our findings reveal that the evolution of transposon defense mechanisms involved exaptation of the nuclear RNA export machinery. Results Nxf2 is a TGS factor that interacts with Panx To identify proteins associated with Panx, we immunoprecipitated a GFP-Panx fusion protein expressed from its endogenous promoter (Handler et al., 2013; Figure 1—figure supplement 1A) from ovary lysates and identified co-purifying proteins by quantitative mass spectrometry. Three proteins showed the strongest enrichment and significance: Panx, Nxf2 and Nxt1 (Figure 1A, Figure 1—source data 1). Nxf2 is a homolog of the general messenger RNA (mRNA) export factor Nxf1 but was reported previously as being dispensable for canonical mRNA transport in S2 cells (Herold et al., 2001; Herold et al., 2003). Nxf2 contains all domains present in the family defined by Nxf1, namely an amino-terminal region (NTR), an RNA-binding domain (RBD), leucine-rich repeats (LRR), the NTF2-like domain (NTF2) and a Ubiquitin-associated (UBA) domain (Figure 1B and Figure 1—figure supplement 1B) (Herold et al., 2001). While the NTR, LRRs and RBD are typically involved in cargo binding, the NTF2 and UBA domains mediate binding to the Nuclear Pore Complex (NPC) and are required for Nxf1-mediated RNA export (Braun et al., 2001; Fribourg et al., 2001). Nxt1, also known as p15, is a co-factor of Nxf1 responsible for interaction with the NPC, specifically through the NTF2-fold (Fribourg et al., 2001; Lévesque et al., 2001). Interestingly, Nxt1 was also reported to interact with Nxf2 (Herold et al., 2001; Herold et al., 2000). Both Nxf2 and Nxt1 were previously identified in screens for piRNA-guided silencing in somatic and germline cells, and their depletion resulted in female sterility (Czech et al., 2013; Handler et al., 2013; Muerdter et al., 2013). Contrary to expectations based upon previous findings (Sienski et al., 2015; Yu et al., 2015), we saw no enrichment for Piwi by mass spectrometry (Figure 1A), results that are consistent with another recent study (Batki et al., 2019). However, co-immunoprecipitation experiments detected weak but reproducible interactions between Piwi and Panx, Nxf2, and Nxt1, but not with a negative control (Figure 1—figure supplement 1D), suggesting that low amounts of transposon substrates in unperturbed cells and/or transient associations might push Piwi below the limit of detection by less sensitive approaches. Figure 1 with 2 supplements see all Download asset Open asset 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. https://doi.org/10.7554/eLife.47999.003 Figure 1—source data 1 List of proteins recovered in GFP-Panx IP-MS. https://doi.org/10.7554/eLife.47999.006 Download elife-47999-fig1-data1-v2.txt Figure 1—source data 2 List of proteins recovered in GFP-Nxf2 IP-MS. https://doi.org/10.7554/eLife.47999.007 Download elife-47999-fig1-data2-v2.txt Using CRISPR/Cas9, we generated flies that express a GFP-Nxf2 fusion protein from the endogenous nxf2 locus. GFP-Nxf2 is expressed in follicle and germline cells of the ovary and localizes predominantly to nuclei (Figure 1C and Figure 1—figure supplement 1C). Mass spectrometry of GFP-Nxf2-associated proteins identified Panx, Nxf2, and Nxt1, as binding partners (Figure 1D, Figure 1—source data 2), implying the existence of a complex containing these three factors, which we named the Panx-induced co-transcriptional silencing (PICTS) complex. We therefore generated two nxf2 mutant alleles, nxf2F10* and nxf2∆1*, which harbor premature stop codons that disrupt the nxf2 open reading frame from amino acid 10 onwards (Figure 1B and Figure 1—figure supplement 1E). Trans-heterozygous mutants were female sterile, with fewer eggs laid and none hatching (Figure 1—figure supplement 1F). nxf2 mutants were severely compromised in the repression of soma- and germline-specific transposons, in a highly similar manner to panx mutants (Figure 1E), with no change in piRNA levels or Piwi localization, despite compromised silencing (Figure 1—figure supplement 1G–H). To assess the specificity of the impact of nxf2 mutations on the transcriptome, we performed RNA-seq from total RNA of heterozygote and mutant ovaries, using panx mutants for comparison. As reported previously, the expression of protein-coding genes was not generally affected in nxf2 mutants, with only 16 out of 7252 (r2 = 0.963) being changed more than 4-fold (Figure 1F) (Herold et al., 2001; Herold et al., 2003). In contrast, 28 out of 60 transposon families (that were above the expression threshold of 1 rpm) were de-repressed by more than 4-fold in nxf2 mutants. Similar results were obtained for panx mutants: 16 out of 60 transposons were de-repressed and only 6 out of 7252 genes mis-regulated (r2 = 0.991). ChIP-seq for the H3K9me3 mark from nxf2 mutant ovaries showed reduced methylation levels at the same transposon families that were de-repressed according to RNA-seq, such as Het-A, while randomly chosen genomic intervals were not changed (Figure 1—figure supplement 2A–B). Ovarian somatic cells (OSCs), cultured in vitro, express a functional piRNA-guided co-transcriptional gene silencing machinery and provide a convenient context for mechanistic studies (Saito et al., 2009). RNA-seq from OSCs depleted of Piwi, Panx or Nxf2 showed marked de-repression of soma-specific (e.g. mdg1, gypsy, 297) and intermediate transposon families (e.g. blood) when compared to control cells treated with GFP siRNAs (Figure 1G left; Figure 1—figure supplement 2C). The de-repression of transposon families strongly correlates with the reduction in H3K9me3 levels mapped over their consensus sequences in ChIP-seq samples generated from the same knockdowns (Figure 1G right). In contrast, no major changes in H3K9me3 were detected over transposons that show no de-repression in these cells upon piwi, panx, or nxf2 knockdown. We next focused on a set of 233 individual, Piwi-regulated genomic transposon insertions in OSCs (see Materials and methods for details). This enabled analysis of chromatin states on individual loci, including flanking regions, rather than averaging contributions over a consensus sequence (Figure 1—figure supplement 2D–H). Piwi depletion resulted in the accumulation of H3K4me2 at these loci and spreading of the mark, indicative of active transcription, beyond the transposon into downstream regions (Figure 1—figure supplement 2D,F), similar to earlier reports (Dönertas et al., 2013; Klenov et al., 2014; Sienski et al., 2012). Knockdown of panx or nxf2 showed similar, though less pronounced, effects. H3K9me3 marks were strongly reduced upon Piwi depletion, with panx and nxf2 knockdowns showing a similar but milder impact (Figure 1—figure supplement 2E,G). H3K4me2 spreading typically correlates with increased RNA output and a decrease in H3K9me3 levels, as evident for a euchromatic gypsy insertion located within an intron of the 5' UTR of the gene ex on chromosome 2L (Figure 1—figure supplement 2H). Panx and Nxf2 proteins are interdependent for their stability Proteins that form complexes are often interdependent for either localization or stability, and there are abundant examples of such interactions in the piRNA pathway (Dönertas et al., 2013; Ohtani et al., 2013). To test for such dependencies among TGS factors, we depleted Piwi, Panx or Nxf2 in germ cells of flies expressing GFP-Nxf2 (Figure 2A) or GFP-Panx (Figure 2B). Germline knockdown of piwi had no effect on the localization of either Nxf2 or Panx. Depletion of Panx, however, led to a pronounced loss of nuclear GFP-Nxf2 in nurse cell nuclei (Figure 2A). The reciprocal was also true, with GFP-Panx nuclear signal being reduced upon nxf2 knockdown in nurse cells (Figure 2B). Similarly, the individual depletion of Panx or Nxf2 in follicle cells resulted in a reduction of both proteins (Figure 2—figure supplement 1A–B). To assess whether the observed reduction reflects protein stability rather than mislocalization, we performed western blots on ovarian lysates. Panx protein level was strongly reduced in nxf2 mutant ovaries (Figure 2C) and GFP-Nxf2 signal was completely lost in homozygous panx mutants (Figure 2D). Of note, mRNA levels of Nxf2 and Panx were not affected when the other factor was mutated (Figure 1F), implying regulation at the protein level. Considered together, the localization and stability of Nxf2 and Panx are interdependent in vivo. Figure 2 with 1 supplement see all Download asset Open asset Protein stability and localization of Nxf2 and Panx is reciprocally co-dependent. (A) Expression and localization of Piwi and GFP-Nxf2 in egg chambers upon germline-specific knockdown (GLKD) of the indicated factors are shown by immunofluorescence (GFP, green; Piwi, magenta; DNA, blue). Scale bar, 10 µm. Plot profiles showing