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RESEARCH COMMUNICATION

Loss of l(3)mbt leads
to acquisition of the ping-pongcycle in Drosophila ovariansomatic cells
Tetsutaro Sumiyoshi,1,3 Kaoru Sato,1,3
Hitomi Yamamoto,1 Yuka W. Iwasaki,2Haruhiko Siomi,2 and Mikiko C. Siomi1
1

Department of Biological Sciences, Graduate School of Science,
The University of Tokyo, Tokyo 113-0032, Japan; 2Department of
Molecular Biology, Keio University School of Medicine, Tokyo
162-8582, JapanIn Drosophila germ cells, PIWI-interacting RNAs (piRNAs) are amplified through a PIWI slicer-dependent
feed-forward loop termed the ping-pong cycle, yielding
secondary piRNAs. However, the detailed mechanism remains poorly understood, largely because an ex vivo model system amenable to biochemical analyses has not been
available. Here, we show that CRISPR-mediated loss offunction of lethal (3) malignant brain tumor [l(3)mbt]
leads to ectopic activation of the germ-specific pingpong cycle in ovarian somatic cells. Perinuclear foci resembling nuage, the ping-pong center, appeared followingl(3)mbt mutation. This activation of the ping-pong machinery in cultured cells will greatly facilitate elucidationof the mechanism underlying secondary piRNA biogenesis in Drosophila.
Supplemental material is available for this article.
Received May 6, 2016; revised version accepted June 15, 2016.

PIWI-interacting RNAs (piRNAs) form piRNA-induced
silencing complexes (piRISCs) with PIWI proteins that repress transposons to maintain the integrity of the germline genome (Aravin et al. 2007; Brennecke et al. 2007;
Ghildiyal and Zamore 2009; Juliano et al. 2011; Iwasaki
et al. 2015). Somatic primary piRNA biogenesis in Drosophila has been extensively studied, mainly genetically andbiochemically, using ovaries and a cultured ovariansomatic cell (OSC) line (Niki et al. 2006; Malone et al.
2009; Saito et al. 2009). According to the current model,
piRNA precursors arising from piRNA clusters accumulate at specific granules, termed the Yb body and Dot
COM, and are then processed at Yb bodies (Saito et al.
2010; Dennis et al. 2013; Murota et al. 2014). In parallel,
Yb centralizes multiple primary piRNA factors to Yb bodies, including Armitage (Armi), Vreteno (Vret), Sister of Yb(SoYb), and Shutdown (Shu) (Iwasaki et al. 2015). Yb bodies are often surrounded by mitochondria (Murota et al.
2014). This unique spatial arrangement in the cytoplasm[Keywords: piRNA; PIWI; ping-pong cycle; l(3)mbt; CRISPR; Drosophila]
3
These authors contributed equally to this work.
Corresponding author: siomim@bs.s.u-tokyo.ac.jp
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.283929.
116. Freely available online through the Genes & Development Open Access option.

