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• W2807631303 abstract "•Maelstrom represses canonical transcription within fly dual-strand piRNA clusters•Maelstrom also represses canonical transcription in conventional heterochromatin•Maelstrom can be guided to its targets both by piRNAs and piRNA-independent mechanisms In Drosophila, 23–30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins. In Drosophila, 23–30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. Here, we report that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. We propose that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins. In Drosophila melanogaster, 23–30 nt long PIWI-interacting RNAs (piRNAs) direct transposon silencing by serving as guides for Argonaute3 (Ago3), Aubergine (Aub), and Piwi, the three fly PIWI proteins (Aravin et al., 2001Aravin A.A. Naumova N.M. Tulin A.V. Vagin V.V. Rozovsky Y.M. Gvozdev V.A. Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline.Curr. Biol. 2001; 11: 1017-1027Abstract Full Text Full Text PDF PubMed Scopus (569) Google Scholar, Vagin et al., 2006Vagin V.V. Sigova A. Li C. Seitz H. Gvozdev V. Zamore P.D. A distinct small RNA pathway silences selfish genetic elements in the germline.Science. 2006; 313: 320-324Crossref PubMed Scopus (975) Google Scholar). In the germ cell cytoplasm, Aub and Ago3 increase the abundance of their guide piRNAs via the ping-pong cycle, an amplification loop in which cycles of piRNA-directed cleavage of sense and antisense transposon-derived long RNAs generate new copies of the original piRNAs in response to transposon transcription (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1089-1103Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar, Gunawardane et al., 2007Gunawardane L.S. Saito K. Nishida K.M. Miyoshi K. Kawamura Y. Nagami T. Siomi H. Siomi M.C. A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila.Science. 2007; 315: 1587-1590Crossref PubMed Scopus (875) Google Scholar). In addition to amplifying piRNAs, this “ping-pong” pathway also produces long 5′ monophosphorylated RNA that enters the phased piRNA pathway, generating head-to-tail strings of piRNAs bound to Piwi and, to a lesser extent, Aub (Han et al., 2015aHan B.W. Wang W. Li C. Weng Z. Zamore P.D. Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production.Science. 2015; 348: 817-821Crossref PubMed Scopus (231) Google Scholar, Mohn et al., 2015Mohn F. Handler D. Brennecke J. Noncoding RNA. piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis.Science. 2015; 348: 812-817Crossref PubMed Scopus (205) Google Scholar, Wang et al., 2015Wang W. Han B.W. Tipping C. Ge D.T. Zhang Z. Weng Z. Zamore P.D. Slicing and binding by Ago3 or Aub trigger piwi-bound piRNA production by distinct mechanisms.Mol. Cell. 2015; 59: 819-830Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Unlike Ago3 and Aub, Piwi acts in both the germline and the adjacent somatic follicle cells to repress transposon transcription rather than to cleave their transcripts (Li et al., 2009aLi C. Vagin V.V. Lee S. Xu J. Ma S. Xi H. Seitz H. Horwich M.D. Syrzycka M. Honda B.M. et al.Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies.Cell. 2009; 137: 509-521Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, Malone et al., 2009Malone C.D. Brennecke J. Dus M. Stark A. McCombie W.R. Sachidanandam R. Hannon G.J. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary.Cell. 2009; 137: 522-535Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Nuclear Piwi is believed to bind nascent RNA transcripts and, through the protein Panoramix, tether the histone methyltransferase SETDB1 to transposon-containing loci. SETDB1, in turn, trimethylates histone H3 on lysine 9 (H3K9me3), a modification required to create repressive heterochromatin (Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, Muerdter et al., 2013Muerdter F. Guzzardo P.M. Gillis J. Luo Y. Yu Y. Chen C. Fekete R. Hannon G.J. A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.Mol. Cell. 2013; 50: 736-748Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Klenov et al., 2014Klenov M.S. Lavrov S.A. Korbut A.P. Stolyarenko A.D. Yakushev E.Y. Reuter M. Pillai R.S. Gvozdev V.A. Impact of nuclear Piwi elimination on chromatin state in Drosophila melanogaster ovaries.Nucleic Acids Res. 2014; 42: 6208-6218Crossref PubMed Scopus (58) Google Scholar, Sienski et al., 2015Sienski G. Batki J. Senti K.A. Dönertas D. Tirian L. Meixner K. Brennecke J. Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery.Genes Dev. 2015; 29: 2258-2271Crossref PubMed Scopus (105) Google Scholar, Yu et al., 2015Yu Y. Gu J. Jin Y. Luo Y. Preall J.B. Ma J. Czech B. Hannon G.J. Panoramix enforces piRNA-dependent cotranscriptional silencing.Science. 2015; 350: 339-342Crossref PubMed Scopus (128) Google Scholar). piRNA precursor RNAs are transcribed from piRNA clusters, heterochromatic loci that comprise transposons and transposon fragments, thereby recording a species’ evolutionary history of transposon invasion (Brennecke et al., 2007Brennecke J. Aravin A.A. Stark A. Dus M. Kellis M. Sachidanandam R. Hannon G.J. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila.Cell. 2007; 128: 1089-1103Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar). Drosophila piRNA clusters can be uni-strand, transcribed from one genomic strand, or dual strand, transcribed from both genomic strands. Uni-strand clusters, such as the ∼180 kbp flamenco (flam) locus, silence transposons in somatic follicle cells (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, Mével-Ninio et al., 2007Mével-Ninio M. Pelisson A. Kinder J. Campos A.R. Bucheton A. The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis.Genetics. 2007; 175: 1615-1624Crossref PubMed Scopus (91) Google Scholar), whereas dual-strand clusters, such as the ∼250 kbp 42AB locus, predominate in the germline (Malone et al., 2009Malone C.D. Brennecke J. Dus M. Stark A. McCombie W.R. Sachidanandam R. Hannon G.J. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary.Cell. 2009; 137: 522-535Abstract Full Text Full Text PDF PubMed Scopus (616) Google Scholar). Some uni-strand clusters, such as cluster 2, are active in both tissues. Canonical, promoter-initiated, RNA polymerase II (Pol II) transcription generates spliced, polyadenylated precursor piRNAs from flam (Mével-Ninio et al., 2007Mével-Ninio M. Pelisson A. Kinder J. Campos A.R. Bucheton A. The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis.Genetics. 2007; 175: 1615-1624Crossref PubMed Scopus (91) Google Scholar, Goriaux et al., 2014Goriaux C. Desset S. Renaud Y. Vaury C. Brasset E. Transcriptional properties and splicing of the flamenco piRNA cluster.EMBO Rep. 2014; 15: 411-418Crossref PubMed Scopus (72) Google Scholar). In contrast, most dual-strand clusters do not use standard promoters. Instead, the heterochromatin protein 1 paralog Rhino (Rhi) binds to H3K9me3 present on the piRNA cluster chromatin and, in conjunction with its protein partners, drives non-canonical transcription (Volpe et al., 2001Volpe A.M. Horowitz H. Grafer C.M. Jackson S.M. Berg C.A. Drosophila rhino encodes a female-specific chromo-domain protein that affects chromosome structure and egg polarity.Genetics. 2001; 159: 1117-1134PubMed Google Scholar, Klattenhoff et al., 2009Klattenhoff C. Xi H. Li C. Lee S. Xu J. Khurana J.S. Zhang F. Schultz N. Koppetsch B.S. Nowosielska A. et al.The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters.Cell. 2009; 138: 1137-1149Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, Zhang et al., 2012aZhang F. Wang J. Xu J. Zhang Z. Koppetsch B.S. Schultz N. Vreven T. Meignin C. Davis I. Zamore P.D. et al.UAP56 couples piRNA clusters to the perinuclear transposon silencing machinery.Cell. 2012; 151: 871-884Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, Zhang et al., 2014Zhang Z. Wang J. Schultz N. Zhang F. Parhad S.S. Tu S. Vreven T. Zamore P.D. Weng Z. Theurkauf W.E. The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing.Cell. 2014; 157: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Le Thomas et al., 2014Le Thomas A. Stuwe E. Li S. Du J. Marinov G. Rozhkov N. Chen Y.C. Luo Y. Sachidanandam R. Toth K.F. et al.Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing.Genes Dev. 2014; 28: 1667-1680Crossref PubMed Scopus (135) Google Scholar, Mohn et al., 2014Mohn F. Sienski G. Handler D. Brennecke J. The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila.Cell. 2014; 157: 1364-1379Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). One Rhi-associated protein, Moonshiner (Moon), is a germline-specific TFIIA-L paralog that allows Pol II to initiate transcription without promoter sequences, allowing every bound Rhi to be a site of potential transcriptional initiation (Andersen et al., 2017Andersen P.R. Tirian L. Vunjak M. Brennecke J. A heterochromatin-dependent transcription machinery drives piRNA expression.Nature. 2017; 549: 54-59Crossref PubMed Scopus (125) Google Scholar). Another Rhi-binding protein, Cutoff (Cuff), suppresses splicing and transcriptional termination (Pane et al., 2011Pane A. Jiang P. Zhao D.Y. Singh M. Schüpbach T. The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline.EMBO J. 2011; 30: 4601-4615Crossref PubMed Scopus (82) Google Scholar, Mohn et al., 2014Mohn F. Sienski G. Handler D. Brennecke J. The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila.Cell. 2014; 157: 1364-1379Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Zhang et al., 2014Zhang Z. Wang J. Schultz N. Zhang F. Parhad S.S. Tu S. Vreven T. Zamore P.D. Weng Z. Theurkauf W.E. The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing.Cell. 2014; 157: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Chen et al., 2016Chen Y.A. Stuwe E. Luo Y. Ninova M. Le Thomas A. Rozhavskaya E. Li S. Vempati S. Laver J.D. Patel D.J. et al.Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors.Mol. Cell. 2016; 63: 97-109Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Thus, Rhi promotes non-canonical transcription: RNA synthesis initiating at many sites throughout both strands of a dual-strand cluster, in contrast to the canonical, promoter-dependent transcription of flam and conventional protein-coding genes. Maelstrom (Mael), a protein with HMG (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 MAEL (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) domains, has been suggested to play multiple roles in Drosophila oogenesis and mouse spermatogenesis, including repression of the microRNA miR-7 (Pek et al., 2009Pek J.W. Lim A.K. Kai T. Drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microRNA-7.Dev. Cell. 2009; 17: 417-424Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), transposon silencing (Lim and Kai, 2007Lim A.K. Kai T. Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster.Proc. Natl. Acad. Sci. U S A. 2007; 104: 6714-6719Crossref PubMed Scopus (272) Google Scholar, Soper et al., 2008Soper S.F. van der Heijden G.W. Hardiman T.C. Goodheart M. Martin S.L. de Boer P. Bortvin A. Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis.Dev. Cell. 2008; 15: 285-297Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), heterochromatin formation (Pek et al., 2009Pek J.W. Lim A.K. Kai T. Drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microRNA-7.Dev. Cell. 2009; 17: 417-424Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), and piRNA production (Castañeda et al., 2014Castañeda J. Genzor P. van der Heijden G.W. Sarkeshik A. Yates 3rd, J.R. Ingolia N.T. Bortvin A. Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice.EMBO J. 2014; 33: 1999-2019Crossref PubMed Scopus (67) Google Scholar). Here, we report that Mael suppresses canonical transcription both within and outside dual-strand piRNA clusters. In mael-mutant ovaries, piRNA cluster heterochromatin organization is largely unaltered, but transcription initiates from previously silent canonical Pol II promoters, including sites within dual-strand clusters. Transcriptional repression mediated by Mael occurs at many sites across the genome; at some, Mael collaborates with the piRNA pathway, while at others Mael repression is piRNA independent. We propose that Mael represses promoter-driven transcription of individual, potentially active transposons, allowing Rhi to transcribe such transposon sequences into intron-containing piRNA precursors with little potential to be translated into proteins required for transposition. Without Mael, both somatic and germline transposons produce long RNA transcripts (Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, Muerdter