Transcriptional Silencing of Transposons by Piwi and Maelstrom and Its Impact on Chromatin State and Gene Expression

Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression. Eukaryotic genomes are colonized by transposons whose uncontrolled activity causes genomic instability. The piRNA pathway silences transposons in animal gonads, yet how this is achieved molecularly remains controversial. Here, we show that the HMG protein Maelstrom is essential for Piwi-mediated silencing in Drosophila. Genome-wide assays revealed highly correlated changes in RNA polymerase II recruitment, nascent RNA output, and steady-state RNA levels of transposons upon loss of Piwi or Maelstrom. Our data demonstrate piRNA-mediated trans-silencing of hundreds of transposon copies at the transcriptional level. We show that Piwi is required to establish heterochromatic H3K9me3 marks on transposons and their genomic surroundings. In contrast, loss of Maelstrom affects transposon H3K9me3 patterns only mildly yet leads to increased heterochromatin spreading, suggesting that Maelstrom acts downstream of or in parallel to H3K9me3. Our work illustrates the widespread influence of transposons and the piRNA pathway on chromatin patterns and gene expression. Piwi-RISC guides transcriptional silencing of transposons in trans Piwi-mediated silencing triggers H3K9me3 heterochromatin formation Maelstrom is required for transcriptional silencing, but not for H3K9 trimethylation Transposon silencing by the piRNA pathway broadly affects gene expression A major selection force during evolution is the maintainance of genomic integrity over generations. Transposable elements (TEs) are threatening genomic stability due to their mobile character and their creating repetitive sequence islands that can initiate ectopic recombination (Kazazian, 2004Kazazian Jr., H.H. Mobile elements: drivers of genome evolution.Science. 2004; 303: 1626-1632Crossref PubMed Scopus (1386) Google Scholar). Small-RNA-based silencing pathways are universally employed by eukaryotes to silence TEs (Slotkin and Martienssen, 2007Slotkin R.K. Martienssen R. Transposable elements and the epigenetic regulation of the genome.Nat. Rev. Genet. 2007; 8: 272-285Crossref PubMed Scopus (1350) Google Scholar). In animals, this is of particular importance in germ cells. The PIWI-interacting RNA (piRNA) pathway serves as the main line of defense in the animal gonad, and defects in it result in TE derepression, genomic instability, and sterility (Malone and Hannon, 2009Malone C.D. Hannon G.J. Small RNAs as guardians of the genome.Cell. 2009; 136: 656-668Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar; Siomi et al., 2011Siomi M.C. Sato K. Pezic D. Aravin A.A. PIWI-interacting small RNAs: the vanguard of genome defence.Nat. Rev. Mol. Cell Biol. 2011; 12: 246-258Crossref PubMed Scopus (929) Google Scholar). At the core of the pathway is the piRNA-induced silencing complex (pi-RISC) that consists of a single-stranded piRNA bound by a PIWI family protein. piRNAs are typically processed from TE RNAs and so-called piRNA cluster transcripts that are enriched in TE sequences. Thus, by virtue of their sequence, piRNAs guide the specific silencing of TEs. (Senti and Brennecke, 2010Senti K.A. Brennecke J. The piRNA pathway: a fly's perspective on the guardian of the genome.Trends Genet. 2010; 26: 499-509Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar; Siomi et al., 2011Siomi M.C. Sato K. Pezic D. Aravin A.A. PIWI-interacting small RNAs: the vanguard of genome defence.Nat. Rev. Mol. Cell Biol. 2011; 12: 246-258Crossref PubMed Scopus (929) Google Scholar). Conceptually, two major silencing modes are distinguished, namely transcriptional silencing (TGS) and posttranscriptional silencing (PTGS). Most animals express multiple PIWI proteins, and these might employ different silencing modes. The Drosophila genome encodes the PIWI proteins Piwi, Aubergine (Aub), and Argonaute 3 (AGO3). Aub and AGO3 piRISCs are cytoplasmic, possess slicer activity, and are the major players in a piRNA amplification loop that requires reciprocal cleavage of TE RNAs and piRNA cluster transcripts (Senti and Brennecke, 2010Senti K.A. Brennecke J. The piRNA pathway: a fly's perspective on the guardian of the genome.Trends Genet. 