enables Zucchini (Zuc), an endonuclease located on thesurface of mitochondria with its active site facing the cytosol, to process piRNA intermediates into mature piRNAs (Ipsaro et al. 2012; Nishimasu et al. 2012). PrimarypiRNAs form piRISCs with Piwi but not with the other
PIWI members Aub and AGO3 because of their absence
in somatic cells. Piwi–piRISCs translocated to the nucleus
transcriptionally repress transposons (Iwasaki et al. 2015).
Germ cells in Drosophila express Aub, AGO3, and Piwi
(Saito et al. 2006; Brennecke et al. 2007; Gunawardane
et al. 2007; Nishida et al. 2007). Of those, Aub and Piwi,
but not AGO3, are loaded with primary piRNAs derived
from piRNA clusters (Li et al. 2009; Malone et al. 2009).
Piwi–piRISCs are then transported to the nucleus, where
they repress transposons transcriptionally. In contrast,
Aub–piRISCs remain in the cytoplasm and repress transposons by cleaving their mRNAs in a slicer-dependent
manner. This Aub slicer-dependent RNA cleavage gives
rise to secondary piRNAs, which are loaded onto AGO3.
Aub–piRISCs and AGO3–piRISCs then operate a slicerdependent feed-forward loop termed the ping-pong cycle,
yielding a substantial amount of secondary piRNAs (Brennecke et al. 2007; Gunawardane et al. 2007). However, thedetailed mechanism of the germ-specific process remains
poorly understood, largely because an ex vivo Drosophilamodel system amenable to biochemical analyses has notbeen available. BmN4 cells (Kawaoka et al. 2009) havebeen used to study the mechanism underlying the pingpong cycle but are of lepidopteran origin. BmN4 cells express two PIWI members but lack a homolog of Drosophila Piwi.
In Drosophila, mutations in lethal (3) malignant brain
tumor [l(3)mbt] generate malignant brain tumors withmetastatic potential (Gateff et al. 1993; Janic et al. 2010)
due to deregulation of l(3)mbt signature (MBTS) genes(Janic et al. 2010). Multiple MBTS genes encode proteins
with germline functions, and mutations in these genes(for instance, piwi) rescue the brain tumor phenotype(Janic et al. 2010). MBTS genes with germline functions
include ping-pong factors such as aub and vasa (Janicet al. 2010). A few small RNAs in l(3)mbt mutant brain tumors were annotated as piRNAs (Janic et al. 2010). Thesefindings of l(3)mbt mutations resulting in ectopic acquisition of germline traits in the brain prompted us to examine whether depletion of l(3)mbt in OSCs initiates the
ping-pong cycle and causes the accumulation of secondary piRNAs in cells.
In this study, we found that both RNAi- and CRISPRmediated loss of function of l(3)mbt lead to ectopic expression of Aub, AGO3, and Vasa—the core factors necessary for operating the germ-specific ping-pong cycle—in
OSCs. Deep sequencing and comparison of mRNAs in
the CRISPR-mediated mutant OSCs (Δmbt-OSCs) and parental OSCs revealed that other ping-pong factors such as
qin were up-regulated in Δmbt-OSCs. Both Aub and
AGO3 copurified with piRNAs in Δmbt-OSCs. Deep sequencing these small RNA populations demonstrated
that Aub- and AGO3-bound piRNAs show a typicalping-pong signature. Perinuclear foci resembling nuage,© 2016 Sumiyoshi et al. This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.

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Sumiyoshi et al.

the ping-pong center (Brennecke et al. 2007; Gunawardane
et al. 2007; Lim and Kai 2007; Malone et al. 2009), also appeared following l(3)mbt mutation. Depletion of Vasa in
Δmbt-OSCs disrupted the ping-pong pathway and Aub localization to the nuage-like structure. Use of Δmbt-OSCs
will greatly facilitate elucidation of the mechanism underlying secondary piRNA biogenesis and nuage formation in Drosophila.
Results and Discussion
Loss of l(3)mbt leads to ectopic expression of ping-pong
factors in OSCs
OSCs
expressl(3)mbt(http://flybase.org/reports/
FBrf0221009.html). We therefore treated OSCs withl(3)mbt siRNA duplexes (Supplemental Fig. S1A). The
mRNA level of l(3)mbt was significantly decreased upon
RNAi treatment (Supplemental Fig. S1B). In contrast,
ago3 and vasa were significantly up-regulated afterl(3)mbt siRNA treatment, while changes in the level ofpiwi mRNA were negligible (Supplemental Fig. S1B).
Western blotting detected AGO3, Vasa, and Aub in theRNAi-treated cells, while the levels of Piwi, Yb, Armi,
and Krimper (Krimp) (Saito et al. 2009; Iwasaki et al.
2015; Sato et al. 2015) remained unchanged (Supplemental
Fig. S1C).
We next set out to ablate l(3)mbt function by editing the
genome in OSCs using the CRISPR/Cas9 system. Withreference to two mutant alleles, l(3)mbt GM76 andl(3)mbt E2 (Wismar et al. 1995; Yohn et al. 2003; Janicet al. 2010), small guide RNAs (sgRNAs) were designed
to target exon 5, which contains MBT domains (Fig. 1A).
The two mutant alleles have an effect on tumorigenesis
in the fly brain (Wismar et al. 1995; Janic et al. 2010). A genomic PCR fragment amplified with a primer set flanking
the sgRNA targeting sites was shortened after CRISPR/
Cas9 treatment (Fig. 1B). Sequencing the fragment revealed that a 558-nucleotide (nt) deletion occurred at thegene locus (Fig. 1A). Full-length l(3)mbt mRNA was notdetectable by quantitative RT–PCR (qRT–PCR) in the
mutant cells (data not shown). We refer to these cells
here as Δmbt-OSCs.
Deep sequencing and comparison of mRNAs in ΔmbtOSCs and the parental OSCs revealed that, in addition
to ago3 and vasa, other ping-pong factors such as aub,
qin, tapas, and tejas (Specchia et al. 2010; Gangaraju
et al. 2011; Czech et al. 2013; Handler et al. 2013; Patilet al. 2014; Iwasaki et al. 2015) were markedly up-regulated in Δmbt-OSCs (Fig. 1C; Supplemental Fig. S2A). Western blotting confirmed increased levels of Aub, AGO3,
and Vasa in Δmbt-OSCs (Fig. 1D). The levels of Piwi andother piRNA factors examined, such as Yb and Armi (Iwasaki et al. 2015), were nearly identical between the twocell lines (Fig. 1D), although a few factors showed slightchanges in their mRNA levels (Fig. 1C; Supplemental
Fig. S2A). Germ-specific genes tended to be more sensitive
to loss of l(3)mbt than germ + soma and soma-specificgenes (Fig. 1C; Supplemental Fig. S2A). The appearanceof Yb in Δmbt-OSCs suggests that CRISPR-mediatedl(3)mbt loss had little effect on the elimination of somatic
traits from the mutant cells. Focusing on MBTS genes revealed that many, if not all, were up-regulated in ΔmbtOSCs (Supplemental Fig. S2B). Among non-MBTS genesthat are up-regulated by mutations in mbt genes otherthan MBTS (Janic et al. 2010), the highest up-regulation
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was observed for ago3 (21.5-fold in Δmbt-OSCs). Resultsof transcriptome-wide analysis are summarized in Supplemental Tables S1 and S2. We confirmed that loss ofl(3)mbt had very little effect on the proliferation rate of
OSCs (Supplemental Fig. S2C).

