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RESEARCH COMMUNICATION

DmGTSF1 is necessaryfor Piwi–piRISC-mediatedtranscriptional transposon
silencing in the Drosophila
ovary
Hitoshi Ohtani,1 Yuka W. Iwasaki,1 Aoi Shibuya,1Haruhiko Siomi,1 Mikiko C. Siomi,2,3
and Kuniaki Saito1,3
1

Department of Molecular Biology, Keio University Schoolof Medicine, Tokyo 160-8582, Japan; 2Department of Biophysics
and Biochemistry, Graduate School of Science,
The University of Tokyo, Tokyo 113-0032, JapanThe Piwi–piRNA (PIWI-interacting RNA) complex (Piwi–
piRISC) in Drosophila ovarian somatic cells represses
transposons transcriptionally to maintain genome integrity; however, the underlying mechanisms remain
obscure. Here, we reveal that DmGTSF1, a Drosophila
homolog of gametocyte-specific factor 1 (GTSF1) (whichis required for transposon silencing in mouse testes), is
necessary for Piwi–piRISC to repress target transposons
and neighboring genes. DmGTSF1 depletion affected
neither piRNA biogenesis nor nuclear import of Piwi–
piRISC. DmGTSF1 mutations caused derepression oftransposons and loss of ovary follicle layers, resulting infemale infertility. We suggest that DmGTSF1, a nuclear
Piwi interactor, is an integral factor in Piwi–piRISCmediated transcriptional silencing.
Supplemental material is available for this article.
Received May 8, 2013; revised version accepted June 26,
2013.

Mobilization of transposable elements (TEs) can lead tonatural insertion mutations that generally have negative
effects on the host genome (Lynch 2007). Thus, hostspecies have evolved control mechanisms that restrict
TE activity. One such mechanism is RNAi/RNA silencing, in which small RNAs 20–30 nucleotides (nt) in length
trigger multiple forms of sequence-specific gene silencing
by guiding Argonaute complexes or RNA-induced silencing complexes (RISCs) to target RNAs by means of basepairing (Ketting et al. 1999; Tabara et al. 1999; Slotkin andMartienssen 2007; Siomi and Siomi 2009).
In the Drosophila germline, PIWI-interacting RNAs
(piRNAs) are produced from single-stranded long noncoding RNAs that are transcribed from intergenic piRNA
clusters on the genome. Mature piRNAs are loaded onto
germline-specific Argonaute proteins or PIWI proteins to[Keywords: DmGTSF1; piRNA; Piwi; transposon; RNA silencing; Drosophila]
3Corresponding authors
E-mail saito@z6.keio.jp
E-mail siomim@biochem.s.u-tokyo.ac.jp
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.221515.113.