2010; 26: 499-509Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar; Siomi et al., 2011Siomi M.C. Sato K. Pezic D. Aravin A.A. PIWI-interacting small RNAs: the vanguard of genome defence.Nat. Rev. Mol. Cell Biol. 2011; 12: 246-258Crossref PubMed Scopus (929) Google Scholar). As TE sense RNAs are consumed during this amplification loop, Aub/AGO3-mediated silencing represents a PTGS process. The third family member Piwi, however, is enriched in the nucleus, and its silencing mode is much less understood. Genetically, Piwi-mediated TE silencing depends on its nuclear localization, but not on its slicer activity (Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar; Saito et al., 2010Saito K. Ishizu H. Komai M. Kotani H. Kawamura Y. Nishida K.M. Siomi H. Siomi M.C. Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila.Genes Dev. 2010; 24: 2493-2498Crossref PubMed Scopus (234) Google Scholar). These observations indicate that Piwi might induce TGS via triggering repressive chromatin modifications. Indeed, changes in chromatin marks and nascent RNA levels have been observed for some TEs in piRNA pathway mutants (Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar; Shpiz et al., 2011Shpiz S. Olovnikov I. Sergeeva A. Lavrov S. Abramov Y. Savitsky M. Kalmykova A. Mechanism of the piRNA-mediated silencing of Drosophila telomeric retrotransposons.Nucleic Acids Res. 2011; 39: 8703-8711Crossref PubMed Scopus (74) Google Scholar; Wang and Elgin, 2011Wang S.H. Elgin S.C. Drosophila Piwi functions downstream of piRNA production mediating a chromatin-based transposon silencing mechanism in female germ line.Proc. Natl. Acad. Sci. USA. 2011; 108: 21164-21169Crossref PubMed Scopus (161) Google Scholar). On the other hand, a specific chromatin association of PIWI proteins in germ cells has not been demonstrated. Some studies even challenge a role for Piwi in chromatin regulation. For example, no significant changes in heterochromatin protein 1 (HP1) occupancy on TEs were observed upon Piwi knockdown (KD) (Moshkovich and Lei, 2010Moshkovich N. Lei E.P. HP1 recruitment in the absence of argonaute proteins in Drosophila.PLoS Genet. 2010; 6: e1000880Crossref PubMed Scopus (59) Google Scholar), and a genetic study in flies concluded that Piwi triggers PTGS rather than TGS (Dufourt et al., 2011Dufourt J. Brasset E. Desset S. Pouchin P. Vaury C. Polycomb group-dependent, heterochromatin protein 1-independent, chromatin structures silence retrotransposons in somatic tissues outside ovaries.DNA Res. 2011; 18: 451-461Crossref PubMed Scopus (8) Google Scholar). A systematic understanding of the silencing mode employed by nuclear PIWI proteins is therefore a major open question in the field. All three Drosophila PIWI family proteins are coexpressed in germline cells. Due to their interdependence in terms of piRNA biogenesis and TE silencing, the precise genetic and mechanistic dissection of Piwi's nuclear role is challenging. Somatic support cells of the ovary, however, express a simplified piRNA pathway based exclusively on nuclear Piwi. Importantly, a stable cell line derived from ovarian somatic cells (these cultured cells are called OSSs or OSCs) has been established (Niki et al., 2006Niki Y. Yamaguchi T. Mahowald A.P. Establishment of stable cell lines of Drosophila germ-line stem cells.Proc. Natl. Acad. Sci. USA. 2006; 103: 16325-16330Crossref PubMed Scopus (96) Google Scholar; Saito et al., 2009Saito K. Inagaki S. Mituyama T. Kawamura Y. Ono Y. Sakota E. Kotani H. Asai K. Siomi H. Siomi M.C. A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila.Nature. 2009; 461: 1296-1299Crossref PubMed Scopus (318) Google Scholar). These cells harbor a piRNA pathway that, in every aspect analyzed, mirrors the pathway acting in ovarian somatic cells. In OSCs, piRNAs antisense to TEs are derived from piRNA clusters such as flamenco. piRNA biogenesis depends on several cytoplasmic factors, and defects in it result in loss of Piwi, presumably due to destabilization of unloaded Piwi (Siomi et al., 2011Siomi M.C. Sato K. Pezic D. Aravin A.A. PIWI-interacting small RNAs: the vanguard of genome defence.Nat. Rev. Mol. Cell Biol. 2011; 12: 246-258Crossref PubMed Scopus (929) Google Scholar). Upon loss of Piwi-RISC, several TEs, which are normally silenced by the piRNA pathway, are derepressed. We took advantage of this linear piRNA pathway and dissected the underlying silencing process in detail. Our data demonstrate that Piwi-RISC mediates TE silencing at the transcriptional level and that this is accompanied by local heterochromatin formation. Remarkably, most euchromatic H3K9me3 islands are due to piRNA-mediated silencing of TE insertions, and spreading of this heterochromatic mark