Loss of l(3)mbt activates the ping-pong cycle in OSCs
To compare Piwi–piRNA loading between Δmbt-OSCs
and OSCs, Piwi was immunoisolated from both lines,
and extracted RNAs were 32P-labeled. piRNAs copurified
with Piwi similarly from both cell lines (Fig. 2A). We thendeep-sequenced Piwi-bound piRNAs in Δmbt-OSCs andcompared the reads with those from OSCs (Fig. 2B–D;
Supplemental Fig. S3A–D; Saito et al. 2009). This revealed
that both sets of piRNAs show strong bias toward 1U andantisense orientation. The levels piRNAs from ΔmbtOSCs and parental OSCs mapped to each transposon
were highly similar (R 2 = 0.872). piRNAs were mapped
similarly to flamenco ( flam), a soma-specific singlestrand piRNA cluster; traffic jam (tj), a genic piRNAsource; and transposons such as mdg1 (Brennecke et al.
2007; Malone et al. 2009; Robine et al. 2009; Saito et al.
2009). piRNAs corresponding to 42AB, a germ-specificdual-strand piRNA cluster (Brennecke et al. 2007; Malone
et al. 2009), were not contained in Piwi-bound piRNAs.
This was expected because a component of the Rhino–
Deadlock–Cutoff (RDC) complex, cutoff, which is necessary for producing piRNAs from 42AB and other germspecific dual-strand piRNA clusters (Mohn et al. 2014;
Zhang et al. 2014), was undetected in both OSC lines (Supplemental Fig. S2A). Another component, rhino, whose
expression was as low as ago3 in OSCs, was even lower
in Δmbt-OSCs, while deadlock was expressed moderately
in both OSCs and Δmbt-OSCs (Supplemental Fig. S2A).
In Δmbt-OSCs, both Aub and AGO3 copurified with
piRNAs (Fig. 2E). Deep sequencing these small RNA populations clarified that Aub-bound and AGO3-bound piRNAs show distinct features (Fig. 2B–D; Supplemental
Fig. S3A–D). While Aub–piRNAs showed significant nucleotide bias toward 1U and were mostly antisense,
AGO3–piRNAs had strong 10A and sense biases. The correlation between levels of Piwi-bound and AGO3-boundpiRNAs mapped to each transposon was low (R 2 =0.149), while the correlation was significantly high between levels of Piwi-bound and Aub-bound piRNAs (R 2
= 0.874). Both flam–piRNAs and tj–piRNAs were foundwith Aub but not with AGO3. 42AB–piRNAs were not detected with Aub or AGO3. Additionally, plotting the distribution of Aub-bound and AGO3-bound piRNAs totransposons DM412, DM297, and mdg1 indicated the production of piRNAs from the same coordinate but in the
opposite orientation.
We previously showed that both Myc-Aub and MycAGO3 exogenously expressed in OSCs by transfection
are loaded with primary piRNAs, including flam–piRNAs
and tj–piRNAs, which are normally loaded onto endogenous Piwi (Olivieri et al. 2012; Sato et al. 2015). This emphasized the compatibility of AGO3 with primary piRNAloading. However, in Δmbt-OSCs, AGO3 avoided binding
primary piRNAs; this was especially apparent when tj–
piRNAs were compared (Supplemental Fig. S3C; Satoet al. 2015). In OSCs, Krimp sequesters unloaded AGO3
to Krimp bodies and blocks AGO3–primary piRNA loading (Olivieri et al. 2012; Sato et al. 2015). Krimp is crucial