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yield piRISCs. piRNAs are amplified by the cytoplasmic‘‘ping-pong’’ cycle, in which TE transcripts are consumed
as both the source of piRNAs and the targets of PIWI
cleavage, thereby repressing TEs in the cytoplasm (Karginov
and Hannon 2010; Senti and Brennecke 2010; Siomi et al.
2011). It is becoming clear that piRISCs also mediate TE
silencing in the nucleus but in a PIWI cleavage-independent manner (Saito et al. 2010; Klenov et al. 2011; Wangand Elgin 2011; Sienski et al. 2012; Darricarrère et al.
2013).
Although three PIWI proteins—AGO3, Aubergine (Aub),and Piwi—are expressed in the Drosophila ovary (Brennecke
et al. 2007), only Piwi is expressed in ovarian somatic cells(OSCs), such as follicle cells, where piRNA intermediates
are digested by a single-stranded nucleic acid-specificendonuclease, Zucchini (Ipsaro et al. 2012; Nishimasu
et al. 2012). Zucchini products are further processed in
the cytoplasmic Yb bodies into mature piRNAs (Olivieri
et al. 2010; Saito et al. 2010). Piwi–piRISC formation also
takes place at Yb bodies, where several piRNA biogenesis
factors, including Armitage (Armi), accumulate (Olivieri
et al. 2010; Saito et al. 2010). piRNAs in OSCs are notamplified because these cells lack the core factors (i.e.,
AGO3 and Aub). Piwi–piRISCs are imported into the
nucleus, where they mediate TE repression. Nuclear
silencing by Piwi–piRISCs does not require cleavage ofTE transcripts but involves chromatin modification of
TE loci on the genome (Saito et al. 2010; Wang and Elgin2011; Sienski et al. 2012; Huang et al. 2013; Le Thomas
et al. 2013; Rozhkov et al. 2013). However, the underlying
mechanism remains completely unknown.
To identify genes required for the TE silencing mediated by Piwi–piRISCs, we used a candidate approach. An
earlier study showed that mouse gametocyte-specificfactor 1 (GTSF1) is required for TE silencing in the testis(Yoshimura et al. 2009). GTSF1 knockout male mice show
infertility owing to a lack of both DNA methylation andsubsequent TE silencing during meiosis in spermatocytes,
phenocopying mice lacking Mili or Miwi2 (Yoshimura
et al. 2007, 2009; Aravin et al. 2008; Kuramochi-Miyagawa
et al. 2008; Pillai and Chuma 2012), both of which encode
mouse PIWI proteins. In the present study, we knocked
down four homologs of GTSF1 in a cultured Drosophila
OSC line (Saito et al. 2009) and found that depletion of one
particular homolog, CG3893, results in derepression of
TEs and neighboring genes, although the level and localization of Piwi–piRISCs were unchanged. We term CG3893
as DmGTSF1 hereafter. DmGTSF1 is a nuclear protein thatinteracts with Piwi, and a lack of DmGTSF1 results instrong enrichment of RNA polymerase II (Pol II) and lessefficient enrichment of trimethylated histone H3 Lys 9(H3K9me3) at TE loci on the genome, suggesting that
DmGTSF1 is an integral factor in Piwi–piRISC-mediatedtranscriptional silencing.
Results and Discussion
OSCs were transfected with individual siRNAs against
DmGTSF1 and its paralogs, CG14036, CG32625, andCG34283 (Supplemental Fig. S1A), and Northern blotting
and RT-qPCR were performed to examine the levels of
piRNAs and mdg1 transcripts, respectively, in the transfected cells. None of the siRNAs changed the expression

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DmGTSF1 in Piwi-mediated silencing

levels of piRNAs, Idefix-piR-1, and/or traffic jam-piR-1(Fig. 1A; Supplemental Fig. S1B). However, knockdown of
DmGTSF1, but not of its paralogs, increased the level of
mdg1 (Fig. 1B; Supplemental Fig. S1C), a long terminal
repeat element, suggesting that only DmGTSF1 is required
for TE silencing. Depletion of Maelstrom (Mael), a known
piRNA factor (Lim and Kai 2007), led to almost identicalphenotypes (Fig. 1A,B). A recent study showed that Maelis required for transcriptional TE silencing mediated by
Piwi–piRISC but not for piRNA biogenesis (Sienski et al.
2012), suggesting the existence of functional parallels
between Mael and DmGTSF1. Immunofluorescence staining for Myc-DmGTSF1 in OSCs revealed that DmGTSF1
shows a nuclear localization, as does Piwi (Fig. 1C). Thisfurther supports the idea that DmGTSF1 functions in
nuclear TE silencing. Depletion of HP1a derepressed
mdg1 (Fig. 1B), consistent with the earlier observationthat HP1a functions in TE silencing in the ovaries (Wangand Elgin 2011). The expression levels of piRNAs were
unaffected by a lack of HP1a (Fig. 1A; Saito et al. 2010).
Thus, piRNA biogenesis does not require HP1a. Loss ofArmi, a piRNA biogenesis factor (Olivieri et al. 2010;
Saito et al. 2010), resulted in down-regulation of piRNAs,
verifying our results.
To investigate the roles of DmGTSF1 in fertility, femalesof the P-element insertion line P{w[+mC]=GSV6}GS12962(Supplemental Fig. S2A), in which DmGTSF1 mRNA andits protein product are expressed below detection levels(Fig. 2A; Supplemental Fig. S2B), were crossed with
Oregon R male flies. DmGTSF1 / females laid no eggs
(data not shown). In contrast, females in the reciprocal
cross laid eggs (data not shown). The DmGTSF1 transcript
was barely detected in testes (Graveley et al. 2011), andFigure 1. DmGTSF1, a nuclear factor, is necessary for TE silencing
but not piRNA biogenesis in OSCs. (A) DmGTSF1 and with the
known piRNA factors Piwi, Armi, HP1a, Mael, and Spn-E weredepleted in OSCs by RNAi and Northern blotting performed on total
RNAs isolated from the cells 48 h post-transfection. The levels of
Idefix-piRNA and tj-piRNA were unchanged by DmGTSF1 depletion, suggesting the dispensability of DmGTSF1 in piRNA biogenesis in OSCs. (B) RT-qPCR showed that mdg1 is derepressed by
DmGTSF1 depletion, as it is by Piwi depletion. (C) Immunofluorescence for Myc-DmGTSF1 (green) in OSCs. DAPI staining (blue)shows the locations of the nuclei.