the relative signal intensity of Piwi (magenta), GFP-Nxf2 fusion protein (green) and DAPI (blue background box) were computed from the indicated sections. (B) as (A) but showing GFP-Panx. (C) Western blot showing Panx protein levels in ovaries from panx and nxf2 heterozygotes and mutants compared to w1118 control flies. His3 served as loading control. (D) as (C) but showing levels of GFP-Nxf2 protein. https://doi.org/10.7554/eLife.47999.008 PICTS complex formation is required for TGS To map the domains of Nxf2 and Panx that mediate their interaction, we expressed various combinations of full-length, truncated, or mutant proteins in S2 cells or in OSCs where native protein expression had been reduced by RNAi (Figure 3A). Interactions were tested by co-immunoprecipitation and western blot analyses, and the subcellular localization analyzed by immunofluorescence staining. In OSCs, the ability of each construct to rescue transposon de-repression was monitored by qPCR. Figure 3 with 2 supplements see all Download asset Open asset Requirements for the formation and function of the PICTS complex. (A) Cartoons displaying the Nxf2 and Panx protein structures and domain mutants used (see also Figure 3—figure supplement 1A and Figure 3—figure supplement 2A). NTR, amino-terminal region; LRR, leucine rich repeats; RBD, RNA-binding domain; NTF2, NTF2-like domain; UBA, Ubiquitin associated domain; CC, coiled coil domain; HA, Hemagglutinin tag. (B) Western blot analyses of FLAG-tag co-immunoprecipitation from lysates of S2 cells transfected with the indicated expression constructs (IN, input; UB, unbound; IP, immunoprecipitate). Asterisk indicates unspecific band from anti-HA antibody (see also Figure 3—figure supplement 1C, Figure 3—figure supplement 2B and Figure 3—figure supplement 2E). (C) Expression and localization of the indicated Nxf2 (3xFLAG-tagged, top, shown in red in the merge) and Panx (HA-tagged, bottom, shown in green in the merge) expression constructs in transfected S2 cells are shown by immunofluorescence (see also Figure 3—figure supplement 1D, Figure 3—figure supplement 2C and Figure 3—figure supplement 2F). Lamin staining (red was used to the of the nuclear Scale bar, µm. (D) Bar graphs showing of (B) and Figure 3—figure supplement and Figure 3—figure supplement was from over (see also Figure 3—figure supplement (E) Bar graphs showing fold changes in steady-state RNA levels of the transposon in total RNA from OSCs transfected with siRNAs against Panx (left) and Nxf2 (right) and the indicated expression constructs (relative to rescue construct and normalized to rp49). Error bars indicate standard deviation n = Nxf2, n = 3). (F) As in (B) but showing co-immunoprecipitation of with and (G) As in (B) but showing co-immunoprecipitation of and Nxt1 recovered with the indicated Nxf2 expression indicate an unspecific band from Nxf2 and Panx (Figure and in S2 cell nuclei (Figure the of Panx a protein that while Nxf2 and these proteins no a complex (Figure expressed alone in S2 cells, Nxf2 predominantly in Figure suggesting that interaction with Panx is necessary for nuclear localization of Nxf2. in contrast, the ability to interact with Nxf2 but to to the nucleus (Figure Strikingly, localization of to the nucleus also nuclear localization of Nxf2 (Figure and Figure 3—figure supplement only either the coiled coil domain or which together most of the of Panx, reduced co-purification with Nxf2 (Figure and Figure 3—figure supplement with construct being to rescue repression upon panx knockdown (Figure these results that the part of Panx its nuclear localization signal that proper localization of Nxf2 via interaction with the Panx To the regions of Nxf2 that are for its we in the of Panx, Nxf2 proteins that either the regions required for RNA cargo binding or the region for its with the (Braun et al., 2001; Fribourg et al., 2001; Herold et al., (Figure and Figure 3—figure supplement to with Panx, and this was by an increased protein localization (Figure In contrast, the region of Nxf2 had less impact on its co-purification with Panx or the nuclear localization of either protein (Figure This mutant was to interact with but not as (Figure of Nxf2, which had individual domains to rescue nxf2 knockdown in OSCs (Figure 3—figure supplement all but still co-purified with Panx (Figure and Figure 3—figure supplement We also generated point mutants