Article

Somatic Primary piRNA Biogenesis Driven by cisActing RNA Elements and trans-Acting YbGraphical Abstract

Authors
Hirotsugu Ishizu, Yuka W. Iwasaki,
Shigeki Hirakata, ..., Wataru Iwasaki,
Haruhiko Siomi, Mikiko C. Siomi

Correspondence
siomim@bs.s.u-tokyo.ac.jp

In Brief
piRNAs protect the germline genome
from transposons, although theunderlying mechanism remains elusive.
Ishizu et al. focused on understanding
how piRNA sources are selectively
determined from all cellular RNAs. They
successfully identified the particular RNA
elements necessary and sufficient for
producing piRNAs and determined Yb
protein as the trans-acting partner.

Highlights
d

d

d

d

cis elements for producing somatic primary piRNAs wereidentified
Yb primary binding to cis elements in piRNA precursors
initiates piRNA production
Yb secondary binding downstream of cis elements does notinitiate piRNA production
Artificial piRNAs from the Yb-cis-element system elicit
transcriptional silencing

Ishizu et al., 2015, Cell Reports 12, 429–440
July 21, 2015 ª2015 The Authors
http://dx.doi.org/10.1016/j.celrep.2015.06.035

Accession Numbers
GSE69625

Cell Reports

ArticleSomatic Primary piRNA Biogenesis Driven
by cis-Acting RNA Elements and trans-Acting Yb
Hirotsugu Ishizu,1 Yuka W. Iwasaki,2 Shigeki Hirakata,1 Haruka Ozaki,3 Wataru Iwasaki,1,3 Haruhiko Siomi,2and Mikiko C. Siomi1,*
1Department 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 160-8582, Japan
3Department of Computational Biology, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo 113-0032, Japan*Correspondence: siomim@bs.s.u-tokyo.ac.jp
http://dx.doi.org/10.1016/j.celrep.2015.06.035
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

Primary piRNAs in Drosophila ovarian somatic cells
arise from piRNA cluster transcripts and the 30
UTRs of a subset of mRNAs, including Traffic jam(Tj) mRNA. However, it is unclear how these RNAs
are determined as primary piRNA sources. Here, we
identify a cis-acting 100-nt fragment in the Tj 30
UTR that is sufficient for producing artificial piRNAs
from unintegrated DNA. These artificial piRNAs
were effective in endogenous gene transcriptionalsilencing. Yb, a core component of primary piRNAbiogenesis center Yb bodies, directly bound the Tjcis element. Disruption of this interaction markedly
reduced piRNA production. Thus, Yb is the transacting partner of the Tj-cis element. Yb-CLIP revealed that Yb binding correlated with somatic
piRNA production but Tj-cis element downstream
sequences produced few artificial piRNAs. We thus
propose that Yb determines primary piRNA sources
through two modes of action: primary binding to cis
elements to specify substrates and secondary binding to downstream regions to increase diversity in
piRNA populations.
INTRODUCTIONPIWI-interacting RNAs (piRNAs) interact with PIWI proteins
to form piRNA-induced silencing complexes (piRISCs), which
repress target genes, mostly transposons, either transcriptionally or at the post-transcriptional level by cleaving transcripts inthe cytoplasm (Aravin et al., 2007; Brennecke et al., 2007; Ghildiyal and Zamore, 2009; Ishizu et al., 2012; Juliano et al., 2011;
Khurana and Theurkauf, 2010; Siomi et al., 2011). Interestingly,
not all cells in the gonads use both mechanisms. Follicle cells
in Drosophila ovaries use transcriptional silencing but lack
piRISC-mediated post-transcriptional silencing, while germ cells
possess both transcriptional and post-transcriptional piRISC
machineries (Ishizu et al., 2012). In Bombyx ovaries, only posttranscriptional silencing occurs (Kawaoka et al., 2009). This variation largely depends on which PIWI proteins are expressed in agiven cell type; transcriptional silencing requires nuclear PIWI
proteins while post-transcriptional silencing requires cytoplasmic PIWI proteins (Huang et al., 2013; Malone et al., 2009).
Primary piRNAs are produced from single-stranded long noncoding RNAs transcribed from piRNA clusters in a Dicer-independent manner (Houwing et al., 2007; Vagin et al., 2006). The
Drosophila genome contains 142 piRNA clusters (Brennecke
et al., 2007), whose expression is regulated differently in different
cell types. flamenco (flam), a representative of unidirectional
piRNA clusters, is expressed only in follicle cells, whereas thebidirectional cluster 42AB is expressed specifically in nurse cells(Brennecke et al., 2007). The types of transposon fragments inserted in individual piRNA clusters also vary; therefore, piRNA
populations differ among cell types. piRNAs in nurse cells arerather complex because primary piRNAs are amplified throughthe amplification loop, yielding secondary piRNAs (Ishizu et al.,2012). Recent studies showed that secondary piRNAs further
produce phased trailer piRNAs (Han et al., 2015; Mohn et al.,2015). Follicle cells do not use this amplification system andthus only contain primary piRNAs.
The biogenesis of somatic primary piRNAs has been studied
using ovaries and an ovarian somatic cell (OSC) line (Olivieri
et al., 2010; Saito et al., 2009). A current model suggests thatupon transcription flam-piRNA precursors are localized to perinuclear Flam bodies (Murota et al., 2014) and processed at adjacent Yb bodies (Olivieri et al., 2010; Saito et al., 2010). Yb bodies
contain many piRNA factors besides Yb (Haase et al., 2010;
Olivieri et al., 2012; Qi et al., 2011; Saito et al., 2009; Saito
et al., 2010; Zamparini et al., 2011). Zucchini (Zuc), an endonuclease required for processing piRNA intermediates into maturepiRNAs, is localized on the surface of mitochondria (Choi et al.,2006; Han et al., 2015; Ipsaro et al., 2012; Mohn et al., 2015;
Nishimasu et al., 2012; Olivieri et al., 2012). Yb bodies tend tobe observed in inter-mitochondrial regions (Murota et al.,2014; Nishimasu et al., 2012; Szakmary et al., 2009). This
arrangement of organelles appears crucial for accelerating
piRNA processing because it centralizes all the necessary factors in the cytoplasm. Upon maturation, piRNAs associatewith Piwi, a Drosophila PIWI protein, to form piRISCs, which
are then translocated to the nucleus to implement nuclear transposon silencing through chromatin modifications on target
transposon loci with support from co-factors such as GTSF1/
Asterix and Maelstrom (Dönertas et al., 2013; Ishizu et al.,Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 429

the piRNA cluster act as antisense oligos to repress parental
transposons (Brennecke et al., 2007; Malone et al., 2009; Saito
et al., 2009). Some protein-coding genes such as Traffic jam(Tj) also act as primary piRNA sources (Robine et al., 2009; Saito
et al., 2009), and genic piRNA sources express proteins in OSCs
and follicle cells. The TJ protein, encoded by Tj, is a large Maftranscriptional factor necessary for controlling gonad morphogenesis (Li et al., 2003). Loss of Tj function abolishes Piwi
expression in follicle cells. However, Piwi expression in nurse
cells is not influenced by TJ loss. Thus, the dependence ofPiwi expression on TJ differs between follicle cells and germ cells(Saito et al., 2009).
Only a limited number of transcripts serve as somatic primarypiRNA precursors. However, the mechanism underlying therecognition and selection of these transcripts as piRNA precursors is poorly understood. To better understand the mechanism,
we used the Tj 30 UTR as representative of somatic primarypiRNA sources to identify a cis element and its trans-acting partner necessary for producing primary piRNAs in OSCs.
RESULTS