DmGTSF1 / testes appeared normal (data not shown).
Thus, DmGTSF1 is specifically required for female fertility.
DmGTSF1 / ovaries appeared rather small and severely
deformed, similar to those observed in Piwi mutants (Linand Spradling 1997). A comparison of Aub immunostaining
images with images showing DAPI-stained nuclei revealed
that DmGTSF1 / ovaries lack a follicle cell layer, which
encapsulates each egg chamber in DmGTSF1+/ ovaries(Fig. 2B).
Immunostaining was also performed using antibodies
against the polar follicle cell marker FasIII (Ruohola et al.
1991) and the oocyte marker protein Orb (Christerson and
McKearin 1994). In DmGTSF1+/ ovaries, FasIII is detectedin monolayered follicle cells in both the germarium andearly stage egg chambers, but later, its expression becomes
restricted to two polar follicle cells in individual egg
chambers, as has been observed in wild-type ovaries(Fig. 2C; Ruohola et al. 1991). However, in DmGTSF1 /ovaries, monolayered FasIII-positive cells were lost at earlystages; rather, FasIII-positive cells were dispersed within
the germarium-like structures. Mutant egg chambers atlater stages contained multiple FasIII-positive cells (Fig.
2C).Polar cell specification is likely disrupted owing to
the loss of DmGTSF1.
Orb expression is normally activated during mitotic
division. Then, Orb gradually accumulates in oocytes
during development (Christerson and McKearin 1994).
This spatiotemporal expression pattern of Orb was observed in DmGTSF1+/ ovaries (Fig. 2D). However, in
DmGTSF1 / ovaries, Orb appeared in all cells within theearly cysts, and its expression was seen in multiple cells
in late egg chambers (Fig. 2D). These DmGTSF1 mutant
phenotypes are very similar, if not identical, to those ofpiRNA pathway mutants: The number of follicle cells
was also reduced in the ovaries of mutant fs(1)Yb, apiRNA biogenesis factor and a key component of Yb
bodies (Szakmary et al. 2009; Olivieri et al. 2010; Saito
et al. 2010). Fusion of egg chambers caused by loss of Maeland fs(1)Yb also resulted in aberrant multiplicity of Orbpositive cells in late egg chambers (Johnson et al. 1995;
Sato et al. 2011). These overlapping phenotypes further
suggest functional parallels between Mael and DmGTSF1.
DmGTSF1 mutant flies appear otherwise normal. This
correlates well with the observation that a subset of TEs
was derepressed in DmGTSF1 / ovaries but not in the
carcasses of DmGTSF1 / flies (Supplemental Fig. S2C).
The RT-qPCR results confirm the requirement of DmGTSF1
for TE silencing not only in OSCs, but also in the ovaries.
To compare the levels of transcripts globally, we performed mRNA sequence (mRNA-seq) analysis of OSCs
transfected with siRNAs against the known piRNA pathway genes Piwi, Mael, and Armi along with DmGTSF1 andnormalized the expression levels to those in OSCs transfected with control siRNAs against EGFP. mRNA-seq
samples were prepared at 48 h post-transfection, yielding
168,369,636 genome-mapped reads in total (Supplemental
Table S1). Reads per kilobase per million mapped reads(RPKM) values for annotated genes were highly correlated
between Piwi knockdown and control knockdown cells(R2 = 0.99). In contrast, several of the 125 annotated
Drosophila melanogaster TE families showed strong increases in RNA levels (Fig. 3A,B). For example, thetranscript levels for the long terminal repeat elements
mdg1, gypsy, and 297 were increased by >156-fold, >32fold, and >17-fold, respectively, upon Piwi knockdown,

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Ohtani et al.