into flanking genomic regions has striking effects on the expression of nearby genes. While the process of piRNA biogenesis within the somatic pathway is being increasingly dissected at the molecular level and multiple involved factors are known, not a single protein has been linked to Piwi-mediated silencing in the nucleus. To identify such factors, we utilized an assay system based on transgenic RNAi and a lacZ reporter that monitors silencing of the gypsy TE in follicle cells (Figure 1A; Olivieri et al., 2010Olivieri D. Sykora M.M. Sachidanandam R. Mechtler K. Brennecke J. An in vivo RNAi assay identifies major genetic and cellular requirements for primary piRNA biogenesis in Drosophila.EMBO J. 2010; 29: 3301-3317Crossref PubMed Scopus (208) Google Scholar; Sarot et al., 2004Sarot E. Payen-Groschêne G. Bucheton A. Pélisson A. Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene.Genetics. 2004; 166: 1313-1321Crossref PubMed Scopus (190) Google Scholar). The evolutionarily conserved maelstrom (mael) gene scored strongly in this assay (Figure 1A). This came as a surprise, as Mael levels are low in ovarian somatic cells (Findley et al., 2003Findley S.D. Tamanaha M. Clegg N.J. Ruohola-Baker H. Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.Development. 2003; 130: 859-871Crossref PubMed Scopus (216) Google Scholar) and a recent study indicated that mael is dispensable for TE silencing in ovarian somatic cells (Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar). To support the gypsy-lacZ results, we induced tissue-specific mael RNAi in soma or germline and analyzed RNA levels of several marker TEs. In both cell types, mael KD resulted in desilencing of TEs to extents comparable to KD of the essential piRNA biogenesis factor Armitage (Armi) (Figures 1B and 1C). Ovaries from mael loss-of-function flies also exhibited derepression of soma and germline transposons (Figure S1A available online; note that Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar did not use mael null alleles).Figure S1Related to Figure 1Show full caption(A) Displayed are fold changes in steady state RNA levels of indicated TEs in mael null mutant ovaries (normalized to mael heterozygote siblings; values are averages of 3 biological replicates (error bars: StDev.).(B) Confocal images of mael heterozygous (top) or mael[M391]/mael[Def] (bottom) egg chambers stained for Piwi, Aub or AGO3.(C) Displayed are fold changes in piwi steady state mRNA levels in OSCs after transfection with indicated siRNAs. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells.(D) Shown are length profiles of small RNAs (normalized to 1 million microRNAs; small insets) isolated from ovaries of mael[M391] heterozygous or mael[391]/mael[Def] flies. siRNA and piRNA populations are indicated.(E) Shown are length profiles of repeat derived ovarian small RNAs (normalized to 1Mio miRNAs) from mael heterozygous and mael[M391]/mael[Def] flies. (red antisense; blue sense). (F) Normalized piRNA profiles (sense up; antisense down; 200nt windows) from mael het. (black) or mael mut. (red) libraries mapping uniquely to the 42AB piRNA cluster.(G) Scatter plot (log2 scale) showing levels of antisense piRNAs mapping to soma dominant (green), intermediate (yellow) or germline dominant (black) TEs in mael het. or mael mut. libraries.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Displayed are fold changes in steady state RNA levels of indicated TEs in mael null mutant ovaries (normalized to mael heterozygote siblings; values are averages of 3 biological replicates (error bars: StDev.). (B) Confocal images of mael heterozygous (top) or mael[M391]/mael[Def] (bottom) egg chambers stained for Piwi, Aub or AGO3. (C) Displayed are fold changes in piwi steady state mRNA levels in OSCs after transfection with indicated siRNAs. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells. (D) Shown are length profiles of small RNAs (normalized to 1 million microRNAs; small insets) isolated from ovaries of mael[M391] heterozygous or mael[391]/mael[Def] flies. siRNA and piRNA populations are indicated. (E) Shown are length profiles of repeat derived ovarian small RNAs (normalized to 1Mio miRNAs) from mael heterozygous and mael[M391]/mael[Def] flies. (red antisense; blue sense). (F) Normalized piRNA profiles (sense up; antisense down; 200nt windows) from mael het. (black) or mael mut. (red) libraries mapping uniquely to the 42AB piRNA cluster. (G) Scatter plot (log2 scale) showing levels of antisense piRNAs