Genes Genet. Syst. (2013) 88, p. 9–17

The epigenetic regulation of transposable elements by
PIWI-interacting RNAs in Drosophila
Kuniaki Saito*
Department of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan(Received 15 January 2013, accepted 26 February 2013)

A mechanism is required to repress the expression and transposition of transposable elements (TEs) to ensure the stable inheritance of genomic information.
Accumulating evidence indicates that small non-coding RNAs are important regulators of TEs. Among small non-coding RNAs, PIWI-interacting RNAs (piRNAs)
serve as guide molecules for recognizing and silencing numerous TEs and work incollaboration with PIWI subfamily proteins in gonadal cells. Disruption of thepiRNA pathway correlates with loss of proper genomic organization, gene expression control and fertility. Moreover, recent studies on the molecular mechanismsof piRNA biogenesis and on piRNA function have shown that piRNAs act asmaternally inherited genic elements, transferring information about repressed
TEs to progeny. These findings enable a molecular explanation of mysterious epigenetic phenomena, such as hybrid dysgenesis and TE adaptation with age. Here,I review our current knowledge of piRNAs derived from biochemical and genetic
studies and discuss how small RNAs are utilized to maintain genome organizationand to provide non-DNA genetic information. I mainly focus on Drosophila but
also discuss comparisons with other species.
Key words: Drosophila, hybrid dysgenesis, piRNAs, PIWI proteins, transposable elements
INTRODUCTION
In eukaryotes, large proportions of genomes consist ofvarious transposable elements (TEs) and their remnant
sequences. For instance, 45% of the human genome and
15–22% of the Drosophila genome contain TE sequences(Biemont and Vieira, 2006; Lander et al., 2001). A majorclass of TEs is the retrotransposons, which are abundantbecause of their “copy and paste” mode of amplification(Finnegan, 2012; Gogvadze and Buzdin, 2009). WhenTEs are inserted into a genomic location, gene expression
patterns may become altered, which may compromise
cell viability (Callinan and Batzer, 2006; Hancks andKazazian, 2012). Some TEs seem to play beneficial roles
in regulating gene expression as distal enhancers, chromatin boundaries and binding sites of many transcription
factors (see the review by Ichiyanagi, 2013), while others
TEs are recognized and repressed to maintain genome
integrity. In various species, TEs are remarkably compartmentalized in the heterochromatin, which is accomplished by histone modification and heterochromatinproteins (Levin and Moran, 2011; Slotkin and Martienssen,
Edited by Hiroshi Iwasaki* Corresponding author. E-mail: saito@z6.keio.jp

2007). In Schizosaccharomyces pombe (S. pombe), a small
RNA-mediated system is involved in heterochromatin formation at the centromere region where small RNAs are
utilized to recognize the centromere region (Moazed,2009). These studies reveal an unsuspected role forsmall RNAs in the regulation of chromatin structure andgenome stability.
Until recently, little was known about how Drosophila
and vertebrate TEs are remarkably compartmentalized inheterochromatin and specifically silenced in cells regardless of their heterogeneity and diversity. However, it is
now proposed that in germ cells, PIWI-interacting RNAs
(piRNAs) act as guides for recognition of TEs by PIWI subfamily proteins (Ishizu et al., 2012; Juliano et al., 2011;
Khurana and Theurkauf, 2010; Senti and Brennecke,2010). Recent studies have shown that piRNAs containnumerous species that cover the entire sequence of a target TE (Ghildiyal and Zamore, 2009; Malone and Hannon,
2009; Siomi et al., 2011). Moreover, accumulating evidence indicates the existence of functional links between
these small RNAs and silencing of TEs and explains epigenetic phenomena such as hybrid dysgenesis, defects ininterspecies crosses and genome imprinting, indicating
the importance of small RNAs as epigenetic regulators(Pillai and Chuma, 2012; Saito and Siomi, 2010).