Figure 1. Artificial piRNA Production from an Inserted Fragment into
the 30 UTR of Tj(A) Top: schematic drawings of EGFP-tj WT and EGFP-tj MT constructs consisting of EGFP CDS (green) and the Tj-30 UTR (orange). The ‘‘hotspot’’element (24 nt) in the Tj 30 UTR of EGFP-tj WT was substituted with a 24-nt
arbitrary sequence (shown by a red box) in EGFP-tj MT. Center: schematic
drawing of Tj mRNA. Tj-piRNAs (green bars) (Saito et al., 2009) are mapped. Tj
30 UTR (415–490) is shown by a black bar. Bottom: alignments of Tj-piRNAs
derived from Tj 30 UTR (415–490). The hotspot sequence is shown in orange.
The 24-nt arbitrary sequence inserted in EGFP-tj MT is shown in red.
(B) Left: scheme of the experiments for detecting piRNAs derived from EGFP-tj
WT and EGFP-tj MT in OSCs. Upper right: total RNAs and piRNAs in the Piwi
complex immunopurified from OSCs were subjected to northern blotting
analysis. WT and MT probes detect natural Tj-piRNAs originating from the first
hotspot in the Tj 30 UTR and artificial piRNAs produced from the 24-nt random
sequence embedded in EGFP-tj MT, respectively. Artificial MT-piRNAs
associate with Piwi in OSCs expressing EGFP-tj MT. Lower right: the amountsof Piwi immunoprecipitated from OSCs expressing EGFP-tj WT and EGFP-tj
MT were examined by western blotting using an anti-Piwi antibody.

2012; Muerdter et al., 2013; Ohtani et al., 2013; Olivieri et al.,2010; Saito et al., 2010)flam is the major source of primary piRNAs in OSCs and follicle
cells in the ovaries (Brennecke et al., 2007; Malone et al., 2009;
Saito et al., 2009). flam is largely occupied by transposon remnants, whose orientation predominantly opposes that of theparental transposons; thus, most primary piRNAs arising from430 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors

Production of Artificial Primary piRNAs from a Random
Sequence Inserted into the Tj 30 UTR
First, we constructed a plasmid, EGFP-tj WT, consisting of
the EGFP coding sequence (CDS; 717 nt) and the Tj 30 UTR(1,467 nt) (Figure 1A). We previously showed that piRNAs are
not produced uniformly along the Tj 30 UTR; rather, it contains
two hotspots that generate substantial amounts of Tj-piRNAs(Saito et al., 2009). In the current study, the first hotspot (24 nt)
in EGFP-tj WT was replaced with a 24-nt arbitrary sequence,yielding a mutant construct, EGFP-tj MT (Figure 1A). The 24-nt
sequence did not match any region in the Drosophila genome(data not shown).
OSCs were transfected individually with EGFP-tj WT and
EGFP-tj MT constructs. After transfection, northern blotting
was performed with two specific probes, WT and MT, designed
to detect natural Tj-piRNAs originating from the first hotspot in
the Tj 30 UTR and artificial piRNAs produced from the 24-nt
random sequence embedded in EGFP-tj MT, respectively. The
MT probe detected small RNAs of 23–29 nt in length among total
RNAs isolated from OSCs after transfection with EGFP-tj MT(Figure 1B). Interestingly, the signals were highly enriched in
the Piwi-piRISC fraction immunopurified from OSCs, demonstrating that the artificial piRNAs produced from the 24-nt
random sequence in EGFP-tj MT were loaded onto Piwi (Figure 1B). These artificial piRNAs (MT-piRNAs) were undetectedin OSCs transfected with EGFP-tj WT, confirming MT probe
specificity (Figure 1B). The WT probe detected Tj-piRNAs in
both cells (Figure 1B). However, the signal intensity appearedslightly higher in OSCs transfected with EGFP-tj WT, likely
because of exogenous expression of Tj-piRNAs from theconstruct.
Identification of a cis-Acting Element in the Tj 30 UTR
To narrow down the region that acts as a cis element in theexpression of MT-piRNAs from EGFP-tj MT, three deletion mutants, MT-1 to MT-3, were produced (Figure 2A). A control

from MT-1 and MT-2 as from EGFP-tj MT (Figure 2B). In sharp
contrast, MT-3 and MT-4 failed to produce MT-piRNAs (Figure 2B). MT-3 and MT-4 mRNA expression levels were nearly
equal to those of MT-1 and MT-2 (Figure S1A), suggesting thatthe 100-nt fragment present in MT-2 but missing from MT-3
serves as the cis element for producing MT-piRNAs from downstream regions.
To confirm that the 100-nt fragment acts as a bona fide cisacting element for generating MT-piRNAs, the element was
replaced with a 100-nt random sequence, yielding MT-2-1 (Figure 2C). Another mutant, MT-2-2, was produced by exchanging
the second 100-nt region of the 30 UTR in MT-2 with the random
sequence inserted into MT-2-1 (Figure 2C). Northern blotting revealed that MT-2-2, but not MT-2-1, expressed MT-piRNAs
similarly to MT-2 (Figure 2D).
Tj encodes Tj-piRNAs and TJ protein, both of which are detected in OSCs and ovaries (Brennecke et al., 2007; Li et al.,
2003; Malone et al., 2009; Saito et al., 2009). We next assessed
if MT-2 mRNA translation is required for primary piRNA production. A hairpin structure that would presumably interfere withtranslation was inserted into the 50 UTR of MT-2, yielding
MT-2-3 (Figure 2C). Western blotting using an anti-EGFP antibody confirmed that no EGFP was expressed from MT-2-3 (Figure 2E). MT-2-3 mRNAs were expressed in OSCs (Figure S1B).
Likewise, MT-piRNAs were expressed from MT-2-3 to a similar
extent as from MT-2 and MT-2-2 (Figure 2D). Thus, translationof piRNA precursors appears to be dispensable for primarypiRNA production.

Figure 2. Identification of a cis-Regulatory Element in the Tj 30 UTR(A) Schematic drawings of EGFP-tj MT and its mutant constructs. MT-4 contains the Actin42A 30 UTR (360 nt) (light blue) instead of the Tj 30 UTR. The 24-nt
artificial sequence (red) exists also in MT-4.
(B) WT-piRNA and MT-piRNA production was monitored by northern blotting
analysis using WT and MT probes as in Figure 1B.
(C) Schematic drawings of MT-2 and its mutant constructs. The 100-nt random
sequence inserted in MT-2-1 and MT-2-2 is shown (purple). A stable stemloop structure was inserted into the 50 UTR of MT-2-3 to inhibit EGFP
translation.
(D) WT-piRNA and MT-piRNA production was monitored by northern blotting
analysis using WT and MT probes as in Figure 2B.
(E) Western blotting shows the expression levels of EGFP protein in OSCs after
transfection. b-tubulin was detected as a loading control.

construct, MT-4, in which the Tj 30 UTR was substituted with theActin42A 30 UTR, was also produced (Figure 2A). The 24-nt insertion was maintained in the Actin42A 30 UTR of MT-4.
Total RNAs isolated from OSCs and RNAs from the PiwipiRISC fraction immunopurified from cells were subjected tonorthern blotting. MT-piRNAs were produced to a similar extent

Deletion of the Tj-cis Element from the Drosophila
Genome using the CRISPR/Cas9 System
To examine whether deletion of the Tj-cis element from the
Drosophila genome would cause defective Tj-piRNA biogenesis, we used the CRISPR/Cas9 system (Hsu et al., 2014). We
constructed three plasmids expressing short-guide RNAs
(sgRNAs) that target specific sites in the Tj locus: targets 1–3(Figure 3A). sgRNAs were expressed in OSCs for targeting
either target 1 + target 2 or target 1 + target 3. We then performed PCR to examine if genomic deletion occurred as
expected. DNA fragments of the expected sizes, 599 nt(target 1 + target 2) and 530 nt (target 1 + target 3), were detected only when sgRNAs had been expressed (Figure S2A).
Single PCR bands were produced after cloning of each cellline (Figure 3A). Sequencing of the PCR fragments determined
the purity of the clones and the deletion sites (Figure S2B), confirming the generation of two deletion mutant cell lines: OSCDtj-cis 1 and OSC-Dtj-cis 2.
We conducted northern blotting using a WT probe (Figure 1B)
to detect endogenous Tj-piRNAs in both wild-type and mutant
OSC lines. This revealed that the piRNAs arising from the first
Tj 30 UTR hotspot (Figure 1A) in normal OSCs were barely detected in OSC-Dtj-cis 1 and OSC-Dtj-cis 2 cells (Figure 3B).
The production of Idefix-piRNAs arising from other genomic
loci was barely affected by Tj-cis-element deletion (Figure 3B). Deep sequencing and genome mapping of piRNAs
co-immunoprecipitated with Piwi showed that the numbers of
Tj-piRNAs, particularly from the 200-nt region neighboring the
Tj-cis element, were severely decreased in OSC-Dtj-cis 1 andCell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 431