within the UBA highly conserved amino at a (Figure 3—figure supplement right). UBA mutant showed a similar to the domain with reduced binding to Panx, increased localization, and to rescue loss of Nxf2 (Figure and Figure 3—figure supplement Thus, the interaction of Nxf2 and Panx on an UBA domain and the portion of Panx. The NTF2-like fold was previously shown to mediate the interaction of proteins with Nxt1 (Herold et al., et al., 2012; et al., 2000). To a for Nxt1 in silencing, we generated NTF2 domain point mutants in involved in the interaction with Nxt1 et al., 2012). point mutants predominantly to the nucleus and of Panx to the control (Figure and Figure 3—figure supplement three mutants and to rescue transposon de-repression upon depletion of Nxf2 (Figure to an of Nxt1 in silencing. reduced amounts of Nxt1 were recovered with the NTF2 mutants and while the NTF2 mutant which transposon expression (Figure as as UBA mutants and showed levels to Nxf2 (Figure and Figure 3—figure supplement Tethering of Nxf2 and Nxt1 to RNA silencing of Panx to nascent RNA or DNA was previously shown to result in co-transcriptional silencing and the accumulation of repressive chromatin marks (Sienski et al., 2015; Yu et al., 2015). To test whether Nxf2 induce we an the promoter an with 9 in its UTR in OSCs (Figure As from previous studies (Sienski et al., 2015; Yu et al., 2015), expression of not lead to reduced RNA or protein levels (Figure and Figure supplement 1A), although it to nuclei (Figure supplement Tethering of in contrast, resulted in repression of RNA and protein as reported (Sienski et al., 2015; Yu et al., 2015). caused an reduction of RNA and protein expression from the reporter (Figure experiments consistent repression by Panx and Nxf2 (Figure supplement 1C). Tethering of also reporter repression (Figure Strikingly, upon of Nxt1, Nxf2 or Panx, we observed increased levels of H3K9me3 over the reporter (Figure These data that Nxf2 and Nxt1, with Panx, as key of co-transcriptional silencing and are each sufficient to recruit the downstream silencing machinery. Figure 4 with 1 supplement see all Download asset Open asset of PICTS components to nascent RNA results in chromatin silencing. (A) Cartoon displaying the RNA construct used in The construct was and the promoter contains an the sequence to the a nuclear localization sequence and the and a UTR containing for are (B) Bar graphs showing fold changes in steady-state RNA levels of the and in total RNA from OSCs transfected with the indicated expression constructs (relative to a construct and normalized to rp49). Bar graphs showing fold changes in protein levels of in lysates from OSCs transfected with the indicated expression constructs (relative to a construct and normalized to denotes P value < (unpaired t-test). Error bars indicate standard deviation (n = (C) Density profiles of normalized reads from H3K9me3 ChIP-seq experiments mapping to the reporter as indicated in the The of reads is shown (D) as in (A) but showing the DNA The construct binding followed by the promoter and the for are (E) as in (B) but showing of DNA * denotes P value < ** denotes denotes (unpaired t-test). Error bars indicate standard deviation (n = 3). (F) as in (C) but showing profiles of normalized reads from the DNA (G) of DNA experiments normalized to or His3 and relative to the same construct used in * denotes P value < ** denotes denotes (unpaired t-test). Error bars indicate standard deviation (n = 3). for piRNA-guided co-transcriptional silencing. Piwi for nascent transposon and upon target recruits the PICTS complex to the RNA via an The of the PICTS complex with target co-transcriptional silencing, with the amino-terminus of Panx being required for silencing PICTS complex recruitment results in chromatin H3K9me3 and depends on general silencing machinery factors such as We also tested whether Nxf2 silence targets upon to DNA rather than nascent construct of the which drives the expression of (Figure While of not resulted in in both RNA and protein levels (Figure and Figure supplement 1D), as previously reported (Sienski et al., 2015). also the both mRNA and protein to a than was to silence the by tethered Panx or Nxf2 resulted in a decrease in H3K4me2 marks over the promoter and while the of the reporter showed low and in the of the mark (Figure supplement 1E). H3K9me3 increased upon repression