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K. SAITO

In this article, I begin by providing a brief overview ofpiRNA expression and function, followed by a summary of
the mechanism of piRNA biogenesis and TE silencing.
With this background, I then describe the epigenetic rolesof Drosophila piRNAs and their impact on genome organization.
EXPRESSION AND FUNCTION OF
ARGONAUTE PROTEINS
Argonaute family members feature a PAZ and a PIWI
domain and associate with small RNAs to act as effector
molecules (Sashital and Doudna, 2010). In the wellestablished small RNA or RNA interference (RNAi) pathway, double-stranded RNAs (dsRNAs) are processed into
small interfering RNAs (siRNAs) of approximately 21
nucleotides by the enzyme Dicer. The resulting siRNAs
associate with Argonaute (Ago) proteins to form the RNAinduced silencing complex (RISC). The siRNA boundAgo protein cleaves the target transcript depending uponbase pairing between the siRNA and the target; this is
known as slicer activity and results in gene silencing. In
an analogous way to RNAi, microRNAs (miRNAs) negatively regulate translation of target mRNAs (Ghildiyal
and Zamore, 2009).
In Drosophila, Argonaute family members include fiveproteins that are further divided into two classes depending upon the tissues in which they are expressed: theArgonaute subfamily proteins, Ago1 and Ago2, are ubiquitously expressed and the PIWI subfamily proteins, Piwi,
Aubergine (Aub) and Ago3, are expressed specifically in
gonadal cells (Williams and Rubin, 2002). SiRNAs arebound by Ago2, whereas Ago1 binds to miRNAs (Okamura
et al., 2004). In S2 cells, a cell-line derived from embryonic soma, Ago2 associates with endo-siRNAs (endogenous siRNAs) consisting of TE derived sequences andsilences TE expression in an analogous way to RNAi
(Czech et al., 2008; Kawamura et al., 2008; Okamura etal., 2008). However, endo-siRNAs are unlikely to be theprimary factor in the system repressing TEs in somatic
cells because disruption of Ago2 does not cause a dramaticeffect on the viability of Drosophila compared with heterochromatin protein mutants (Deshpande et al., 2005;
Eissenberg et al., 1990).
In contrast to somatic cells, the small RNA pathway is
the major system for silencing TEs in germ cells andgonadal somatic cells where PIWI subfamily proteins are
expressed and associate with piRNAs derived from
TEs. Piwi was the first PIWI subfamily protein to bewell characterized in animals (Cox et al., 1998). Piwi
has been shown to be expressed in both germline stem
cells and ovarian somatic cells, such as the follicle cells,
and to be crucial for maintenance of the germline stem
cells (Cox et al., 2000). The other PIWI subfamily members, Aub and Ago3, are exclusively expressed in germline

cells and are required for the formation of pole cells in offspring (Brennecke et al., 2007; Li et al., 2009; Nishida etal., 2007). Sequencing of piRNAs in Drosophila hasrevealed that they are 23–30 nucleotide single-stranded
RNAs that mainly originate from TEs and TE-related
genomic elements (Brennecke et al., 2007; Li et al., 2009;
Saito et al., 2006). Whereas retrotransposon expression
is increased less than 10-fold in ago2 mutant flies, the disruption of Piwi genes can cause a more than 100-foldoverexpression of TEs and sterility in females, indicating
the crucial roles of PIWI subfamily genes in TE silencing
as well as in germline development (Vagin et al., 2006).
PiRNA BIOGENESIS AND TRANSPOSONSILENCING
PiRNA clusters in Drosophila PiRNAs are definedas small RNAs that associate with PIWI subfamily proteins (Aravin et al., 2006; Girard et al., 2006; Lau et al.,2006). In Drosophila, piRNAs were originally describedas rasiRNAs (repeat-associated small interfering RNAs)
that were defined as a subset of small non-coding RNAs
discovered in testis and derived from repetitive genomic
regions, such as suppressor of stellate, satellite DNA and
TEs (Aravin et al., 2003). This suggested roles for the
regulation of repetitive elements. In Drosophila, piRNAs
have several characteristics that are different from thoseof miRNAs and siRNAs, such as their length and sources(Brennecke et al., 2007; Gunawardane et al., 2007; Saito
et al., 2006; Vagin et al., 2006; Yin and Lin, 2007). The
length of piRNAs is slightly longer than miRNAs, ranging
from 24 to 31 nucleotides (Brennecke et al., 2007;
Gunawardane et al., 2007; Saito et al., 2006). Althoughsome piRNAs are drived from genic region of protein coding mRNAs, a large proportion of piRNAs is mapped on
TEs (Saito et al., 2009; Robine et al., 2009). Strikingly,
cloning analyses show the considerable heterogeneity ofpiRNAs (Saito et al., 2006; Brennecke et al., 2007;
Gunawardane et al., 2007). To date, hundreds of thousands of piRNA species have been cloned and more than
70% of piRNAs have been cloned only once, indicating
considerable piRNA heterogeneity and comprehensive
coverage of TE sequences (Brennecke et al., 2007; Yin andLin, 2007). PiRNAs are mapped on several hundred
genomic regions, referred to as piRNA clusters where TEs
and TE remnants are enriched (Fig. 1). The majority ofpiRNAs are generated from two types of cluster: monodirectional clusters, such as the flamenco (flam) locus,
where piRNAs map to only one DNA strand; and dualstrand clusters, such as the 42AB region, where piRNAs
are mapped on both DNA strands (Fig. 1) (Brennecke et al.,2007). The flam locus is composed of antisense-oriented
retrotransposon copies, such as gypsy, idefix, ZAM andmdg1, resulting in the production of antisense piRNAs(Brennecke et al., 2007; Desset et al., 2008; Pelisson et al.,