Figure 3. Deletion of the Tj-cis Element by
CRISPR/Cas9 from the Drosophila Genome(A) Targeting sites of three sgRNAs, sgRNA1,
sgRNA2, and sgRNA3 are shown by purple triangles. Genomic regions deleted from the Tj locus
by CRISPR/Cas9 system are shown by blue bars.
Green bar represents the hotspot in the Tj 30 UTR.
The 599- and 530-nt PCR bands in Figure S2A
became single after cloning of the cell lines OSCDtj-cis 1 and OSC-Dtj-cis 2, respectively.
(B) Northern blotting was performed on total RNAs
isolated from OSC-Dtj-cis 1 and OSC-Dtj-cis 2
using the WT probe. Idefix-piRNA was detected asa control.
(C) Distribution of Tj-piRNAs. Signals are displayed
as reads per million (RPM) values. The lower
panels show enlarged views of the Tj 30 UTR(1–500). Red and green lines represent the Tj-cis
element and the hotspot, respectively. Gray boxes
show deletion sites.
(D) Distribution of genic piRNAs derived from Dm
and Brat.

OSC-Dtj-cis 2 cells (Figure 3C). piRNAs corresponding to thedeletion sites were no longer detected, confirming deletion of
the genomic elements. piRNAs mapped to the Tj 30 UTR showed
little difference between OSC-Dtj-cis 1 and OSC-Dtj-cis 2,
agreeing with the observation that MT-3 failed to produce MTpiRNAs (Figure 2B). Dm and Brat 30 UTRs are known as genic
piRNA sources (Saito et al., 2009) and the levels of Dm-piRNAs
and Brat-piRNAs originating from them were unaffected inboth mutant cell lines (Figure 3D). These results suggest that
the Tj-cis element identified in this study acts as a cis-regulatory
element for producing endogenous Tj-piRNAs from the neighboring region in OSCs.
The Tj-cis Element Is Sufficient for Producing piRNAs
from Downstream Regions
To determine whether the Tj-cis element is sufficient for
producing piRNAs from unintegrated DNA, we constructed
432 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors

plasmid EGFP-tj-cis, consisting of the
EGFP CDS, the Tj-cis element, andfour tandem repeats of a 25-nt fragment from which artificial piRNAs were
designed to be exogenously expressed(Figure 4A). We confirmed that thesequence was not found in the
Drosophila genome (data not shown).
Another mutant, MT-5, was also constructed, in which the Tj-cis element
and tandem repeat positions in EGFPtj-cis were swapped (Figure 4A). Northern blotting showed that in sharp
contrast to EGFP-tj-cis, MT-5 expressed
few piRNAs from the tandem repeats(Figure 4B). The expression levels of
the two constructs were similar (Figure S3). These results verify that the
Tj-cis element is sufficient to express
artificial piRNAs from downstream, but not upstream, arbitrary
elements.
We then deep-sequenced primary piRNAs associated with
Piwi in OSCs expressing EGFP-tj-cis and mapped them on
the construct. This revealed that piRNAs can be generated
throughout the 30 UTR, not only from the random sequence consisting of four identical repeats, but also from the Tj-cis element
and polylinker regions (Figure 4C). Both endogenous and exogenous piRNAs mapped to the Tj-cis element, and only a few
sequence reads corresponding to the EGFP CDS were detected.
These results corroborate that the Tj-cis element expressed
piRNAs from downstream, but hardly at all from upstream elements. It also agrees with the observation that a limited numberof piRNAs mapped upstream of the Tj-cis element, including the
Tj CDS and Tj 50 UTR (Robine et al., 2009). Characteristics of theartificial piRNAs derived from the EGFP-tj-cis construct are summarized (Figures 4D–4F).

Figure 4. The Tj-cis Element Is Sufficient to
Produce Genic piRNAs from Its Downstream Region(A) Schematic drawings of EGFP-tj-cis and its
mutant constructs. The Tj-cis element (100 nt)
is shown in orange. Blue boxes indicate 25-nt
random sequences, which are repeated four times
in each construct.
(B) Artificial piRNAs derived from the random sequences [blue in (A)] were detected by northernblotting using a 50-nt probe complementary to twotandem repeats. The WT probe detected endogenous Tj-piRNAs.
(C) piRNA sequencing reads mapping to the
EGFP-tj-cis construct. Signals are displayed as
RPM values.
(D) The size distribution of piRNAs derived from therepeat region in EGFP-tj-cis.
(E) Bar diagrams indicating nucleotide of 50 end inpiRNAs mapped to the repeat region.
(F) piRNA 50 end coverage over a 25-nt random
sequence (minimal unit of the repeat sequence).

Primary piRNA Production Largely Depends on
Yb Binding to the cis element
We previously performed HITS crosslinking immunoprecipitation(CLIP) experiments in OSCs using an anti-Yb antibody (Murota
et al., 2014). Here, to expand the read numbers, we constructed
two new Yb-CLIP libraries (Figure S4A), which showed a highcorrelation for tag sequences (Figure S4B). Bioinformatic analysis revealed that Yb bound strongly and persistently with
the Tj 30 UTR, but not the Tj CDS or 50 UTR (Figure 5A). The
Tj-cis element resided in one of the strong, if not the strongest,
Yb association sites in the 30 UTR (Figure 5A). Comparison of

Yb-CLIP mapping data with piRNA mapping data obtained in this study (Figure 3C; wild-type) showed that theygreatly overlap (Figure S4C), suggesting
the direct involvement of Yb in determining substrates in somatic primarypiRNA biogenesis.
Unlike MT-2, MT-3 failed to produce
artificial MT-piRNAs in OSCs (Figures 2A
and 2B). However, Yb-binding marks
were observed on regions shared with
MT-2 and MT-3 (Figures 2A and 5A). TjR1, a 121-nt sequence adjacent to the
Tj-cis element, was within the shared regions, so we next investigated whether
Tj-R1 could also produce artificial
piRNAs. The Tj-cis element in EGFP-tjcis (Figure 4A) was replaced with Tj-R1
to yield construct MT-6 (Figure 5B).
Northern blotting revealed that limited
artificial piRNAs were produced fromMT-6 in sharp contrast to EGFP-Tj-cis(Figures 5B and S4D). RNA immunoprecipitation (RIP) experiments followed by
qRT-PCR showed that MT-6 transcripts
only weakly bound Yb (Figure 5C). This suggested that despite
the close proximity of Tj-R1 to the Tj-cis element and Yb-binding
marks determined by CLIP, it was ineffective in driving piRNA
production. We also constructed MT-7, in which Tj-R1 was replaced with Tj-R2, encompassing another 100-nt sequence
following Tj-R1. MT-7 transcripts showed little Yb-binding capacity and produced only a small amount of artificial piRNAs(Figures 5B, 5C, and S4D). Yb-RNA binding determined by
CLIP may therefore reflect primary binding to cis elements thatprovokes piRNA production, and secondary binding that does
not provoke piRNA production but determines the domains to
Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 433

Figure 5. The Tj-cis Element, but Not TjR1 and Tj-R2, Triggers Artificial piRNA
Production(A) A browser view of Yb-CLIP tags mapped onto
the Tj locus. Signals are displayed as read counts.
(B) Schematic of EGFP-tj-cis, MT-6, MT-7, andMT-8. Northern blotting was performed as in
Figure 4B.
(C) RIP-qPCR analysis shows that transcripts from
MT-6, MT-7, and MT-8 only weakly bound Yb in
OSCs. The primers used for qPCR are indicated byarrows in (B). Bars represent means ± SD of three
independent experiments (*p < 0.05).
(D) Yb-CLIP mapping data show that Yb binding of
the Tj-R1 and Tj-R2 regions in the Drosophila
genome is low in OSC-Dtj-cis1 (Figure 3A). Signals
are displayed in RPM.