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Activation of the ping-pong loop in soma

unknown proteins other than Krimp may also be involvedin the regulation of AGO3 sDMA modification along with
Krimp.
If AGO3-bound piRNAs were produced via the pingpong cycle in an Aub slicer-dependent manner, a pingpong signal—i.e., complementary overlap of 10 nt at the
5′ ends—should be observed between Aub-bound andAGO3-bound piRNAs. piRNA production by such aping-pong cycle was evaluated, and a significant pingpong signal was observed between Aub-bound andAGO3-bound piRNAs (Fig. 2G). These results, alongwith mapping data shown in Supplemental Figure S3D,
further support the idea that AGO3-bound piRNAs areproducts of Aub slicer in the ping-pong pathway.
Piwi-bound primary piRNAs in OSCs involve a uniquesubset of piRNAs called phased piRNAs (Han et al. 2015;
Mohn et al. 2015). Examination of piRNAs in Δmbt-OSCs
by focusing on this trait revealed that phased piRNAs
with d = 1 (this means that two piRNAs can be mappedright next to each other on the same genomic strand
without a nucleotide gap between them) are particularly
abundant in Piwi-bound piRNAs (Z1 = 22.3). This wascomparable with Piwi-bound piRNAs (Z1 = 16.8) in
OSCs (Supplemental Fig. S4A,B). Z1 scores of Aub-boundand AGO3-bound piRNAs in OSCs were 11.5 and 2.9, respectively (Supplemental Fig. S4A), suggesting that
AGO3-bound piRNAs tend to exclude phased piRNAs(Han et al. 2015; Mohn et al. 2015). In contrast, piRNAs
loaded onto Myc-AGO3 in OSCs (Sato et al. 2015) showeda significant phasing pattern (Z1 = 12.9) (Supplemental
Fig. S4C). These results further support the notion that
AGO3 in Δmbt-OSCs avoids binding primary piRNAs,in contrast to Myc-AGO3 in OSCs.

Appearance of perinuclear foci resembling nuage
in Δmbt-OSCs
Figure 1. CRISPR-mediated generation of Δmbt-OSCs. (A) Genomic
structure of l(3)mbt in OSCs and Δmbt-OSCs. sgRNAs GM76 and E2
targeting l(3)mbt exon 5 are indicated by arrowheads. Sequences atthe l(3)mbt gene locus in OSCs and Δmbt-OSCs are also shown(sgRNA and PAM sequences are shown in blue and red, respectively).
(CDS) Protein-coding DNA sequence; (MBT domain) malignant brain
tumor domain; (SAM domain) sterile α motif domain; (UTR) untranslated region. (B) Genomic PCR fragments amplified with a primer set,
shown by arrows in A, are indicated by arrowheads (OSC, 772 nt;
Δmbt-OSC, 214 nt). (UM) Upper marker; (LM) lower marker. (C) Scatter plot comparing transcript abundance (mRNA-seq) of small RNA
biogenesis factors in OSCs and Δmbt-OSCs. Classification of the factors to four clades—germ-specific, germ + soma, soma-specific, andRNAi/miRNA—was carried out with reference to Handler et al.
(2013). (FPKM) Fragments per kilobase of exon per million mapped sequence reads. (D) Western blotting shows protein levels of piRNA factors in OSCs and Δmbt-OSCs. βTubulin (βTub) was detected as aloading control.