Epigenetic roles of small RNAs

Supporting this idea, most flam-mapped piRNAs are lostin fruit fly lines with an inserted P element at the distal
end of flam (Brennecke et al., 2007; Klattenhoff et al.,2009).
Many copies of over 100 different kinds of TEs are pres-1994). 42AB is composed of a line of TEs, such as GATE,
Rt1b and roo. In both clusters, the mapped piRNAs
cover tens of thousands of DNA base pairs, suggestingthat piRNAs are generated from long precursor transcripts (Brennecke et al., 2007; Klattenhoff et al., 2009).
<Bidirectional clusters><42AB>

11<Monodirectional clusters><flamenco>

Tandem arrays of TEs(generally oriented antisense)

piRNA
biogenesis
TE antisense
TE sense

piRNAspiRNAspiRNA-mediated TE silencing at
post-transcriptional or transcriptional level

Effector phase

TEs (targeted by piRNAs)
TEs (not targeted by piRNAs)

Fig. 1. The piRNA clusters in Drosophila. The monodirectional clusters, such as the flamenco (flam) locus, generate piRNAs
from only one DNA strand. The bidirectional clusters, such as the 42AB region, where piRNAs are derived from both DNA
strands. In both clusters, the piRNAs cover many different kinds of TEs in the Drosophila genome. The repressed speciesof TE are determined by the complementarities between TEs and piRNAs.

Fig. 2. Proposed role for Piwi in TE silencing and impact on nearby genes. PiRNAs that are bound by Piwi act as guide molecules to silence TEs. The repressive histone mark, H3K9 trimethylation, is predominantly seen in TEs. Upon loss of Piwi,
H3K9me3 marks decrease not only in TEs but also in the regions flanking TEs, which leads to a more open chromatin state,allowing polymerase II to transcribe TEs and genes in the vicinity of TEs, suggesting that the Piwi-piRNA complex represses
TE expression and influences genes adjacent to the target. However, how the Piwi-piRNA complex induces H3K9 trimethylation on TEs remains unclear.