UV crosslinking has been shown tooccur preferentially at U-rich stretches(Sugimoto et al., 2012), it does not
exclude the possibility that Yb showsa U bias in its RNA binding. CIMSs
were found in the Tj-cis element, Tj-R1,
and Tj-R2 (Figure S4F), suggestingthat it would be difficult to distinguishprimary and secondary Yb-RNA binding
via bioinformatic approaches. We sought
to computationally identify any shared
structural motifs in the Yb-CLIP sequence tags but found no obvious motifs(data not shown).

be processed in piRNA production. HITS-CLIP experiments using
OSC-Dtj-cis 1 (Figure 3A) revealed that when the Tj-cis element
was deleted from the Drosophila genome, Yb only slightly boundthe Tj-R1 and Tj-R2 regions within the genome (Figure 5D). These
results agree with the observation that MT-6 and MT-7 produced
a limited amount of piRNAs (Figure 5B) and further support our
original idea that Yb binding to Tj-R1 and Tj-R2 shown by YbCLIP (Figure 5A) is a secondary event depending on the primarybinding of Yb to the Tj-cis element.
We enumerated 57,079 Yb-binding positions by identifying
crosslinking-induced mutation sites (CIMSs) (Zhang and Darnell, 2011), which clarified that the nucleotide composition
around CIMSs showed strong U peaks (Figure S4E). Although
434 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors

cis-Regulatory Elements in flamand Other Genic piRNA Sources
Genome-wide analysis of Yb-CLIP tags
revealed that Yb binds various but particular regions across flam transcripts, andthat the regions greatly overlap withflam-piRNA mapping regions (Figure 6A).
Neither piRNAs nor Yb-CLIP tags were
mapped on the neighboring gene, Dip1
(Figure 6A). This further verifies a pivotal
role for Yb in piRNA biogenesis substrate
determination.
flam exons 1 and 2 are shared with flam isoforms in ovariansomatic sheet cells (equivalent to OSCs in this study) and fly
ovaries (Goriaux et al., 2014). Both exons are also good targets
for Yb binding (Figure 6A), so we examined if they act as cis-regulatory elements for piRNA production. For this, the Tj-cis
element in the EGFP-tj-cis construct was replaced with either
flam exon 1 or exon 2, yielding EGFP-flam-e1 and EGFP-flam-e2(Figure 6B). Both exons produced artificial piRNAs from theirdownstream region (Figures 6C and S5A). We then fragmented
exon 1 into R1 and R2 and repeated the assays. In accordancewith Yb-CLIP results, R1 was predicted to bind Yb only weakly
but it produced few artificial piRNAs. By contrast, R2, predicted
to bind Yb relatively strongly, produced piRNAs similarly toFigure 6. Analysis of cis Elements in flamand CG9257 Transcripts(A) A browser view of Yb-CLIP tags and piRNAsequences in wild-type OSCs (Figure 3C) mapped
onto the flam locus. The bottom two rows show
zoom-in mapping data of the top row. Signals are
displayed as read counts.
(B) Schematic of EGFP-tj-cis, EGFP-flam-e1, and
EGFP-flam-e2.
(C) Northern blotting was performed as in Figure 4B.
(D) A browser view of Yb-CLIP tags and piRNAsequences in wild-type OSCs (Figure 3C) mapped
onto the flam exon 1. Two deletion mutants, R1
and R2, are indicated. Signals are displayed asread counts. Schematic of EGFP-flam-e1, EGFPflam-e1-R1, and EGFP-flam-e1-R2.
(E) Northern blotting was performed as in Figure 4B.
(F) A browser view of Yb-CLIP tags and piRNAsequences in wild-type OSCs (Figure 3C) mapped
onto the CG9257 locus. Three fragments, R1, R2,
and R3, used to produce the constructs are indicated. Signals are displayed as read counts.
Schematic of EGFP-CG9257-R1, EGFP-CG9257R2, and EGFP-CG9257-R3 constructs.
(G) Northern blotting was performed as in Figure 4B.

full-length exon 1 (Figures 6D, 6E, and S5B). Yb-RNA binding
therefore plays a crucial role in driving piRNA production.
We further extended our experiments to the genic piRNA
gene CG9257 (Saito et al., 2009). Yb-CLIP showed that Yb
bound almost the entire 30 UTR of the transcripts (Figure 6F),
and regions R1 and R2 in the CG9257 30 UTR acted as cisregulatory elements similar to the Tj-cis element (Figures 6F
and 6G). However, a deletion mutant of R2, R3, produced few
piRNAs, despite its moderate binding with Yb. R3 may therefore represent a secondary binding site of Yb, as well as Tj-R1
and Tj-R2.
Exogenously Expressed piRNAs Are Sufficiently
Abundant to Elicit the Silencing of Endogenous Genes
Next, we assessed whether artificial piRNAs expressed under
the control of the Tj-cis element were able to repress endoge-

nous genes in OSCs. We chose Krimper(Krimp) as an endogenous gene target
because its knockdown previously had
no effect on piRNA biogenesis or function (Olivieri et al., 2012). We constructedfour plasmids (Krimp-50 , Krimp-CDS-1,
Krimp-CDS-2, and Krimp-30 ) expressing
piRNAs targeting the 50 UTR, CDS-1(1–50), CDS-2 (591–640), and the 30
UTR of Krimp mRNA, respectively (Figures 7A and 7B). All plasmids werebased on the EGFP-tj-cis construct byinserting three identical copies of Krimp
targeting sequences (50 nt each; except
for the sequence targeting the 50 UTR,
which was 72 nt) in front of the tandem repeats. The insertion
was made in an antisense orientation; thus, piRNAs arisingfrom the insertions should act as antisense to Krimp mRNAs.
Northern blotting confirmed the expression of Krimp-piRNAs(Figure 7C).
To determine the efficiency of Krimp-piRNAs in Krimp
silencing, OSCs were transfected with Krimp-50 , Krimp-CDS-1,
Krimp-CDS-2, and Krimp-30 , together with another plasmid containing a blasticidin-resistance gene. On day 3 after transfection,
blasticidin selection was applied to remove untransfected cells(Figure S6A). Western blotting with an anti-Krimp antibody (Nagao et al., 2011) showed that Krimp was significantly repressedin OSCs when Krimp-piRNAs targeting the Krimp CDS (1–50)
and 30 UTR were expressed (Figure S6B). Conversely, KrimppiRNAs targeting the 50 UTR and CDS (591–640) repressed
Krimp to a lesser extent (Figure S6B).
Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 435

Figure 7. Artificial piRNAs Are Driven by the
Tj-cis Element Silence Endogenous Genesin OSCs(A) Schematic of constructs for expressing artificial
Krimp-piRNAs. Genomic DNA fragments corresponding to Krimp 50 UTR, CDS-1, CDS-2, and the
30 UTR (see in B) are inserted three times into
EGFP-tj-cis.
(B) Four regions in the Krimp gene were selected
as Krimp-piRNA generation sites: Krimp-50 (1–72),
Krimp-CDS-1 (1—50), Krimp-CDS-2 (591–640),
and Krimp-30 (1–50).
(C) Production of exogenous Krimp-piRNAs wasmonitored by northern blotting. Artificial piRNAs
expressed from the random sequence repeats(blue) were also detected.
(D) Immunofluorescence using anti-EGFP andanti-Krimp antibodies was performed on OSCs
expressing exogenous Krimp-piRNAs. Krimp
forms cytoplasmic foci called Krimp body (arrowheads), which are not observed in EGFP-positive
OSCs. Scale bars represent 20 mm.
(E) Two regions in the Tj gene were selected as TjpiRNA generation sites: Tj-50 and Tj-CDS.
(F) The production of exogenous Tj-piRNAs wasmonitored by northern blotting using probes corresponding to the particular regions. Artificial
piRNAs expressed from the random sequence
repeats (blue) were also detected.
(G) Immunofluorescence using anti-EGFP andanti-TJ antibodies was performed on OSCs
expressing exogenous Tj-piRNAs. Scale bars
represent 20 mm.