mapping to soma dominant (green), intermediate (yellow) or germline dominant (black) TEs in mael het. or mael mut. libraries. To identify the level at which Mael acts in the piRNA pathway, we monitored Piwi in clones of mael KD cells in the follicular epithelium. Defective piRNA biogenesis (e.g., armi KD) triggers loss of Piwi, presumably as unloaded Piwi is unstable (Figure 1D). In contrast, depletion of Mael had no impact on nuclear Piwi levels (Figure 1E). Similarly, levels and localizations of all PIWI proteins were unaffected in soma and germline of mael null ovaries (Figure S1B). This suggested that Mael does not act in piRNA biogenesis. To test this, we monitored TE expression and piRNA levels in OSCs upon mael KD or armi KD (Figures 1F–1I). Both KDs resulted in derepression of the TEs mdg1 and 412, but not of the germline-specific element HeT-A (Figure 1G), demonstrating an essential role for Mael in the OSC piRNA pathway. However, whereas loss of Armi resulted in reduced Piwi protein (Figure 1F; but not mRNA: Figure S1C) as well as in reduced piRNA levels (Figure 1H), loss of Mael did not. The size of Piwi-bound piRNAs was also unaffected upon mael KD (Figure 1I). We finally sequenced piRNAs from mael mutant ovaries and compared them to heterozygous controls. In agreement with the OSC data and in contrast to known primary biogenesis factors, loss of Mael did not affect piRNAs derived from the soma-dominant flamenco cluster or the traffic jam 3′UTR (Figure 1J). For the global pool of ovarian piRNAs (soma and germline), we observed a slight shift toward sense piRNAs, probably due to abundant derepressed TE messages (Figures S1D and S1E). piRNAs derived from the germline-dominant 42AB cluster were moderately reduced (Figure S1F). At the level of most individual TEs, loss of Mael had only mild impacts on antisense piRNA populations from soma-dominant, intermediate, and many germline-dominant TEs (Figure S1G). The most notable exceptions were the telomeric TEs HeT-A, TAHRE, and TART that exhibited strong piRNA losses. We speculate that desilencing of these TEs interferes with piRNA precursor transcription at the same loci, therefore blocking piRNA production. Taken together, Mael is not required for biogenesis or nuclear accumulation of the Piwi-RISC yet is essential for Piwi-mediated TE silencing. The subcellular localization of the Piwi-RISC suggests a nuclear silencing process. Indeed, experiments in OSCs indicated that Piwi's nuclear localization, but not its slicer activity, is required for silencing (Saito et al., 2010Saito K. Ishizu H. Komai M. Kotani H. Kawamura Y. Nishida K.M. Siomi H. Siomi M.C. Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila.Genes Dev. 2010; 24: 2493-2498Crossref PubMed Scopus (234) Google Scholar). Also in flies, N terminally truncated Piwi is cytoplasmic and piwi[ΔN] flies are defective in TE silencing (Klenov et al., 2011Klenov M.S. Sokolova O.A. Yakushev E.Y. Stolyarenko A.D. Mikhaleva E.A. Lavrov S.A. Gvozdev V.A. Separation of stem cell maintenance and transposon silencing functions of Piwi protein.Proc. Natl. Acad. Sci. USA. 2011; 108: 18760-18765Crossref PubMed Scopus (121) Google Scholar). We reconstructed these findings in vivo by complementing piwi[1]/piwi[2] mutant flies with various GFP-tagged genomic piwi rescue constructs. A nine-amino-acid deletion at the N terminus (ΔNLS) largely prevented nuclear accumulation of Piwi-GFP, whereas both slicer mutant GFP-Piwis (ADK or DAK) localized like wild-type GFP-Piwi to the nucleus (Figures 2A and 2B ; efficient loading of all variants with piRNAs verified by IP-CIP-kinase experiments). Real-time quantitative PCR (RT-qPCR) analysis of TE RNA levels showed derepression of soma- and germline-specific TEs in piwi[ΔNLS], but not in piwi[ADK] or piwi[DAK] ovaries (Figure 2C). Moreover, both slicer mutant flies resembled wild-type flies in fertility, whereas only some eggs laid by piwi[ΔNLS] flies developed into larvae and adults. An involvement of Mael in the silencing process predicts a nuclear localization for this protein. In ovaries, endogenous Mael, as well as GFP-tagged Mael expressed under the mael control regions, is abundant in germline cells and localizes to cytoplasm, nuage, and nucleus (Figures 2D and 2E, upper left; Findley et al., 2003Findley S.D. Tamanaha M. Clegg N.J. Ruohola-Baker H. Maelstrom, a Drosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage.Development. 