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K. SAITO

ent in the Drosophila genome (Biemont and Vieira, 2006;
Kapitonov and Jurka, 2003). In the absence of flamderived piRNAs, gypsy, idefix and ZAM elements arehighly expressed, resulting in defects of oogenesis,
whereas disruption of piRNA biogenesis from dual-strand
clusters causes derepression of TEs, such as GATE andHeT-A, suggesting that piRNAs are involved in the silencing of TEs (Brennecke et al., 2007; Desset et al., 2008;
Klattenhoff et al., 2009; Pelisson et al., 1994).
The role of Piwi In Drosophila ovarian somatic cells,
Piwi mainly associates with piRNAs derived from monodirectional clusters, such as flam (Lau et al., 2009; Saito
et al., 2009). The disruption of Piwi causes desilencingof flam-related TEs and defects in the proliferation ofovarian follicle cells and in the maintenance of germ line
stem cells (Cox et al., 1998; Saito et al., 2010; Vagin et al.,2006). This phenotype is also seen in flam mutant flies,
suggesting the cooperative roles of Piwi and piRNAs(Desset et al., 2008; Pelisson et al., 1994). Interestingly,
mutational analyses and functional studies indicate thatthe nuclear localization of Piwi is required for TE silencing in fruit flies and ovarian somatic cell line, whereas its
slicer activity is not, suggesting that the silencing step
occurs in the nucleus and is not a consequence of the directcleavage of TE mRNAs (Cox et al., 2000; Darricarrere etal., 2013; Klenov et al., 2011; Saito et al., 2010).
Loss of Piwi expression results in a decreased abundance of the stable repressive histone mark (trimethylated
H3K9) and Pol II density for some TEs, suggesting transcriptional gene silencing roles of Piwi (Fig. 2) (Klenov etal., 2011; Shpiz et al., 2011; Sienski et al., 2012; Wangand Elgin, 2011). TEs are remarkably compartmentalized in the heterochromatin, which is accomplished byhistone modification, such as H3K9 and H3K27 trimethylation in Drosophila (Negre et al., 2011). These observations raise the question of whether the Piwi-piRNAcomplex causes H3K9 trimethylation by directly recruiting histone modifiers on TEs. Remarkably, in Piwi
knocked down cells, H3K9 trimethylation marks are also
lost in the euchromatic genes adjacent to TEs, indicatingthat Piwi-mediated epigenetic marks spread and affect
the gene expression patterns of the genomic regions thatflank TEs (Fig. 2) (Sienski et al., 2012). In addition, it
is surprising that H3K9 trimethylation by Piwi-piRNAs
seems to depend upon the transcription of TE mRNAs,
despite TEs being tightly associated with the repressedchromatin state, H3K9 trimethylation (Sienski et al.,2012). This is analogous to the well-characterized RNA
silencing system of heterochromatin formation in S.
pombe. In S. pombe, siRNAs recognize the centromere
region through complementarity between small RNAsand centromere-derived nascent RNA transcripts,
thereby recruiting the histone modifiers to achieve heterochromatinization (Moazed, 2009). It is therefore pos-sible that the piRNA-Piwi complex recruits histone
modifiers on TEs and TE-flanking regions. However, itremains unclear whether Piwi directly induces H3K9
trimethylation on TEs and what molecular mechanisms
and related factors are involved.
The role of Aub and AGO3 In contrast to Piwi, Aub
and Ago3 are exclusively expressed in germ cells andlocalized in the cytoplasm (Harris and Macdonald, 2001;
Li et al., 2009; Nishida et al., 2007). Unlike Piwi, Auband AGO3-associated piRNAs are mainly mapped on bidirectional clusters (Brennecke et al., 2007; Li et al., 2009).
Examination of the nucleotide preferences of piRNAs has
shown that Aub-associated piRNAs have a strong bias for
5’ uracil and an antisense TE transcript orientation andthat Ago3-associated piRNAs have a strong bias for adenosine at the tenth nucleotide from their 5’ end (10th A)
and a sense TE transcript orientation (Brennecke et al.,2007; Gunawardane et al., 2007). Both Aub and Ago3
can cleave target RNA between nucleotides 10 and 11
from the 5’ end of their associated piRNAs in vitro(Gunawardane et al., 2007). Moreover, Aub-boundpiRNAs are markedly decreased and TEs are derepressedin ago3 and aub mutants, indicating that the silencing of
TEs is coupled with piRNA biogenesis by the slicerdependent mechanism in which Aub cleaves TE mRNAand creates the 5’ end of Ago3-associated piRNAs, andAgo3 cleaves antisense TE transcripts and creates the 5’
end of Aub-associated piRNAs, thereby amplifying piRNAs
and enhancing TE silencing. This model is referred to as
the ‘ping-pong cycle’, and was first described in Drosophila
and later reported in mouse (Aravin et al., 2007; Brennecke
et al., 2007; Carmell et al., 2007; Gunawardane et al.,2007; Kuramochi-Miyagawa et al., 2008). In contrast to
the Piwi-mediated silencing mechanism, in which theextent of silencing by Piwi-associated piRNAs spreads tonearby genes, ping-pong-mediated silencing is unlikely to
spread because the cleavage reactions depend on complementarity between the piRNAs and the targets.
Commonalities and differences between Drosophila and mouse While there are some similarities
between mouse and Drosophila piRNAs, there are also
several differences between the two species. Similar to
Drosophila, mouse piRNAs are 24-32 nucleotides inlength and are longer than miRNAs and siRNAs (Aravin
et al., 2006, 2007; Carmell et al., 2007; Girard et al., 2006;
Grivna et al., 2006a, 2006b; Watanabe et al., 2006).
However, it seems that the character of piRNAs changes
corresponding to the stage of spermatogenesis in mouse.
In the fetal and neonatal testis, a large proportion ofpiRNAs known as pre-pachytene piRNAs originated from
TEs is mapped many times in the genome (Aravin et al.,2007). In contrast to the pre-pachytene piRNAs, the proportion of pachytene-piRNAs originated from TEs is lowEpigenetic roles of small RNAsin the adult testis (Aravin et al., 2006; Girard et al., 2006).
In an analogous way to Drosophila, the ping-pong cycle
operates in mouse. In the fetal and neonatal testis, Miliand Miwi2, both of which are PIWI-subfamily proteins,
associate with pre-pachytene piRNAs and are responsible
for the ping-pong cycle and TE silencing (Aravin et al.,2008; Kuramochi-Miyagawa et al., 2008). In the malegametogeneisis, de novo CpG DNA methylation occurs at
the imprinted genes and repetitive elements in mice fetal
testes, which is essential for fertility. DNA methylationis one of several epigenetic mechanisms that cells use torepress gene expression. The epigenetic mark of DNA
methylation, which is not present in Drosophila, might beestablished by piRNAs in fetal mice testes. Cells lacking
either Mili or Miwi2 showed a marked reduction in CpG
DNA methylation across TEs, such as IAP and LINE, andan increase of TE expression. Interestingly, a proportionof Miwi2 is localized in the nucleus, suggesting thatMiwi2 mediates de novo DNA methylation on TEs in fetal
mouse testis (Aravin et al., 2008; Kuramochi-Miyagawa
et al., 2008). More recently, disruption of piRNA biogenesis has been shown to cause the loss of DNA methylationof Rasgrf1, a paternally imprinted gene, suggesting a link
between piRNAs and genomic imprinting (Watanabe etal., 2011). Overall, the biogenesis mechanism and function of piRNAs show many similarities, but also important differences, between Drosophila and mouse.
HYBRID DYSGENESIS AND ADAPTATIONTO TE INVASIONIn Drosophila, the determinants specifying germ cellfate are inherited in pole cells from pole plasm at the posterior of the oocyte (Rongo and Lehmann, 1996). Polecells then become the primordial germ cells in offspring.
Aub but not Ago3 is continuously detected at the posteriorpole of the oocyte and embryo, suggesting that piRNAs as
well as Aub are incorporated into the germ cells of thenext generation (Fig. 3A) (Brennecke et al., 2007; Nishida
et al., 2007). This maternal deposition of piRNAs
explains the molecular basis of the mysterious genetic
phenomenon, hybrid dysgenesis, which was discovered 30
years ago (Fig. 3B) (Kidwell et al., 1977).
Hybrid dysgenesis is a sterility syndrome reported in
the early 1970s (Hiraizumi, 1971; Kidwell et al., 1977).
Laboratory strains of Drosophila melanogaster, separated
about 100 years ago, allowed the isolation of strains carrying the P element, which are referred to as P strains,while strains lacking it are referred to as M strains(Brookfield et al., 1984; Daniels et al., 1990). The sterility syndrome is observed in progeny of crosses between P
strain males and M strain females, whereas the reciprocal
crosses do not show the sterility phenotype, regardless ofgenetic identity (Fig. 3B) (Kidwell et al., 1977; Rubin etal., 1982). Similar observations have been reported for13