et al., 2013Muerdter F. Guzzardo P.M. Gillis J. Luo Y. Yu Y. Chen C. Fekete R. Hannon G.J. A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.Mol. Cell. 2013; 50: 736-748Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Pek et al., 2009Pek J.W. Lim A.K. Kai T. Drosophila maelstrom ensures proper germline stem cell lineage differentiation by repressing microRNA-7.Dev. Cell. 2009; 17: 417-424Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), while protein-coding genes are largely unaffected (Figure S1). For example, in the germline of maelM391/r20 ovaries, steady-state RNA abundance from telomeric transposons increased >360-fold for HeT-A and TART and ∼49-fold for TAHRE (n = 3; Figure S1). Intriguingly, RNA increased >13-fold from two individual gypsy12 long terminal repeat (LTR) transposon insertions: one in the dual-strand piRNA cluster 42AB (at 42A14; hereafter gypsy1242AB) and one in the dual-strand piRNA cluster cluster62 (at 40F7; hereafter gypsy12cluster62) (Figures 1 and S1). The same two gypsy12 elements are also desilenced in Rhi- or Cuff-deficient ovaries (Zhang et al., 2014Zhang Z. Wang J. Schultz N. Zhang F. Parhad S.S. Tu S. Vreven T. Zamore P.D. Weng Z. Theurkauf W.E. The HP1 homolog rhino anchors a nuclear complex that suppresses piRNA precursor splicing.Cell. 2014; 157: 1353-1363Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Mohn et al., 2014Mohn F. Sienski G. Handler D. Brennecke J. The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila.Cell. 2014; 157: 1364-1379Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). As in rhi and cuff, but unlike wild-type, RNA from the two gypsy12 LTRs was spliced in maelM391/r20-mutant ovaries. The increase in steady-state gypsy12 LTR RNA from these two loci in maelM391/r20 mutants reflects a concomitant increase in nascent transcription as measured by global run-on sequencing (GRO-seq; Figure 1; 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). Lysine 4 trimethylation of histone H3 (H3K4me3), a chromatin mark associated with active, promoter-driven transcription (Bernstein et al., 2002Bernstein B.E. Humphrey E.L. Erlich R.L. Schneider R. Bouman P. Liu J.S. Kouzarides T. Schreiber S.L. Methylation of histone H3 Lys 4 in coding regions of active genes.Proc. Natl. Acad. Sci. U S A. 2002; 99: 8695-8700Crossref PubMed Scopus (593) Google Scholar, Santos-Rosa et al., 2002Santos-Rosa H. Schneider R. Bannister A.J. Sherriff J. Bernstein B.E. Emre N.C. Schreiber S.L. Mellor J. Kouzarides T. Active genes are tri-methylated at K4 of histone H3.Nature. 2002; 419: 407-411Crossref PubMed Scopus (1597) Google Scholar, Schneider et al., 2004Schneider R. Bannister A.J. Myers F.A. Thorne A.W. Crane-Robinson C. Kouzarides T. Histone H3 lysine 4 methylation patterns in higher eukaryotic genes.Nat. Cell Biol. 2004; 6: 73-77Crossref PubMed Scopus (615) Google Scholar), also increased at both gypsy1242AB and gypsy12cluster62 (>3-fold and >9-fold, respectively; n = 2; Figure 1). These data suggest that in the absence of Mael, RNA Pol II initiates canonical transcription within the gypsy12 LTR. A combination of canonical and Rhi-dependent transcription has been proposed to produce piRNA precursor RNA from dual-strand cluster 38C1: two promoters flanking the cluster initiate canonical transcription, while Rhi ensures non-canonical, promoter-independent transcription within the cluster (Mohn et al., 2014Mohn F. Sienski G. Handler D. Brennecke J. The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila.Cell. 2014; 157: 1364-1379Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, Andersen et al., 2017Andersen P.R. Tirian L. Vunjak M. Brennecke J. A heterochromatin-dependent transcription machinery drives piRNA expression.Nature. 