2003; 130: 859-871Crossref PubMed Scopus (216) Google Scholar). As levels in follicle cells were low, we turned to OSCs in which endogenous Mael, as well as N- and C-tagged GFP-Mael, localized throughout the cell but were clearly enriched in the nucleus (Figure 2E, upper right; data not shown). Based on Mael's domain architecture, we tested the requirement of HMG and MAEL domains for nuclear localization and TE silencing in complementation assays using mael loss-of-function alleles and GFP-tagged mael rescue constructs. Whereas the wild-type construct rescued sterility and TE derepression nearly completely, two constructs harboring point mutations in conserved residues of the MAEL domain (Zhang et al., 2008aZhang D. Xiong H. Shan J. Xia X. Trudeau V.L. Functional insight into Maelstrom in the germline piRNA pathway: a unique domain homologous to the DnaQ-H 3′-5′ exonuclease, its lineage-specific expansion/loss and evolutionarily active site switch.Biol. Direct. 2008; 3: 48Crossref PubMed Scopus (42) Google Scholar) did not (Figures 2F and 2G). In both cases, nuclear accumulation of mutant Mael was strongly reduced in ovaries and OSCs (Figure 2E). Loss of the HMG domain had only mild effects on Mael's subcellular localization (Figure 2E). mael[ΔHMG] flies did lay eggs, but these displayed defects in egg asymmetry, presumably as TE silencing was only partially rescued in these flies (Figures 2F and 2G). We conclude that Piwi-mediated silencing is a nuclear process that is independent of Piwi's slicer activity but requires Mael and, in particular, its MAEL domain. To dissect at which step of TE expression Piwi mediates silencing, we took advantage of cultured OSCs. These cells express a functional linear piRNA pathway and allow gene knockdowns using siRNAs. We profiled gene expression at three hierarchical levels in cells treated with GFP siRNAs (control KD) or with siRNAs targeting key pathway factors (piwi KD, armi KD, mael KD; Figure 3A). We first defined the set of TEs that are repressed by the piRNA pathway by comparing steady-state RNA levels (RNA-seq) between control KD and piRNA pathway KD cells. We then determined transcription rates by measuring RNA polymerase II (Pol II) occupancy (Rpb3 chromatin immunoprecipitation sequencing (ChIP-seq); Adelman et al., 2005Adelman K. Marr M.T. Werner J. Saunders A. Ni Z. Andrulis E.D. Lis J.T. Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.Mol. Cell. 2005; 17: 103-112Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) and nascent RNA polymerase output via global run-on sequencing (GRO-seq; Figures S2A and S2B; Core et al., 2008Core L.J. Waterfall J.J. Lis J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.Science. 2008; 322: 1845-1848Crossref PubMed Scopus (1416) Google Scholar).Figure S2Related to Figure 3Show full caption(A) Metagene profiles of normalized RNA-seq, Pol II ChIP-seq and GRO-seq reads around (±0.5 kb) transcriptional start site (TSS) and polyadenylation site (PAS) from indicated siRNA mediated knockdowns (GFP: blue; piwi: red; mael: green). Only genes meeting the following criteria were used: RNA-seq RPKM ≥ 5, length > 1 kb, no overlaps with flanking gene up to 200 nt upstream of annotated TSS; n = 2628; note the strong TSS bias for Pol II occupancy and GRO-seq; note also that only RNA-seq shows the expected drop at the PAS, while Pol II occupancy and GRO-seq do not as expected as they continue to transcribe downstream of the PAS.(B) Heatmap showing TSS data presented in (A) at single gene-resolution. Profiles were sorted for decreasing signal of Pol II ChIP-seq in control knockdowns.(C) Table listing RNA-seq RPKM values for indicated TEs (upper part) or a set of highly expressed genes (lower part) upon indicated siRNA knockdowns in OSC.(D) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in armi KD versus piwi KD OSCs.(E) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in mael KD versus piwi KD OSCs.(F) Displayed are fold changes in steady state RNA levels of indicated TEs and genes upon piwi KD or mael KD or piwi+mael KD in OSCs. Note that the gypsy primer pair spans a splice junction, which explains the higher de-repression values compared to the RNA-seq data. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells.(G) Box plot analysis indicating fold changes (log2) in RNA-seq RPKM values (left) or Pol II occupancy values (right) for indicated TE groups in armi KD or mael KD cells; for the RNA-seq analysis, reads mapping sense or antisense to TEs were contrasted; p-values were computed with Wilcoxon rank-sum test. Box plots show median (line), 25th–75th percentile (box) ± 1.5 interquartile range; circles represent outliers.