Considering that blasticidin treatment might not fully eliminate
untransfected cells, leading to an apparent weakness in thesilencing effect, we performed immunofluorescence using ananti-Krimp antibody. This determined the silencing effect onlyin cells in which Krimp-piRNAs had been generated, because
cells considered EGFP-positive showed high EGFP mRNA
expression, suggesting a high level of Krimp-piRNA expression.
As expected, Krimp bodies (Nagao et al., 2011; Olivieri et al.,2012), cytoplasmic granules in which Krimp strongly accumulates, disappeared in EGFP-positive cells regardless of theconstructs used for transfection (Figures 7D and S6C). This indicated that Krimp silencing occurred regardless of where KrimppiRNAs targeted Krimp mRNA.
Krimp-piRNAs exogenously expressed from the constructs
were loaded onto Piwi to form piRISCs (Figure 7C). To examine
if Krimp silencing occurred Piwi-dependently, the efficiency of
Krimp silencing was assessed in Piwi-depleted OSCs. When
Piwi was present in OSCs, Krimp expression was strongly downregulated by Krimp-piRNAs (Figure S6D). However, when Piwi
was depleted, Krimp expression was restored (Figure S6D).
436 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors

Thus, Krimp silencing mediated byexogenous piRNAs appears to be Piwi
dependent.
To assess whether Krimp silencing
mediated by exogenous Krimp-piRNAs
is transcriptional, we performed chromatin-immunoprecipitation (ChIP). The association of RNA
pol II with Krimp was significantly reduced by Krimp-piRNAs
targeting the Krimp CDS (1–50) and 30 UTR (Figure S6E). Under
these conditions, the accumulation of H3K9me3 at Krimp
increased (Figure S6E). The expression of Krimp-piRNAs inthe sense orientation only slightly affected RNA pol lI and
H3K9me3 binding to Krimp (Figure S6F). These results suggestthat exogenously expressed piRNAs trigger transcriptionalsilencing in OSCs.
We also set out to determine the silencing effect of artificial
piRNAs against Tj. Primary piRNAs targeting Tj mRNAs (Figure 7E) were expressed in OSCs. Two plasmids were produced
based on the EGFP-tj-cis construct, as in Figure 7A, by inserting
three identical Tj targeting sequences (Tj-50 and Tj-CDS; 50 nteach) in front of the tandem repeats. Northern blotting confirmed
the expression of corresponding piRNAs (Figure 7F). Immunofluorescence using anti-Tj antibodies showed that EGFP-positive
cells expressing artificial Tj-50 -piRNAs and Tj-CDS-piRNAs
lacked TJ signals in the nucleus (Figure 7G) (note that these artificial piRNAs were ‘‘antisense’’ to Tj mRNA while endogenous

Tj-piRNAs are ‘‘sense’’ to Tj mRNA). Thus, artificial piRNAs
produced under control of the Tj-cis element appear capableof silencing endogenous genes.
DISCUSSION
Yb bodies and Flam bodies in OSCs are considered to be thecenters for primary piRNA maturation/piRISC formation andpiRNA intermediate storage, respectively, and exist in close
proximity (Murota et al., 2014; Olivieri et al., 2010; Saito et al.,2010). The formation of both bodies depends on the Yb protein,
particularly its RNA-binding activity (Murota et al., 2014). In theabsence of this, piRNA processing fails, resulting in piRNAloss, although piRNA intermediates and processing factors arepresent in the cytosol. Thus, Yb binding to piRNA sources centralizes all necessary ingredients for piRNA biogenesis, whichis crucial for primary piRNA production (Murota et al., 2014). In
this study, we continued the study on Yb and discovered thatthe direct association of Yb with a specific 100-nt element(i.e., cis element) within the piRNA precursors provokes somatic
primary piRNA biogenesis from downstream regions. Insertion of
the Yb-binding element within RNA molecules that do not otherwise serve as piRNA precursors converts the RNA transcripts
into piRNA sources. Artificial primary piRNAs were mapped
only downstream, but not upstream, of regions of the Yb-binding
element. Previous studies demonstrated that natural genic
piRNAs mostly arise from 30 UTRs rather than mRNA CDS or 50
UTRs (Robine et al., 2009; Saito et al., 2009). The present study
also showed that few Tj-piRNAs mapped to the Tj CDS (Figure 3C), and that few Yb-CLIP tags were also found in the Tj
CDS (Figure 5A). Thus, Yb determines not only substrate specificity but also processing directionality in the somatic primarypiRNA biogenesis pathway. This may occur through the Ybcontrolled recruitment of other piRNA factors, such as anotherputative RNA helicase Armi and endonuclease Zuc, only todownstream sequences. We are currently investigating thispossibility.
Yb-CLIP tags greatly overlap with primary piRNA-producing
loci in the genome. This strongly supports the idea that Yb
is the central player in determining substrates in the piRNA
pathway. An unexpected but intriguing observation in our study
is that Tj-R1 and Tj-R2 in the Tj 30 UTR show strong Yb-binding
marks, as does the Tj-cis element, but provoked very little artificial piRNA production in contrast to the Tj-cis element (Figure 5B). Yb-CLIP experiments showed that Yb binding to Tj-R1
and Tj-R2 within the Drosophila genome largely depends on
Yb binding to its upstream Tj-cis element (Figure 5D). We therefore propose a model in which Yb determines primary piRNAsources by two sequential modes of action: primary binding to
cis elements that represents selection of piRNA precursors
among cellular RNAs, then secondary binding to downstream regions, representing the defining domains to be processed by
precursors (Figure S7). This complexity in determining piRNA
precursors could ensure the high diversity in piRNA populations,which is a unique feature of piRNAs (Aravin et al., 2007; Brennecke et al., 2007; Lau et al., 2009).
The RNA-binding activity of Yb is required for primary piRNA
production in OSC. Yb mutants carrying a point mutation within