2017; 549: 54-59Crossref PubMed Scopus (125) Google Scholar). Loss of Mael led to increased use of the two canonical cluster 38C1 promoters: in maelM391/r20 ovaries, transcription initiating at the TATA-box sequences flanking the cluster increased for both plus (mean increase mael/control = 3 ± 1; p = 0.046) and minus (mean increase mael/control = 5 ± 2; p = 0.014) genomic strands (Figure 1). Similarly, the steady-state abundance of cluster 38C1 RNA >150 nt long increased ∼15-fold in maelM391/r20 ovaries (mean mael/control = 15 ± 5; p = 3.1 × 10−4). However, the density of the active chromatin mark H3K4me3 at the flanking promoters of cluster 38C1 was essentially unchanged in maelM391/r20 ovaries (mean mael/control = 1.2 ± 0.2; p = 0.34 for the left promoter; mean mael/control = 1.4 ± 0.4; p = 0.26 for the right promoter; Figure 1). We speculate that the absence of a change in H3K4me3 in maelM391/r20-mutant ovaries reflects the pre-existing, canonical, promoter-driven transcription at cluster 38C1 and its accompanying high levels of H3K4me3. Together, these data suggest that Mael is required for repression of both transposons outside piRNA clusters and canonical transcription within dual-strand piRNA clusters. piRNA clusters are highly repetitive, complicating bioinformatic analyses. To test the idea that Mael represses canonical transcription within dual-strand clusters, we used a fly strain (P{GSV6}42A18) bearing a GAL4-responsive gfp transgene inserted into cluster 42AB. The transgene contains five tandem UAS repeats and a core promoter derived from Hsp70Bb (Figure S2A; Toba et al., 1999Toba G. Ohsako T. Miyata N. Ohtsuka T. Seong K.H. Aigaki T. The gene search system. A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster.Genetics. 1999; 151: 725-737PubMed Google Scholar). In the presence of the germline-specific transcriptional activator nanos-Gal4, the non-repetitive gfp sequence provides a proxy for canonical euchromatic transcription within piRNA clusters. Both gfp mRNA and protein were undetectable even in the presence of nanos-GAL4-VP16 (Figures 2A and 2B ). Like 42AB itself, the gfp transgene has a high density of H3K9me3, HP1a, and Rhi across its sequence. Moreover, the reporter construct produces more piRNAs from the (+) genomic strand, as is true for the region of 42AB into which it is inserted. Finally, production of sense and antisense piRNAs from the gfp transgene requires Rhi, Cuff, Piwi, and Armi, proteins all required for piRNA production from dual-strand clusters (Figure S2B; Han et al., 2015aHan B.W. Wang W. Li C. Weng Z. Zamore P.D. Noncoding RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production.Science. 2015; 348: 817-821Crossref PubMed Scopus (231) Google Scholar). In maelM391/r20-mutant ovaries, the P{GSV6}42A18 transgene driven by GAL4-VP16 produced correctly spliced gfp mRNA that terminated at a canonical polyadenylation signal sequence; the appearance of gfp mRNA was accompanied by increased transcription (mean mael/control = 80 ± 60; n = 3; p = 3.0 × 10−3) and H3K4me3 (>3-fold increase) across the gfp transgene (Figure 2A). Moreover, the gfp mRNA in maelM391/r20 mutants was translated into full-length GFP protein (Figures 2B and S2C). Finally, a transgene encoding FLAG-Mael restored repression of gfp in maelM391/r20, demonstrating that loss of Mael, not a secondary mutation, caused inappropriate GFP expression from the transgene inserted in 42AB (Figure 2B). The number of piRNAs antisense to gfp mRNA decreased ∼13-fold (n = 3, p = 10−4) in maelM391/r20 (Figure 2A). In theory, gfp derepression in maelM391/r20 could be explained by the loss of piRNA-directed silencing. Moreover, the P{GSV6}42A18 transgene contains 248 bp of Hsp70 5′ UTR sequence, which is complementary to endogenous antisense piRNAs from the endogenous Hsp70 locus (DeLuca and Spradling, 2018DeLuca S.Z. Spradling A.C. Efficient expression of genes in the Drosophila germline using a UAS promoter free of interference by Hsp70 piRNAs.Genetics. 2018; 209: 381-387Crossref PubMed Scopus (27) Google Scholar, Huang et al., 2018Huang Y.C. Moreno H. Row S. Jia D. Deng W.M. Germline silencing of UASt depends on the piRNA pathway.J. Genet. Genomics. 