ing of various TEs in OSCs (Fig. 3B), the TE
derepression pattern was rather different in
HP1a-depleted cells (R2 = 0.49) (Supplemental Fig. S3B). Depletion of DmGTSF1 does
not affect the levels or cellular localization of
Piwi and Mael in OSCs (Supplemental Fig.
S3C,D; data not shown), confirming that theobserved defective phenotypes were not due
to a secondary effect of DmGTSF1 depletion.
Likewise, loss of Mael and HP1a did notchange the cellular localization of DmGTSF1(Supplemental Fig. S3E).
Piwi–piRISCs influence the expressionof protein-coding genes near TE insertions(Sienski et al. 2012). To assess the role of
DmGTSF1 in this effect, we analyzed ourmRNA-seq data for 28 genes shown in theprevious study (Fig. 3C,D; Sienski et al.
2012). As expected, the expression levels ofthese neighboring genes were comparable
between DmGTSF1 and Piwi knockdown
cells. The results for some genes (for example,
bab1 and CG12477) did not coincide withthose of the previous study (Sienski et al.
2012), although transposon insertions near
the genes were confirmed by PCR (Supplemental Fig. S4). This variegation may be due
to subtle unexpected differences in knockdown and/or cell manipulation conditions.
Collectively, all of these bioinformatic datastrongly support the notion that DmGTSF1 isan integral factor in the Piwi–piRNA pathway.
Both Piwi and DmGTSF1 are localized to
the nucleus (Fig. 1C; Supplemental Fig. S3D).
We then asked whether DmGTSF1 forms acomplex with Piwi in vivo. Myc-DmGTSF1
fused C-terminally with EGFP (Myc-DmGTSF1Figure 2. DmGTSF1 is essential for development of the ovaries. (A, left) RT–PCR
EGFP) was expressed in OSCs, and the comresults showing that the homozygous P{w[+mC]=GSV6}GS12962 mutant does notplex immunoprecipitated with an anti-Myc
express DmGTSF1 mRNAs but does express CG6843, a neighboring gene. A schematic
drawing of DmGTSF1 (green) and CG6843 (magenta) genes on the Drosophila genome isantibody was probed with an anti-Piwi anshown in Supplemental Figure S2A. (+/ ) P{w[+mC]=GSV6}GS12962/TM3; ( / )tibody. Myc-DmGTSF1-EGFP and endogeP{w[+mC]=GSV6}GS12962/P{w[+mC]=GSV6}GS12962. (Right) Western blot shownous Piwi coprecipitated (Fig. 4A) even in the/ing that DmGTSF1
flies do not express DmGTSF1 protein but do express Piwi. A
presence of RNase A (Supplemental Fig. S5A).
monoclonal antibody against DmGTSF1 was generated, and the specificity is shown in
An in vitro pull-down assay using recombiSupplemental Figure S2B. (B, left) Immunostaining images showing that Aub (red) is
expressed only in germline cells that are encapsulated by somatic follicle cells, which arenant DmGTSF1 fused to glutathione S-trans+//
positive by DAPI-staining in DmGTSF1
flies. (Right) In contrast, the DmGTSF1
ferase (GST-DmGTSF1) and crude OSC lysates
egg chamber lacks follicular epithelium morphogenesis. (C) Polar cell specification is
confirmed their association (Fig. 4B; Suppledefective in DmGTSF1 / ovaries. (Left) Ovaries were immunostained with antibodies+/
mental Fig. S5B): Piwi was detected with GSTagainst a polar follicle cell marker, FasIII (red). Enlarged images of the DmGTSF1
egg
DmGTSF1 to an extent similar to that withchamber (middle) and the DmGTSF1 / egg chamber (right) are shown. FasIII-positive
cells in the mutant are indicated by white arrowheads. (D) Oocyte polarity and
GST-Pimet/DmHen1, which interacts withspecification are defective in DmGTSF1 / ovaries. (Left) Ovaries were immunostained
Piwi and 29-O-methylates piRNAs upon Piwi
with antibodies against an oocyte marker protein, Orb (red). Enlarged images of theloading (Horwich et al. 2007; Saito et al. 2007).
DmGTSF1+/ egg chamber (middle) and the DmGTSF1 / egg chamber (right) are shown.
Under the conditions we used, Mael was not
Several Orb-positive cells are detected in the DmGTSF1 / ovary (arrowheads).
detected in the DmGTSF1 complex (Supplemental Fig. S5C), although endogenous Maelsupporting the recent results of Sienski et al. (2012). Almost
was partially localized to the nucleus (Supplemental Fig.
identical results were obtained upon knockdown of Armi,
S5D). Moreover, neither Armi nor AGO1, another ArgoMael, and DmGTSF1 (Fig. 3B; Supplemental Fig. S3B). This
naute protein and a component of the microRNA pathway
suggests that not only Armi (Haase et al. 2010; Olivieriin Drosophila (Okamura et al. 2004), was detected withet al. 2010; Saito et al. 2010) and Mael (Sienski et al. 2012)
GST-DmGTSF1 (Fig. 4B). Armi is cytoplasmic and inbut also DmGTSF1 function in the Piwi–piRISC pathway
teracts with Piwi (Olivieri et al. 2010; Saito et al. 2010),
in OSCs. RT-qPCR confirmed that mdg1, but not othersuggesting that Piwi changes protein interactors uponTEs such as I-element and Het-A, was markedly derenuclear import.
pressed in Piwi- and DmGTSF1-depleted cells (SuppleDmGTSF1 consists of 167 amino acid residues andmental Fig. S3A). Although HP1a is required for silenccontains two U11-48K-like CHHC-type Zn finger motifs