the DEAD box showed little RNA binding activity (Murota et al.,2014). When these Yb mutants were expressed individually in
OSC lacking endogenous Yb, piRNA precursors were not accumulated in Flam bodies, and few piRNAs were produced. As aconsequence, transposons were de-silenced. Therefore, there
is little doubt that the RNA-binding activity of Yb through the
DEAD-box is indispensable for primary piRNA production.
HITS-CLIP experiments clarified direct interaction of Yb with
piRNA sources, including Tj mRNA. Insertion of a particular
Yb-bound RNA element within Tj mRNA, i.e., the Tj-cis element,
upstream of any given RNA molecule enables the arbitrary
sequences to produce artificial piRNAs. Deletion of the Tj-cis
element from the Drosophila genome significantly abolishedpiRNA production from its downstream region spanning at least200 nt. These observations strongly support our model (Figure S7), in which Yb is the trans-acting factor that recognizes
and binds cis elements within piRNA precursors to provoke
primary piRNA biogenesis in ovarian somatic cells. However, it
does not exclude the possibility that Yb collaboratively achieves
this task with unknown factors. Moreover, we are not certain if Yb
is the uppermost factor in the cytoplasmic phase of the biogenesis pathway upon nuclear transport of piRNA precursors. We
are currently engaged in addressing these challenging questionsin the laboratory.
EXPERIMENTAL PROCEDURES
Cell Culture and Transfection
OSCs were cultured as described previously (Saito et al., 2009; Niki et al.,2006). OSC transfection was carried out using Xfect Transfection Reagent(Clontech) as described previously (Murota et al., 2014). Blasticidin (Life Technologies) was added to the media at 50 mg/ml upon co-transfection usinga plasmid carrying a blasticidin-resistant gene. RNAi was performed as
described previously (Murota et al., 2014).
Plasmid Construction
To generate the EGFP-tj WT construct, the full-length Tj 30 UTR was first PCRamplified with the primers tj-30 UTR F/R (Table S1, all oligonucleotides werepurchased from Invitrogen) from OSC cDNA samples using KOD plus DNA polymerase (Toyobo) and then cloned between XhoI and BamHI sites of pEGFPC1 (Clontech). The polylinker region between EGFP and Tj 30 UTR was removed
by inverse PCR using the primers delta-mcs F/R. The EGFP-tj 30 UTR insert was
subcloned into the NheI and BamHI sites of pAcM (Saito et al., 2009). The Cas9
plasmid, phsp70-Cas9, and the sgRNA plasmid, pU6-BbsI-chiRNA, were purchased from Addgene. Target sequences were synthesized as oligomers and inserted into the BbsI site of pU6-BbsI-chiRNA. Detailed methods for constructing
other plasmids are described in the Supplemental Information.
Derivation of the Tj-cis Element Deletion Mutant OSCs
OSCs were transfected with 4 mg of Cas9 plasmid, 4 mg of sgRNA plasmids, and
400 ng of the blasticidin resistance gene plasmid as described above. Cells
were incubated for 48 hr post-transfection prior to blasticidin selection. Threedays after addition of blasticidin, 5.0 3 103 cells were passaged in 6-cm dishes
and allowed to grow in blasticidin containing medium. After 6–7 days of culture,colonies were picked and passaged in single wells of 24-well plates andallowed to grow to confluence in blasticidin free medium. Genomic DNA wasextracted using QuickExtract DNA Extraction Solution (Epicenter) following
the manufacturer’s protocol. The genomic region flanking the CRISPR target
site was PCR amplified and analyzed by electrophoresis on 1% agarose gels.
Immunoprecipitation
Immunoprecipitation from OSCs was performed as previously described(Saito et al., 2009). Anti-Piwi antibody (Saito et al., 2006) was immobilized on

Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 437

Dynabeads protein G (Invitrogen). Total RNAs were isolated from the immunoprecipitates with phenol-chloroform and precipitated with ethanol.
Northern Blotting
Total RNA was isolated from OSCs using RNAzol RT (Molecular ResearchCenter). Any contaminating DNA was digested by TURBO DNase (Ambion).
For small RNA detection, northern blotting analysis was carried out using
5 mg of total RNAs as described previously (Saito et al., 2006). The DNA oligonucleotides used are summarized in Table S1. mRNAs were purified from20 mg of total RNA using Oligotex-dT30 (Takara) according to the manufacturer’s instructions, and northern blotting analysis was carried out asdescribed previously (Murota et al., 2014). DNA probes were synthesizedusing a Random Primer DNA Labeling Kit Ver. 2 (Takara) in the presence of
32
P-dCTP. Templates of random-primed probes were generated by PCR.
For the EGFP probe, the EGFP ORF was amplified using the primers EGFP
F/R with EGFP-C1 vector as template. For the GAPDH probe, the GAPDH
ORF was amplified using the primers GAPDH F/R with OSC cDNA as template.
Western Blotting
Western blotting was performed as described previously (Saito et al., 2006).
Anti-Piwi (Saito et al., 2006) (1:1,000 dilution), anti-EGFP (MBL) (1:1,000 dilution), anti-Yb (Murota et al., 2014) (1:1,000 dilution), and anti-tubulin (DSHB)(1:2,000 dilution) antibodies were used.
Small RNA Cloning and Sequencing
For piRNA cloning, coimmunoprecipitated piRNA was extracted from Piwi immunoprecipitates using phenol and chloroform, and gel-purified piRNAs werecloned using the NEBNext Small RNA Library Sample Prep Set (NEB). piRNAlibraries were analyzed on a HiSeq2000 (Illumina).
Small RNA Sequencing Data Analysis
Sequencing of small RNA libraries generated from Piwi immunoprecipitates
were performed using HiSeq2000. Adaptor sequences were removed from obtained reads and mapped to the Release 5 assembly of the Drosophila genome
using Bowtie (Langmead et al., 2009), allowing zero mismatch and extracting
the reads mapped uniquely to the genome. Reads originating from EGFP-tj-cis
construct were mapped against construct sequence using Bowtie, allowing
zero mismatch and default parameters. The reads were normalized to RPM(read per million) by the number of Drosophila genome-mapped reads.
HITS-CLIP
CLIP was performed basically as described previously (Jaskiewicz et al.,2012). OSCs (1–2 3 108) were UV crosslinked by irradiating uncovered
with 200 mJ/cm2 of 254 nm UV, followed by lysis with lysis buffer (20 mM
HEPES-KOH [pH 7.3], 1 mM EDTA, 1 mM DTT, 150 mM sodium chloride,
2 mg/ml pepstatin, 2 mg/ml leupeptin, 0.5% aprotinin, 0.5% NP40), and celldebris was removed by centrifugation. Immunoprecipitation was performed
using 20 mg of anti-Yb antibody (Murota et al., 2014), followed by the treatmentof 10 U/ml RNaseT1 (Roche). Dephosphorylation of RNA segments crosslinked
to immunoprecipitated proteins was performed by calf intestinal alkaline
phosphase (CIP form NEB), and the samples were 50 end labeled, followedby NuPAGE (Life Technologies) and transfer to the membrane. Protein-bound
RNA signal was cut out from the membrane, and RNA was isolated by phenol/
chloroform extraction. For library construction, preadenylated 30 adaptor was
ligated to the RNA samples using T4 RNA Ligase 2, truncated KQ (NEB), followed by 50 adaptor ligation using T4 RNA Ligase 1 (NEB). Adaptor-ligated
RNA samples were reversed transcribed using SuperScript III (Life Technologies) and PCR amplified using Q5 High-Fidelity DNA Polymerase (NEB).
Bioinformatic Analysis
CLIP tags were mapped to Release 5 assembly of the Drosophila genome aspreviously described (Murota et al., 2014). CIMSs were detected according toZhang and Darnell (2011). For each genomic position where at least one deletion was observed on the mapped CLIP tags, the number of mapped CLIP tags(k) and that of tags with deletions (m) were counted. The null distribution of mfor given k was obtained by permuting positions of the deletions. The permutation was conducted by preserving the distribution of the CLIP tags on the