for AGO3 function in ovary germ cells (i.e., symmetricaldimethyl arginine [sDMA] modification and secondary
piRNA association) (Sato et al. 2015; Webster et al.
2015); therefore, it would be expected that, in ΔmbtOSCs, AGO3 loading with secondary piRNAs might beunder the control of Krimp. Indeed, AGO3 was sDMAmodified in Δmbt-OSCs, as in ovaries, while Myc-AGO3
in OSCs was sDMA-free (Fig. 2F; Supplemental Fig.
S3E). However, this does not exclude the possibility that

Vasa, a germ-specific DEAD-box RNA helicase, plays acrucial role in the ping-pong cycle by displacing cleaved
RNAs from Aub–piRISCs (or its counterpart, Siwi–piRISCs, in silkworms) in an ATP hydrolysis-dependent
manner (Xiol et al. 2014; Nishida et al. 2015). Immunofluorescence in Drosophila ovaries showed that Aub, AGO3,
and Vasa colocalize to nuage (Lim and Kai 2007; Malone
et al. 2009; Iwasaki et al. 2015). Immunostaining of
Δmbt-OSCs revealed that Aub and Vasa mostly coincide
at perinuclear foci that look similar to nuage (Fig. 3A).
Myc-Aub exogenously expressed in OSCs was uniformly
distributed in the cytosol and did not form speckles(Sato et al. 2015). Signals of Myc-Vasa in OSCs were slightly stronger at the perinuclear region in the cytoplasm but
not punctate (Fig. 3B). Myc-Aub coexpressed with Vasa in
OSCs was distributed almost evenly in the cytoplasm (Fig.
3B). These results suggest that some factors whose expression is up-regulated by loss of l(3)mbt function promote
Aub to form nuage-like structures with Vasa in ΔmbtOSCs.
Aub and AGO3 also colocalized to nuage-like structures(Fig. 3C). Krimp in OSCs sequesters unloaded AGO3 to
Krimp bodies, which are generally present at a frequency
of one per cell (Fig. 3D; Olivieri et al. 2012; Sato et al.
2015). However, in Δmbt-OSCs, AGO3-positive fociwere more numerous than one per cell, and some could
be superimposed with Aub-positive foci. This led us to examine the cellular localization of Krimp in Δmbt-OSCs,
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Figure 2. The ping-pong pathway in Δmbt-OSCs. (A) 32P-labeled piRNAs (top panel) associated with Piwi (bottom panel) in OSCs and ΔmbtOSCs. (n.i.) Nonimmune IgG. (B) Heat maps of piRNA strand bias. Strand bias of transposon-derived piRNAs in Piwi/Aub/AGO3 small RNA libraries from Δmbt-OSCs is shown. Strand bias of Piwi-bound piRNAs in parental OSCs (Ishizu et al. 2015) is also shown. (C) Sequence logos of
Piwi/Aub/AGO3-bound piRNAs mapped to transposons in Δmbt-OSCs. Sequence logos of Piwi-bound piRNAs in parental OSCs (Ishizu et al.
2015) are also shown (OSC Piwi at the top). (D, left) Scatter plot showing the correlation of antisense Piwi-bound piRNA reads in OSCs (percentage;
Y-axis) with those in Δmbt-OSCs (percentage; X-axis). (Right) The correlation of antisense Aub-bound (pink dots) and antisense AGO3-bound (bluedots) piRNA reads in Δmbt-OSCs (percentage; Y-axis) with antisense Piwi-bound piRNA reads in Δmbt-OSCs (percentage; X-axis) is also shown.
Each dot corresponds to the ratio of piRNAs mapped to transposons (shown in B). (E) 32P-labeled piRNAs (top panel) associated with Aub and
AGO3 (bottom panel) in Δmbt-OSCs. (n.i.) Nonimmune IgG. (F ) Western blotting shows that AGO3 in Δmbt-OSCs is dimethylated, as in ovaries.
(G) Depiction of the ping-pong signature between antisense Aub-bound piRNAs and sense AGO3-bound piRNAs, defined as the value at position
10 nt. Graphs indicate the relative frequency that a complementary piRNA exists with a 5′ end (Y-axis) at the indicated distance (X-axis).

