Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery

Abstract

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWIpiRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwimediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged PiwipiRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.[Keywords: H3K9 methylation; Piwi; transposon silencing; heterochromatin formation; piRNA pathway; transcriptional silencing] Supplemental material is available for this article. In fungi, plants, and animals, nuclear Argonaute proteins target nascent transcripts-RNAs that are still attached to their originating DNA locus via the transcribing RNA polymerase. This provides an opportunity for small RNA silencing pathways to connect to the various cellular chromatin-modifying activities for the sequence-specific formation of heterochromatin, typically at transposable element (TE) insertions (Castel and Martienssen 2013).Pioneering work in fission yeast has identified methylation of histone H3 at Lys9 (H3K9me2/3) and histone deacetylation as two major hallmarks that are required for small RNA-guided silencing and heterochromatin formation (Nakayama et al. 2001;Hall et al. 2002;Volpe et al. 2002;Yamada et al. 2005). A sequential order of events downstream from the recruitment of the Schizosaccharomyces pombe Ago1/siRNA complex to nascent RNA of centromeric repeats has been described. According to this, the Ago1/siRNA complex recruits the H3K9 methyltransferase Clr4 to chromatin, which results in H3K9 methylation (Noma et al. 2004;Verdel et al. 2004). This is believed to be a binding platform for the chromodomain-containing protein Swi6/HP1, which in turn recruits the major histone deacetylase and remodeling complex SHREC (Sugiyama et al. 2007). The final outcome of these events is the establishment of a nucleosome-dense region with low histone turnover, which effectively prevents the recruitment of RNA polymerase II (Pol II) to transcription initiation sites and hence transcription as such (e.g., Aygun et al. 2013).H3K9 methylation is also a hallmark of small RNAguided heterochromatin formation in plants, ciliates, and multicellular animals. In animal gonads, many TE insertions are methylated at...

piRNA-mediated regulation of transposon alternative splicing in the soma and germ line

“…Drosophila melanogaster stocks used: w 1118 (R. Lehmann); Harwich (Bloomington Drosophila Stock Center, BDSC# 4264); w;; P nos ::egfp-moe::nos -3’UTR 31 , w;; hsp83-IVS3-LacZ-neo/TM3 Sb 20 , w;; nos-GAL4::VP16 32 , P{UAS-Dcr-2.D}1 w 1118 (BDSC# 24646), w 1118 ; piwi 1 /CyO, w + (BDSC# 43637), w 1118 ; piwi 2 /CyO, w + (BDSC# 43319), w 1118 ; aub QC42 cn 1 bw 1 /CyO, P{sevRas1.V12}FK1 (BDSC# 4968), aub HN2 cn 1 bw 1 /CyO (gift from P. Macdonald), bw 1 ; st 1 ago3 t2 /TM6B , bw 1 ; st 1 ago3 t3 /TM6B (gifts from P. Zamore), ru 1 st 1 spn-E 1 e 1 ca 1 /TM3 Sb 1 Ser 1 (BDSC# 3327), w;; FRT82B spn-E 100.37 e 1 /TM3 Sb (R. Lehmann), vas PH165 /CyO 33 , w 1118 ; Df(2L)osp29, Adh UF osp 29 pr 1 cn 1 /CyO, P{sevRas1.V12}FK1 (BDSC# 3078), w 1118 ; CG9754 [m1-9] /CyO (VDRC# v313502; Panx/Silencio mutant from J. Brennecke 9 ), w 1118 ; CG9754 [m3-4] /CyO (VDRC# v313500; Panx/Silencio mutant from J. Brennecke 9 ), w m4h ;; ago2 414 /TM6b (gift from R. Carthew), w 1118 ;; Df(3L)BSC558/TM6c Sb (BDSC# 25120), dcr2 L811fsX , P{GMR-w.IR}; Df(2R)BSC45/CyO (gifts from E. Lai), aub -shRNA (Vienna Drosophila Resource Center, VDRC# v313413), piwi -dsRNA (VDRC# v101658), arx/Gtsf1 -dsRNA (VDRC# v40408), Panx/Silencio -dsRNA (VDRC# v102702), mael -dsRNA (VDRC# v100907), Su(var)205/HP1a -shRNA (BDSC# 36792), white -dsRNA (dsRNA control, VDRC# v30033), mCherry -shRNA (shRNA control, BDSC# 35785).…”

“…Unexpectedly, we show that the piRNA pathway components do not act to reduce P -element transposon transcript levels during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila 3,4 . Instead, splicing regulation is mechanistically achieved in concert with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix/Gtsf1 5–7 and Panoramix/Silencio 8,9 , as well as Heterochromatin Protein 1a (Su(var)205/HP1a). Furthermore, we show that this machinery, together with the piRNA Flamenco cluster 10 , not only controls the accumulation of Gypsy retrotransposon transcripts 11 but also regulates splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events 12,13 .…”

“…Piwi-interacting proteins such as Asterix/Gtsf1 (Arx) and Panoramix/Silencio (Panx) are dispensable for piRNA biogenesis but are essential for establishing piwi-mediated chromatin changes, possibly by acting as a scaffold to recruit histone modifying enzymes and chromatin-binding proteins to target loci 8,9 . To test the role of these chromatin regulators on P -element splicing, we performed germline-specific RNAi knockdown experiments in the Harwich background.…”

“…Interestingly, it has been shown that mutations in flamenco not only elicited the accumulation of Gypsy RNA, but also modulated pre-mRNA splicing, favoring the production of the Env protein mRNA and therefore germline infection 10 . To test whether the piRNA pathway, in addition to its role in regulating the accumulation of Gypsy RNA 11 , is also responsible for modulating the splicing of Gypsy elements in somatic tissues, we analyzed publically available RNA sequencing data from poly (A)-selected RNAs extracted from Ovarian Somatic Cell (OSC) in vitro cultures 7,9,25 . Our analysis indicates that piwi knockdown was sufficient to modulate Gypsy splicing, favoring the accumulation of env -encoding mRNA (Fig.…”

Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway

“…Nuclear PIWI proteins, along with cofactors (Asterix and Panoramix in flies), recruit histone-modifying enzymes, including deacetylases and methyltransferases, to establish a chromatin signature correlated with transcriptional repression (Dönertas et al 2013;Muerdter et al 2013;Ohtani et al 2013;Rozhkov et al 2013;Sienski et al 2015;Yu et al 2015).…”