438 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors

genome and the positional distribution of deletions relative to the 50 ends of
the CLIP tags. The false discovery rate (FDR) was estimated using the cumulative distributions of m for a given k, and the positions with FDR <0.01 weredetected as CIMSs.
Assessing Reproducibility of HITS-CLIP Data
Given two biological replicate data sets of HITS-CLIP, their reproducibility wasassessed as follows. For each CIMS, in one data set (query) and in the other(target), we collected reads that were mapped to the CIMS position and to
the same strand using BEDTools (version 2.18.1) and an in-house Perl script.
The boundaries of each read cluster were defined as the left-most and rightmost positions of the collected reads. For each cluster in query, the clusterin target that overlapped with it by at least 1 bp and whose CIMS position
was closest to the query CIMS position was identified. Then, Pearson andSpearman’s rank correlation coefficients between their read numbers were
calculated. If multiple clusters in target satisfied the above criterion (i.e., withthe same CIMS distances), the averaged read number was used.
RIP and qPCR
OSCs (4–5 3 107) were homogenized in lysis buffer (20 mM HEPES-KOH[pH 7.3], 150 mM sodium chloride, 1 mM DTT, 1 mM EDTA, 2 mg/ml pepstatin,
2 mg/ml leupeptin, 0.5% aprotinin, 0.5% NP40, and 40 U/ml RNasin [Promega]), and cell debris was removed by centrifugation. One-tenth of the lysate
was saved as RNA input, and total RNAs were isolated using ISOGEN-LS
reagent (Nippongene). Anti-Yb antibody (Murota et al., 2014) (2 mg) was immobilized on Dynabeads protein G. Lysates were incubated with beads for 2 hr at
4 C before washing three times with IP wash buffer (20 mM HEPES-KOH[pH 7.3], 300 mM sodium chloride, 1 mM DTT, 2 mg/ml pepstatin, 2 mg/ml leupeptin, 0.5% aprotinin, 0.05% NP40) and three times with high-salt wash
buffer (20 mM HEPES-KOH [pH 7.3], 500 mM sodium chloride, 1 mM DTT,
2 mg/ml pepstatin, 2 mg/ml leupeptin, 0.5% aprotinin, and 0.05% NP40). Total
RNAs were isolated from the immunoprecipitates with phenol-chloroform andwere precipitated with ethanol. For qPCR analysis following RNA immunoprecipitation, a fixed volume of RNA isolated from input and immunoprecipitates
was used for reverse transcription. Reverse transcription was performed using
ReverTra Ace qPCR RT Master Mix (Toyobo). qPCRs were performed using
the specific qPCR primers mcs F/R (Table S1) in the StepOnePlus RealTime PCR System (Life Technologies). THUNDERBIRD SYBR qPCR Mix(Toyobo) was used as described in the instruction manual. The specific enrichment was analyzed based on a percent input calculation.
Immunofluorescence
Immunofluorescence of OSCs was performed using anti-EGFP IgG2b (MBL)(1:500 dilution), anti-Krimp IgG1 (Nagao et al., 2011) (1:250 dilution), andanti-TJ IgG1 (1:250 dilution) antibodies as described previously (Murota
et al., 2014). Alexa-Fluor-488-conjugated anti-mouse IgG2b (Molecular
Probes) and Alexa-Fluor-594-conjugated anti-mouse IgG1 (Molecular Probes)
antibodies were used as secondary antibodies (1:1,000 dilution). Anti-TJ
monoclonal antibody was produced by immunizing mice with GST-TJ protein
as previously described (Saito et al., 2009).
ChIP and qPCR
OSCs (1–2 3 108) were crosslinked with 1% formaldehyde for 5 min at room
temperature, and crosslinking was then quenched with 125 mM glycine for5 min at room temperature. Fixed cells were washed twice with PBS, harvested by scraping, lysed with swelling buffer (25 mM HEPES-KOH [pH 7.3],
1.5 mM magnesium chloride, 10 mM potassium chloride, 0.1% NP40, 1 mM
DTT, 1 3 Halt Protease Inhibitor Cocktail [Thermo Scientific]), and pelleted.
Nuclear pellets were extracted by sonication buffer (50 mM HEPES-KOH[pH 7.3], 140 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 0.1% sodium
deoxycholate, 0.1% SDS, 1 3 Halt Protease Inhibitor Cocktail), sonicated in a
Covaris S220 focused ultrasonicator, and diluted. After removing input, theDNA-protein complexes were incubated with 2 mg of nonimmune IgG antibody, anti-Pol II antibody 4H8 (CST), or anti-H3K9me3 antibody (Active motif)
at 4 C overnight. DNA-protein complexes were precipitated using Dynabeads
Protein G for 1 hr. The beads were consequently washed with low-salt wash
buffer (20 mM Tris-HCl [pH 8.0], 150 mM sodium chloride, 2 mM EDTA,

0.1% SDS, 0.1% Triton X-100, 1 mM PMSF), high-salt wash buffer (20 mM
Tris-HCl [pH 8.0], 500 mM sodium chloride, 2 mM EDTA, 0.1% SDS, 0.1%
Triton X-100, 1 mM PMSF), LiCl wash buffer (10 mM Tris-HCl [pH 8.0], 1 mM
EDTA, 250 mM lithium chloride, 1% NP40, 1% sodium deoxycholate,
1 mM PMSF), and twice with TE (10 mM Tris-HCl [pH 8.0], 1 mM EDTA,
1 mM PMSF). After washing, immunoprecipitated chromatin was eluted byelution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 1% SDS). Eluted
chromatin and input samples were reverse crosslinked for 12–16 hr at 65 C.
ChIP DNA was treated with RNase A for 30 min at 37 C, and then treatedwith proteinase K for 60 min at 55 C. DNA was purified by extraction withphenol/chloroform/isoamyl alcohol and ethanol precipitation. Pellet Paint NF
Co-Precipitant (Merck Millipore) was added to the DNA prior to precipitation.
qPCRs were performed using ChIP DNA with specific qPCR primers Krimper
F/R (Table S1) in StepOnePlus Real-Time PCR Systems. THUNDERBIRDSYBR qPCR Mix was used as described in the instruction manual. The specificenrichment was analyzed by percentage input calculation.

Han, B.W., Wang, W., Li, C., Weng, Z., and Zamore, P.D. (2015). Noncoding
RNA. piRNA-guided transposon cleavage initiates Zucchini-dependent,
phased piRNA production. Science 348, 817–821.
Houwing, S., Kamminga, L.M., Berezikov, E., Cronembold, D., Girard, A., vanden Elst, H., Filippov, D.V., Blaser, H., Raz, E., Moens, C.B., et al. (2007). A rolefor Piwi and piRNAs in germ cell maintenance and transposon silencing in
Zebrafish. Cell 129, 69–82.
Hsu, P.D., Lander, E.S., and Zhang, F. (2014). Development and applicationsof CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278.
Huang, X.A., Yin, H., Sweeney, S., Raha, D., Snyder, M., and Lin, H. (2013). A
major epigenetic programming mechanism guided by piRNAs. Dev. Cell 24,
502–516.
Ipsaro, J.J., Haase, A.D., Knott, S.R., Joshua-Tor, L., and Hannon, G.J. (2012).
The structural biochemistry of Zucchini implicates it as a nuclease in piRNAbiogenesis. Nature 491, 279–283.

ACCESSION NUMBERS

Ishizu, H., Siomi, H., and Siomi, M.C. (2012). Biology of PIWI-interacting RNAs:
new insights into biogenesis and function inside and outside of germlines.
Genes Dev. 26, 2361–2373.

Deep sequencing data sets have been deposited in the NCBI GEO and areavailable under accession number GEO: GSE69625.

Jaskiewicz, L., Bilen, B., Hausser, J., and Zavolan, M. (2012). Argonaute
CLIP—a method to identify in vivo targets of miRNAs. Methods 58, 106–112.

SUPPLEMENTAL INFORMATION

Juliano, C., Wang, J., and Lin, H. (2011). Uniting germline and stem cells: thefunction of Piwi proteins and the piRNA pathway in diverse organisms.
Annu. Rev. Genet. 45, 447–469.

Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2015.06.035.
ACKNOWLEDGMENTS
We thank the other members of the M.C.S. laboratories for their discussions
and comments on the manuscript. We also thank T. Fukunaga 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 (MEXT) of Japan to H.I., Y.W.I., W.I., H.S., and M.C.S.
Received: May 19, 2015
Revised: June 8, 2015
Accepted: June 10, 2015
Published: July 9, 2015
REFERENCES
Aravin, A.A., Hannon, G.J., and Brennecke, J. (2007). The Piwi-piRNA pathway
provides an adaptive defense in the transposon arms race. Science 318,
761–764.
Brennecke, J., Aravin, A.A., Stark, A., Dus, M., Kellis, M., Sachidanandam, R., and Hannon, G.J. (2007). Discrete small RNA-generating locias master regulators of transposon activity in Drosophila. Cell 128,
1089–1103.
Choi, S.Y., Huang, P., Jenkins, G.M., Chan, D.C., Schiller, J., and Frohman,
M.A. (2006). A common lipid links Mfn-mediated mitochondrial fusion and
SNARE-regulated exocytosis. Nat. Cell Biol. 8, 1255–1262.
Dönertas, D., Sienski, G., and Brennecke, J. (2013). Drosophila Gtsf1 is an
essential component of the Piwi-mediated transcriptional silencing complex.
Genes Dev. 27, 1693–1705.