and we observed that co-localization of Krimp with AGO3
was apparent (Fig. 3D), as occurs in ovaries where Krimp
normally accumulates at nuage. Krimp bodies are present
only when piRNA biogenesis is defective because of mutations in piRNA factors such as aub. These results support
the intriguing idea that Δmbt-OSCs likely recapitulate formation of the ping-pong center.
We next examined the spatial relationship between Yb
and Aub-positive structures in Δmbt-OSCs. Aub and Yb
signals only occasionally coincide (Fig. 3E), although thenumber of Yb bodies may be higher in Δmbt-OSCs compared with that in OSCs. These results suggest that primary and secondary piRNA biogenesis pathways may occur
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separately at Yb bodies and Aub-positive nuage-like foci,respectively, in Δmbt-OSCs.
In vasa mutant ovaries, Aub does not accumulate in thenuage and is evenly distributed in the cytosol of nurse
cells (Lim and Kai 2007; Malone et al. 2009; Iwasaki
et al. 2015). We therefore asked how depletion of Vasa affects Aub localization in Δmbt-OSCs. Treatment of ΔmbtOSCs with vasa siRNA duplexes caused Aub to be largely
dispersed in the cytosol (Fig. 4A,B). Under such conditions, AGO3 was loaded with far fewer piRNAs, although
Aub piRNA association remained largely unaffected (Fig.
4C). Thus, Vasa depletion disrupted the ping-pong pathway in Δmbt-OSCs, as expected.

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Activation of the ping-pong loop in soma

Figure 3. Nuage-like structures in Δmbt-OSCs. (A) Immunofluorescence shows that Aub and Vasa coincide at nuage-like structures in
Δmbt-OSCs. (B) Coexpression of Myc-Aub with Myc-Vasa in OSCs
by transfection. Immunofluorescence was performed using antiVasa and anti-Aub antibodies. (C) AGO3 colocalizes with Aub inΔmbt-OSCs. (D) AGO3 partially colocalizes with Krimp in ΔmbtOSCs. (E) Aub and Yb only occasionally coincide in Δmbt-OSCs.
Bars, 10 µm.