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DmGTSF1 in Piwi-mediated silencing

would affect Piwi–piRISC targeting to TE locion the genome. Piwi-RNAi and Mael-RNAi
were carried out as control experiments(Sienski et al. 2012). Chromatin complexes
were immunoprecipitated using anti-Pol II
and anti-H3K9me3, from which DNA fragments were prepared and subjected to quantitative PCR (qPCR) for quantification of
mdg1, gypsy, and roo elements in the complexes. Pol II accumulation to specific TEs
and/or genes indicates derepression of the
genomic elements (because they are transcribed by Pol II), whereas H3K9me3 accumulation indicates silencing (H3K9me3 isa silent chromatin marker). We found thata lack of DmGTSF1 resulted in accumulationof Pol II (Fig. 5A; Supplemental Fig. S7A) andless efficient enrichment of H3K9me3 (Fig.
5B) at mdg1 and gypsy loci on the genome,
although accumulation at roo loci washardly affected. These results are consistent
with the mRNA-seq data shown in Figure
3A. Thus, DmGTSF1 is required for Piwi–
piRISCs to regulate target TE loci on the
genome. Depletion of DmGTSF1 did notchange the cellular localization of Piwi (MycPiwi in Supplemental Fig S3D). Likewise, depletion of Piwi did not change the localizationof DmGTSF1 (Myc-DmGTSF1 in Supplemental Fig S7B). We propose that DmGTSF1,
which is a component of nuclear Piwi–
piRISCs, acts as a cofactor during Piwi-meFigure 3. DmGTSF1 regulates expression of TEs and their neighboring genes in the Piwi
diated transcriptional transposon silencing at
pathway. (A) Scatter plot of RPKM values (log2) for TEs in EGFP (control) or Piwi (left)/
DmGTSF1 (right) RNAi knockdown samples based on RNA sequencing (RNA-seq). TEs
the genomic sites where Piwi–piRISCs exert
whose expression level differs from that in the control by more than threefold are coloreda silencing function.
orange. (B) Fold change of TEs to EGFP (control) RNAi knockdown are displayed as heat
Previous studies have shown that Piwi ismaps for Piwi, Armi, DmGTSF1, HP1a, and Mael RNAi knockdown samples. (C) Heat
imported
to the nucleus only after being
maps indicating the fold change of 28 genes, which are mapped TE insertions in either
loaded with mature piRNAs at cytoplasmic
the gene body or close proximity (up to 5 kb away) and up-regulated in Piwi RNAi
Yb bodies in ovarian somas (Saito et al.
knockdown cells (Sienski et al. 2012). Fold change of these genes in Piwi, Armi,
DmGTSF1, HP1a, and Mael RNAi knockdown samples. (D) The results of mRNA-seq2010). In other words, cells use an elaboanalyses are recapitulated by RT-qPCR analysis. The steady-state levels of transcripts
rate system to avoid localization of Piwi tofrom protein-coding genes (as described in C) were measured relative to RP49.
the nucleus when the protein is devoid ofpiRNAs. This ‘‘gatekeeping’’ regulation is
crucial for control of cellular events and gonadal devel(Andreeva and Tidow 2008) in which four cysteines (C4and C24 in the first domain; C37 and C57 in the second
opment because Piwi devoid of piRNAs in the nucleus
would not know what to target. This study and an earlier
domain) are highly conserved among orthologs and paraone (Sienski et al. 2012) showed that two factors, Maellogs of DmGTSF1 (Supplemental Fig. S1A). We mutatedand DmGTSF1, are required for the targeting of Piwi–
these four cysteines individually to alanine and asked
piRISCs to achieve efficient TE silencing in the nucleus.
whether the mutations would abolish the function of
Currently, it is unclear why this type of nuclear regulation
DmGTSF1. We conducted rescue experiments in whichis required and what functions of Mael and DmGTSF1 areendogenous DmGTSF1 was depleted by RNAi, and RNAirequired. To determine this, it will be important to isolate
resistant DmGTSF1 wild type (DmGTSF1r) and DmGTSF1
the nuclear Piwi–piRISC as a full unit, identify the
mutants were individually expressed in DmGTSF1-depleted
components, and unveil their functions and the relationOSCs. RT-qPCR showed that mutations in the Zn finger
ships among them. This would shed new light on nuclear
motifs decreased TE silencing activity (Fig. 4C). All of the
RNA silencing pathways.
DmGTSF1 mutants localized to the nucleus (Supplemental Fig. S6A; data not shown) and associated with Piwi
Materials and methods(Supplemental Fig. S6B), showing that functional Zn finger
motifs are not required for the interaction with Piwi. This
Knockdown, Northern blotting, Western blotting, antibody
also implies that the interaction of DmGTSF1 with Piwi
production, immunoprecipitation, immunofluorescence, RNA
is not sufficient for retrotransposon silencing and that thesequencing (RNA-seq) analysis, plasmid construction, rescue
Zn fingers have a crucial role in this process through as yet
assay, GST pull-down assay, and RT-qPCR analysis
unidentified factors.
We next performed chromatin immunoprecipitation
siRNA transfection, Northern blotting, Western blotting, production(ChIP) analyses to examine how depletion of DmGTSF1of anti-DmGTSF1 antibody, immunoprecipitation, immunofluorescence,