the I element, one of the LINEs in Drosophila. I element-carrying strains are divided into two categories, one
is the I strains, which possess active I elements that cantranspose into genomic regions, and the other is the R
strains, which contain defective copies of I elements(Bucheton et al., 1984). In the offspring of dysgenic
crosses, P or I elements are overexpressed and frequenttranspositions are detected. Interestingly, the repressedstate of a P or I element, referred to as P cytotype or I
cytotype, respectively, is maternally but not paternallyinherited, suggesting that maternal components determine repression of TEs in offspring. However, it was not
until 2008 that the maternally inherited piRNA-Aub complex was identified as the molecular entity of the cytotype(Brennecke et al., 2008). Regarding P-M hybrid dysgenesis, P elements do not transpose efficiently in P strains,because of the presence of high amounts of piRNAs thatare amplified by the ping-pong cycle between hetero- andeuchromatic P element transcripts in the female germline.
Importantly, the amplification of piRNAs is not observed
without maternal piRNA deposition even if the P element
is paternally inherited, suggesting that maternal deposition of piRNAs is required for the accumulation of piRNAs
in progeny. In other words, maternally deposited piRNAs
act as the trigger that starts the piRNA amplification in
offspring germ cells (Fig. 3) (Brennecke et al., 2008).
Similar mechanism was proposed for I element, indicating that piRNAs serve as transgenerational informationof the repressed TEs (Chambeyron et al., 2008).
As described above, maternal deposition of piRNAs isrequired for the ping-pong cycle and TE silencing in
offspring. In addition, a supply of piRNA precursor isalso required for the sustained ping-pong cycle in the offspring (Brennecke et al., 2008). Although the sense
piRNAs, namely TE mRNAs, are produced from both bidirectional piRNA clusters, such as 42AB, and euchromatic TEs, the antisense piRNA precursors are onlytranscribed from bidirectional clusters, which are highly
enriched for transposon fragments (Klattenhoff et al.,2009). Therefore, the repression of TEs is determined by
not only the maternally inherited piRNAs acting as triggers but also by the TEs in bidirectional clusters acting
as enhancers. From these characteristics of piRNAs,
they can be considered as part of an ‘immune system’ forTEs. Therefore, it is not surprising that loss of TEs inbidirectional clusters raises the possibility of new TE
invasion and that, in contrast, acquisition of new TE
sequence in bidirectional clusters raises the immunity
against new TE invasion. Interestingly, the observationthat P element insertion into bidirectional piRNA clusters
occurs within a single generation and restores fertility indysgenic crosses indicates the existence of an adaptive
system, allowing the dysgenic genome to overcome invasive TEs (Khurana et al., 2011). This adaptation efficiency increases as the hybrids of P element dysgenic