Loss of l(3)mbt fails to activate the ping-pong cycle
in S2 cells
The Drosophila cell line S2 is of embryonic and somaticorigin; therefore, PIWI proteins and piRNAs are below
detection levels (Saito et al. 2006, 2009). l(3)mbt mRNAwas detected in S2 cells by qRT–PCR (data not shown);
therefore, we examined whether l(3)mbt knockdown has
an impact on piRNA production in S2 cells. Upon
RNAi, Aub was slightly up-regulated (Fig. 4D). However,very low levels of other factors, including Piwi, AGO3,
and Vasa, were detected (Fig. 4D). Indeed, we failed todetermine PIWI–piRNA loading by immunoprecipitation(data not shown). Although Drosophila brains and S2 cells
are both nongonadal and somatic, the mechanism bywhich l(3)mbt controls downstream genes in these cells
seems to be distinct.
In summary, our results show that CRISPR-mediatedl(3)mbt knockout in OSCs leads to the ectopic expressionof ping-pong factors, resulting in the accumulation of secondary piRNAs that are otherwise undetected in OSCs. In
the mutant cells, AGO3-loaded secondary piRNAs were
mostly 10A-biased and sense-oriented (Fig. 2; Supplemental Fig. S3). piRNA mapping revealed that the piRNAs
originate from transposon mRNAs (Supplemental Fig.
S3D). However, Piwi was expressed in Δmbt-OSCs to an
extent similar to that in parental OSCs (Fig. 2A). Piwibound piRNAs were highly comparable in the two celllines in all aspects, including piRNA origins and nucleotide and strand biases (Fig. 2; Supplemental Fig. S3).
Thus, Piwi in Δmbt-OSCs should be similarly functionalin repressing transposons compared with Piwi in OSCs.
Indeed, Piwi was localized in the nucleus in both ΔmbtOSCs and OSCs (Supplemental Fig. S5A). The levels oftransposon mRNAs were not drastically changed by lossof l(3)mbt (R 2 = 0.988) (Supplemental Fig. S5B), suggestingthat l(3)mbt is dispensable in transposon silencing in ovarian somas. The question then arises of where transposon
mRNAs that give rise to sense AGO3-bound piRNAs
came from in Δmbt-OSCs. Our previous study showedthat overexpression of Myc-Aub in OSCs caused a slightbut significant increase in the levels of mdg1 andDM297, which are targets of Piwi in OSCs (Sato et al.
2015). This derepression of transposons could be because
Myc-Aub competes with endogenous Piwi for primarypiRNAs, impairing the Piwi-mediated transcriptional silencing activity. Indeed, AGO3-bound piRNAs included
piRNAs originating from mdg1 and DM297 mRNAs(Fig. 2B). These results raise the intriguing idea that bothtranscriptional and post-transcriptional transposon silencing (i.e., the ping-pong cycle) occur in Δmbt-OSCs,
mediated by nuclear Piwi–piRNA and cytoplasmic Aub–piRNA complexes, respectively, as in germ cells in fly ovaries. Thus, this newly made cell line, Δmbt-OSC, is thefirst cell line of any animal that accommodates both transcriptional and post-transcriptional silencing events andhence would also be useful in exploring the relationshipof the two pathways.
Materials and methods
Establishment of Δmbt-OSCs
Gene knockout in OSCs using the CRIPSR/Cas9 system and isolation ofknockout OSCs were performed essentially as described previously (Ishizu
et al. 2015). In brief, OSCs were transfected with 0.2 μg of a plasmid expressing both pBS-Hsp70-Cas9 (Addgene, 46294) and the blasticidin resistance gene and 4 µg of two sgRNA-expressing plasmids (GM76 and E2)
using Xfect transfection reagent (TaKaRa Clontech). After incubation for
24 h, blasticidin (Thermo Fisher Scientific, Inc., R210-01) was added tothe culture medium at 50 µg/mL. The next day, 5.0 × 103 cells were passaged in 3.5-cm dishes and allowed to grow in blasticidin-containing medium. During culturing, cells were washed with the medium to remove
nonadherent dead cells. After 6–7 d of culture, 5.0 × 103 cells were passagedin 6-cm dishes and allowed to grow in blasticidin-containing medium. After further incubation for a few days, colonies were picked and passaged insingle wells of 24-well plates. The next day, these colonies were suspended
with a Tip Strainer (Bel-Art) and allowed to grow to confluence. To detect
the genomic deletion, genomic DNA was extracted using QuickExtract

Figure 4. The effect of Vasa depletion in Δmbt-OSCs. (A) RNAi efficiently reduced the amount of Vasa in Δmbt-OSCs. siLuc was used asa negative control. (B) Depletion of Vasa in Δmbt-OSCs caused Aub tobe scattered in the cytoplasm in Δmbt-OSCs. Bars, 10 µm. (C ) AGO3,
but not Aub, associates with fewer secondary piRNAs in Δmbt-OSCs
upon Vasa RNAi treatment. siEGFP was used as a negative control. (n.
i.) Nonimmune IgG. (D) Expression levels of piRNA factors in S2 cells
upon l(3)mbt depletion by RNAi. dsEGFP (siEGFP) was used as a negative control.

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Sumiyoshi et al.

DNA extraction solution (Epicenter) following the manufacturer’s protocol, and the genomic region flanking the CRISPR target site was amplified
by PCR and then analyzed using MultiNa (Shimadzu).

Acknowledgments
We thank D. Chen and T. Kai for providing antibodies. We also thank K.M.
Nishida, A. Nagao, and H. Ohtani for producing antibodies, and the othermembers of the Siomi laboratories, particularly K. Saito and S. Yamanaka,
for discussions and comments on the manuscript. We are also thankful to
T. Ono and W. Iwasaki for bioinformatics assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan to K.S.,
Y.W.I., H.S., and M.C.S.

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Loss of l(3)mbt leads to acquisition of the ping-pong cycle inDrosophila ovarian somatic cells
Tetsutaro Sumiyoshi, Kaoru Sato, Hitomi Yamamoto, et al.
Genes Dev. 2016 30: 1617-1622
Access the most recent version at doi:10.1101/gad.283929.116

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