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Ohtani et al.

GST pull-down assay, and RT-qPCR analysis were performed as previously described (Supplemental Material; Saito et al. 2007, 2010). DNA
oligonucleotides used in RT-qPCR are summarized in Supplemental Table
S2. RNA-seq analysis, plasmid construction, and rescue assay are shownin the Supplemental Material.

Drosophila strains and brooming analysis
Yellow white (y w) and Oregon R were employed as wild-type strains. The
DmGTSF1 allele used was P{w[+mC]=GSV6}GS12962/TM3 (Drosophila
Genomics Resource Center stock no. 204406). All stocks were maintained
at 25°C. Mating tests were done in batches of test males and at least 10
virgin females per vial. The presence or absence of progeny was scoredafter at least 7 d. For brooding tests, virgin DmGTSF1+/ and DmGTSF1 /females were mated to Oregon R males in yeasted apple agar plates. The
females were left in the plate to continue to lay eggs until the 10th day aftermating. Laid eggs on the plate were counted on the fourth, seventh, and 10thdays under a microscope.

ChIP assay
Cells (2 3 108) were cross-linked with 1% formaldehyde for 10 min atroom temperature, and cross-linking was then stopped with 125 mM
glycine for 5 min at room temperature. The DNA–protein complexes werelysed with 1% lysis buffer (50 mM HEPES-KOH at pH 7.4, 150 mM
sodium chloride, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, protease inhibitors). The cross-linked chromatin was sheared into
;200 base pairs using Covaris S2 ultrasonicators. The DNA–protein
complexes were incubated with nonimmune IgG antibody, anti-Pol II
antibody 8WG16 (Santa Cruz Biotechnology), or anti-H3K9me3 antibody(Abcam, 8898) immobilized on Dynabeads-Protein G for 2 h at 4°C. The
beads were washed with high-salt lysis buffer (50 mM HEPES-KOH at pH
7.4, 450 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 0.1%
sodium deoxycholate, protease inhibitors) and wash buffer (50 mM
Tris-HCl at pH 8.0, 1 mM EDTA, 250 mM lithium chloride, 0.5% NP-40,
0.5% sodium deoxycholate) followed by TE (10 mM Tris-HCl at pH 8.0, 1 mM
EDTA). After reversing the cross-linking for 12–16 h at 65°C, samples weretreated with RNase A for 30 min at 37°C, and then DNA was recovered. ShortDNA fragments corresponding to retrotransposons were amplified using