Kawaoka, S., Hayashi, N., Suzuki, Y., Abe, H., Sugano, S., Tomari, Y.,
Shimada, T., and Katsuma, S. (2009). The Bombyx ovary-derived cell line
endogenously expresses PIWI/PIWI-interacting RNA complexes. RNA 15,
1258–1264.
Khurana, J.S., and Theurkauf, W. (2010). piRNAs, transposon silencing, andDrosophila germline development. J. Cell Biol. 191, 905–913.
Langmead, B., Trapnell, C., Pop, M., and Salzberg, S.L. (2009). Ultrafast andmemory-efficient alignment of short DNA sequences to the human genome.
Genome Biol. 10, R25.
Lau, N.C., Robine, N., Martin, R., Chung, W.J., Niki, Y., Berezikov, E., and Lai,
E.C. (2009). Abundant primary piRNAs, endo-siRNAs, and microRNAs in a
Drosophila ovary cell line. Genome Res. 19, 1776–1785.
Li, M.A., Alls, J.D., Avancini, R.M., Koo, K., and Godt, D. (2003). The large Maf
factor Traffic Jam controls gonad morphogenesis in Drosophila. Nat. Cell Biol.
5, 994–1000.
Malone, C.D., Brennecke, J., Dus, M., Stark, A., McCombie, W.R., Sachidanandam, R., and Hannon, G.J. (2009). Specialized piRNA pathways act ingermline and somatic tissues of the Drosophila ovary. Cell 137, 522–535.
Mohn, F., Handler, D., and Brennecke, J. (2015). Noncoding RNA. piRNAguided slicing specifies transcripts for Zucchini-dependent, phased piRNAbiogenesis. Science 348, 812–817.
Muerdter, F., Guzzardo, P.M., Gillis, J., Luo, Y., Yu, Y., Chen, C., Fekete, R.,
and Hannon, G.J. (2013). A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila. Mol.
Cell 50, 736–748.
Murota, Y., Ishizu, H., Nakagawa, S., Iwasaki, Y.W., Shibata, S., Kamatani,
M.K., Saito, K., Okano, H., Siomi, H., and Siomi, M.C. (2014). Yb integrates
piRNA intermediates and processing factors into perinuclear bodies toenhance piRISC assembly. Cell Rep. 8, 103–113.

Ghildiyal, M., and Zamore, P.D. (2009). Small silencing RNAs: an expanding
universe. Nat. Rev. Genet. 10, 94–108.

Nagao, A., Sato, K., Nishida, K.M., Siomi, H., and Siomi, M.C. (2011). Genderspecific hierarchy in nuage localization of PIWI-interacting RNA factors in
Drosophila. Front. Genet. 2, 55.

Goriaux, C., Desset, S., Renaud, Y., Vaury, C., and Brasset, E. (2014). Transcriptional properties and splicing of the flamenco piRNA cluster. EMBO
Rep. 15, 411–418.

Niki, Y., Yamaguchi, T., and Mahowald, A.P. (2006). Establishment of stable
cell lines of Drosophila germ-line stem cells. Proc. Natl. Acad. Sci. USA 103,
16325–16330.

Haase, A.D., Fenoglio, S., Muerdter, F., Guzzardo, P.M., Czech, B., Pappin,D.J., Chen, C., Gordon, A., and Hannon, G.J. (2010). Probing the initiationand effector phases of the somatic piRNA pathway in Drosophila. Genes
Dev. 24, 2499–2504.

Nishimasu, H., Ishizu, H., Saito, K., Fukuhara, S., Kamatani, M.K., Bonnefond,L., Matsumoto, N., Nishizawa, T., Nakanaga, K., Aoki, J., et al. (2012). Structure and function of Zucchini endoribonuclease in piRNA biogenesis. Nature
491, 284–287.

Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors 439

Ohtani, H., Iwasaki, Y.W., Shibuya, A., Siomi, H., Siomi, M.C., and Saito, K.
(2013). DmGTSF1 is necessary for Piwi-piRISC-mediated transcriptionaltransposon silencing in the Drosophila ovary. Genes Dev. 27, 1656–1661.
Olivieri, D., Sykora, M.M., Sachidanandam, R., Mechtler, K., and Brennecke, J.
(2010). An in vivo RNAi assay identifies major genetic and cellular requirementsfor primary piRNA biogenesis in Drosophila. EMBO J. 29, 3301–3317.
Olivieri, D., Senti, K.A., Subramanian, S., Sachidanandam, R., and Brennecke,
J. (2012). The cochaperone shutdown defines a group of biogenesis factors
essential for all piRNA populations in Drosophila. Mol. Cell 47, 954–969.
Qi, H., Watanabe, T., Ku, H.Y., Liu, N., Zhong, M., and Lin, H. (2011). The Ybbody, a major site for Piwi-associated RNA biogenesis and a gateway for
Piwi expression and transport to the nucleus in somatic cells. J. Biol. Chem.
286, 3789–3797.

Saito, K., Ishizu, H., Komai, M., Kotani, H., Kawamura, Y., Nishida, K.M.,
Siomi, H., and Siomi, M.C. (2010). Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila. Genes Dev. 24,
2493–2498.
Siomi, M.C., Sato, K., Pezic, D., and Aravin, A.A. (2011). PIWI-interacting
small RNAs: the vanguard of genome defence. Nat. Rev. Mol. Cell Biol.
12, 246–258.
Sugimoto, Y., König, J., Hussain, S., Zupan, B., Curk, T., Frye, M., and Ule, J.
(2012). Analysis of CLIP and iCLIP methods for nucleotide-resolution studies ofprotein-RNA interactions. Genome Biol. 13, R67.
Szakmary, A., Reedy, M., Qi, H., and Lin, H. (2009). The Yb protein defines anovel organelle and regulates male germline stem cell self-renewal in
Drosophila melanogaster. J. Cell Biol. 185, 613–627.

Robine, N., Lau, N.C., Balla, S., Jin, Z., Okamura, K., Kuramochi-Miyagawa,S., Blower, M.D., and Lai, E.C. (2009). A broadly conserved pathway generates
3’UTR-directed primary piRNAs. Curr. Biol. 19, 2066–2076.

Vagin, V.V., Sigova, A., Li, C., Seitz, H., Gvozdev, V., and Zamore, P.D. (2006).
A distinct small RNA pathway silences selfish genetic elements in the germline.
Science 313, 320–324.

Saito, K., Nishida, K.M., Mori, T., Kawamura, Y., Miyoshi, K., Nagami, T.,
Siomi, H., and Siomi, M.C. (2006). Specific association of Piwi with rasiRNAs
derived from retrotransposon and heterochromatic regions in the Drosophila
genome. Genes Dev. 20, 2214–2222.

Zamparini, A.L., Davis, M.Y., Malone, C.D., Vieira, E., Zavadil, J., Sachidanandam, R., Hannon, G.J., and Lehmann, R. (2011). Vreteno, a gonad-specificprotein, is essential for germline development and primary piRNA biogenesis
in Drosophila. Development 138, 4039–4050.

Saito, K., Inagaki, S., Mituyama, T., Kawamura, Y., Ono, Y., Sakota, E., Kotani,
H., Asai, K., Siomi, H., and Siomi, M.C. (2009). A regulatory circuit for piwi by
the large Maf gene traffic jam in Drosophila. Nature 461, 1296–1299.

Zhang, C., and Darnell, R.B. (2011). Mapping in vivo protein-RNA interactions
at single-nucleotide resolution from HITS-CLIP data. Nat. Biotechnol. 29,
607–614.

440 Cell Reports 12, 429–440, July 21, 2015 ª2015 The Authors