2018; 45: 273-276Crossref PubMed Scopus (3) Google Scholar). To test the possibility that reporter derepression reflects impaired piRNA-directed silencing, we examined the effect of loss of Mael on gfp expression in a fly strain bearing an identical gfp transgene inserted in the first intron of the heterochromatic, subtelomeric protein-coding gene, zip (P{GSV6}zip; Figure 2C). zip is expressed in the germline, and P{GSV6}zip reflects the expression of zip, producing full-length, spliced mRNA in control ovaries (Figure 2B). Like P{GSV6}42A18, expression of P{GSV6}zip increased without Mael: P{GSV6}zip produced >10-fold more gfp mRNA in maelM391/r20 ovaries (mean mael/control = 11 ± 2; n = 3; p = 7.9 × 10−5). Unlike P{GSV6}42A18, P{GSV6}zip generated more rather than fewer gfp piRNAs without Mael (mean mael/control = 14 ± 3; n = 3; p = 4.4 × 10−4). Nearly all of these piRNAs derived from the gfp mRNA; few were from the intron in the gfp 3′ UTR. These data suggest that without Mael, increased transcription of P{GSV6}zip provides more mRNA both for translation into GFP and for processing into piRNAs. We conclude that loss of piRNA-directed silencing alone cannot explain the derepression of either P{GSV6}42A18 or P{GSV6}zip in maelM391/r20. The arrangement of piRNAs within the gfp mRNA from P{GSV6}zip suggests that piRNA production is initiated by endogenous antisense Hsp70 piRNAs: the majority of phased, sense gfp piRNAs map immediately downstream of sites predicted to be cleaved by initiator Hsp70 piRNAs in both maelM391/r20 and control ovaries (Figure 2C). To test whether Hsp70 piRNAs initiate phased piRNA production from spliced P{GSV6}zip transcripts, we measured the distance from the 5′ ends of piRNAs that both have a ping-pong partner on the opposite genomic strand and arise from the Hsp70 region of the reporter to the 5′ ends of piRNAs without a ping-pong partner, that is, the 5′-to-5′ distance from the putative responder piRNAs to the putative phased piRNAs (Gainetdinov et al., 2018Gainetdinov I. Colpan C. Arif A. Cecchini K. Zamore P.D. A single mechanism of biogenesis, initiated and directed by PIWI proteins, explains piRNA production in most animals.Mol. Cell. 2018; 71: 775-790.e5Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). As expected if Hsp70 piRNAs initiate phased piRNA production from the reporter transcript, the probability plot exhibited periodic peaks corresponding to the 5′ ends of phased piRNAs downstream of initiating Hsp70 ping-pong pairs (Figure 2D). Together, our data demonstrate that gfp derepression in maelM391/r20-mutant ovaries does not reflect a loss of piRNAs targeting the gfp reporter but rather corresponds to an independent function of Mael in repressing canonical transcription. Without Mael, RNA accumulates from both individual transposons outside clusters (Sienski et al., 2012Sienski G. Dönertas D. Brennecke J. Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression.Cell. 2012; 151: 964-980Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar, Muerdter et al., 2013Muerdter F. Guzzardo P.M. Gillis J. Luo Y. Yu Y. Chen C. Fekete R. Hannon G.J. A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila.Mol. Cell. 2013; 50: 736-748Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) and transposon sequences within heterochromatic piRNA dual-strand clusters (Figures S1 and S3A). To further test the idea that Mael represses canonical transcription at sites of Rhi-driven non-canonical transcription within and outside dual-strand clusters, we examined in more detail changes in the transcription of transposons in maelM391/r20 ovaries. Among those individual transposons whose steady-state mRNA level changed significantly (increased or decreased ≥2-fold; false discovery rate [FDR] ≤ 0.05) in maelM391/r20 ovaries, the overw" @default.
• W2807631303 created "2018-06-13" @default.
• W2807631303 creator A5020225652 @default.
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• W2807631303 creator A5038473436 @default.
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• W2807631303 creator A5091631127 @default.
• W2807631303 date "2019-01-01" @default.
• W2807631303 modified "2023-10-16" @default.
• W2807631303 title "Maelstrom Represses Canonical Polymerase II Transcription within Bi-directional piRNA Clusters in Drosophila melanogaster" @default.
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• W2807631303 doi "https://doi.org/10.1016/j.molcel.2018.10.038" @default.
• W2807631303 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6551610" @default.

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