Figure 5. Depletion of DmGTSF1 increases the association of Pol II
with retrotransposons and decreases the levels of H3K9me3 onretrotransposons targeted by Piwi–piRISC. The associations of Pol II(A) and H3K9me3 (B) with mdg1, gypsy, and roo were assayed by ChIP
and qPCR. The results represent the percentage input values of three
independent samples. Each experiment was repeated at least threetimes. Error bars indicate SEM values. P-values were calculated using
the two-tailed Student’s t-test for comparisons with control siRNAtransfected groups (EGFP). (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

a LightCycler 480 Real-Time PCR Instrument II with SYBR Premix Ex Taq(Takara). The primers used are listed in Supplemental Table S2. Pol II ChIP
sequencing (ChIP-seq) libraries for EGFP, Piwi, Mael, and DmGTSF1
knockdown OSCs were analyzed using a single lane of Illumina MiSeq.
This yielded ;2.6 million to 3.4 million genome-mapped reads in eachtransfected sample. For computational analyses, we trimmed for adaptor
sequences, mapped the reads to Drosophila genome release dm3, andcalculated reads per million genome mappers (RPM) values as describedwithin the RNA-seq computational analysis section. Sequencing data are
available through Gene Expression Omnibus (accession no. GSE47006).

Acknowledgments

Figure 4. DmGTSF1 associates with Piwi in vivo and in vitro. (A)Immunoprecipitation and Western blotting show that endogenous
Piwi immunoprecipitated with Myc-DmGTSF1-EGFP but not with
Myc-EGFP. (B) DmGTSF1 associates with PIWI but not with Armi or
AGO1. Bound fractions with GST-Pimet/DmHen1 and GST-DmGTSF1
were probed with antibodies against Piwi, Armi, and AGO1. (Bottom
panel) An arrowhead shows where full-length GST-DmGTSF1 migrates
on the Coomassie Brilliant Blue-stained gel. (C, top) A schematic
drawing of DmGTSF1. (Bottom) RNAi-resistant Myc-tagged full-length
DmGTSF1r fused with EGFP (Myc-DmGTSF1r-EGFP) and Zn finger
mutants were expressed in DmGTSF1-depleted OSCs. The expression
levels of mdg1 were monitored by RT-qPCR. DmGTSF1 Zn finger
mutants failed to rescue mdg1 silencing lost following depletion of
DmGTSF1 in OSCs.

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We thank H. Ishizu, K. Sato, S. Yamanaka, and M. Kamatani for technicalassistance, and other members of the Siomi laboratory for discussions andcomments on the manuscript. We thank S. Inagaki for technical suggestions and comments regarding the immunofluorescence analyses. This
work was supported by the Funding Program for Next-Generation WorldLeading Researchers (LS109) to K.S.; Core Research for Evolutional Scienceand Technology (CREST) from the Japan Society for the Promotion of
Science and Technology (JSPS) to M.C.S.; a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science, andTechnology (MEXT) of Japan to H.S. and M.C.S.; and a Grant-in-Aid for the
Global COE program from MEXT to Keio University to H.S.

Note added in proof
While this manuscript was under revision, Muerdter et al. (2013)
reported that DmGTSF1/CG3893 is required for TE silencing. The investigators termed DmGTSF1/CG3893 as Asterix (Muerdter et al. 2013).

Downloaded from genesdev.cshlp.org on February 2, 2015 - Published by Cold Spring Harbor Laboratory Press

DmGTSF1 in Piwi-mediated silencing

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GENES & DEVELOPMENT

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DmGTSF1 is necessary for Piwi−piRISC-mediated transcriptionaltransposon silencing in the Drosophila ovary
Hitoshi Ohtani, Yuka W. Iwasaki, Aoi Shibuya, et al.
Genes Dev. 2013 27: 1656-1661
Access the most recent version at doi:10.1101/gad.221515.113

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