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Metagene profiles of normalized RNA-seq, Pol II ChIP-seq and GRO-seq reads around (±0.5 kb) transcriptional start site (TSS) and polyadenylation site (PAS) from indicated siRNA mediated knockdowns (GFP: blue; piwi: red; mael: green). Only genes meeting the following criteria were used: RNA-seq RPKM ≥ 5, length > 1 kb, no overlaps with flanking gene up to 200 nt upstream of annotated TSS; n = 2628; note the strong TSS bias for Pol II occupancy and GRO-seq; note also that only RNA-seq shows the expected drop at the PAS, while Pol II occupancy and GRO-seq do not as expected as they continue to transcribe downstream of the PAS. (B) Heatmap showing TSS data presented in (A) at single gene-resolution. Profiles were sorted for decreasing signal of Pol II ChIP-seq in control knockdowns. (C) Table listing RNA-seq RPKM values for indicated TEs (upper part) or a set of highly expressed genes (lower part) upon indicated siRNA knockdowns in OSC. (D) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in armi KD versus piwi KD OSCs. (E) Scatter plot showing RNA-seq RPKM values (log2) of group I-IV TEs in mael KD versus piwi KD OSCs. (F) Displayed are fold changes in steady state RNA levels of indicated TEs and genes upon piwi KD or mael KD or piwi+mael KD in OSCs. Note that the gypsy primer pair spans a splice junction, which explains the higher de-repression values compared to the RNA-seq data. Values are averages of 3 biological replicates (error bars: StDev.) and normalized to GFP siRNA treated cells. (G) Box plot analysis indicating fold changes (log2) in RNA-seq RPKM values (left) or Pol II occupancy values (right) for indicated TE groups in armi KD or mael KD cells; for the RNA-seq analysis, reads mapping sense or antisense to TEs were contrasted; p-values were computed with Wilcoxon rank-sum test. Box plots show median (line), 25th–75th percentile (box) ± 1.5 interquartile range; circles represent outliers. To determine steady-state RNA levels, we sequenced total RNA after removal of ribosomal RNA. Reads per kilobase per million mapped reads (RPKM) values for annotated genes were highly correlated between piwi KD and control KD cells (Pearson correlation coefficient 0.95). In contrast, several out of the 125 annotated D. melanogaster TE families showed strong increases in RNA levels (Figure 3B). For example, the LTR elements mdg1 or gypsy increased by >200- or >30-fold upon piwi KD, respectively. With RPKM values of >1,000, both TEs were among the most abundant coding transcripts in OSCs (Figure S2C). Almost identical results were obtained upon knockdown of Armi (Figure S2D; Pearson correlation coefficient piwi KD/armi KD 0.99). Thus, loss of the Piwi-RISC led to highly reproducible increases in the RNA levels of a subset of TEs. Based on these data, we classified TEs into four groups (Figure 3B). Group I elements exhibited RNA increases >10-fold; group II elements. 3- to 10-fold; group III elements, <3-fold; and group IV elements were expressed below an RPKM cutoff of five in any of the analyzed libraries (no or very low expression and not further analyzed). Knockdown of Mael resulted in slightly weaker but otherwise very similar derepression of TEs at the RNA level (Figures 3C and S2E). This strongly supports the notion that Mael is an integral piRNA pathway factor. Indeed, piwi+mael double-KD cells exhibited TE derepression similar to piwi KD cells, indicating that both proteins act in the same pathway (Figure S2F). Strikingly, changes in steady-state RNA levels were highly correlated with changes in RNA Pol II occupancy (Pol II ChIP-seq; piwi KD and mael KD), as well as with changes in nascent RNA levels (GRO-seq; piwi KD; Figure 3D). Upon piRNA pathway KD, both measures indicative of active transcription were strongly increased in the LTR regions (containing the TSS), as well as in the internal portions of regulated TEs, but not of nonregulated TEs (Figure 3E). For example, overall Pol II occupancy and nascent RNA levels for mdg1 increased 6.5-fold and 22-fold, respectively. For all three assays, the respective controls (antisense reads for RNA-seq; input for Pol II ChIP-seq; antisense reads for GRO-seq) showed no difference among the three TE groups (Figures 3F–3H and S2G). This argues against a general increase in repeat or heterochromatin transcription upon pathway loss