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K. SAITOA

Aub-piRNA complexes are
inherited from mother to offspring

Posterior PoleEgg chamber

Nurse cell

Aub

EmbryopiRNAEmbryoPole cells

Pole plasm

Oocyte<Mother><>B
Non-dysgenic cross
M cytotype

Dysgenic crossP cytotype

P cytotype

M cytotype

Aub
Aub Aub

Aub

Aub

Aub

Fig. 3. P-M Hybrid dysgenesis and piRNA inheritance. (A) Maternal inheritance of Aub-associated piRNAs. Aub is accumulated at the posterior pole of oocytes, and can be persistently detected in embryonic pole cells, suggesting that piRNAs are
inherited in progeny germ cells from the mother as a complex with Aub. Antisense but not sense piRNAs are inherited,
because of the absence of Ago3 at the posterior pole of oocytes. (B) Schematic model of phenotypes observed in progeny fromcrosses with incompatible and compatible fly lines. A cross between an M cytotype female and a P cytotype male causes highlevels of P element expression and a sterile phenotype (Dysgenic cross). Conversely, a reciprocal cross between a P cytotype
female and an M cytotype male does not (Non-dysgenic cross). This repressor trait is mediated by piRNAs associated with
Aub, which are maternally inherited. The piRNAs are amplified by the ping-pong cycle in F1 female germ cells, giving rise to
the subsequent piRNAs that are inherited in F2 progeny.

crosses age, which might contribute to genomic evolution
as well as diversity.
CONCLUSION AND PERSPECTIVES
Characterization of the biogenesis and functions of piRNAs demonstrates their unique roles in maintaining
genomic stability and organization. In this regard, small
RNAs have numerous advantages in regulating TEs, aswell as defective TE sequences. First, PIWI-piRNA complexes can act in trans, allowing the repression of inter-

spersed TEs on chromosomes. Second, the fragmentationof TEs into small RNA sequences might contribute to therepression of diverse TEs and to toleration of small numbers of mutations of TE sequences.
Accumulating evidence has started to emerge concerning the roles of protein factors in piRNA biogenesis; however, how piRNA-mediated silencing is achieved and whatmolecules are involved is largely unknown. In thisregard, knowledge of the molecular basis of the small
RNA pathway in other species, such as S. pombe and tetrahymena provides some hints for small RNA mediatedEpigenetic roles of small RNAs
mechanisms (Grewal, 2010; Mochizuki, 2012). For
example, studying the potential roles of the Drosophila
homologues of S. pombe H3K9 methyltransferase andheterochromatin proteins, which have key functions indetermining the heterochromatin structure of the S.
pombe centromere, will be of great interest. Similarly,
the role of the scnRNA-mediated pathway for genomic
rearrangement in tetrahymena is well characterized, anddetermining whether homologues of this machinery are
required in Drosophila will also be an important line of
future research. In addition, the small RNA-dependent
TE silencing is also reported in plants (see the review by
Ito, 2013). Therefore, comparing the silencing systems
among organisms as well as TE species might provide
clues to TE-mediated genome expansion.
In the context of hybrid dysgenesis and P element adaptation with age in Drosophila, the mechanisms how
piRNA clusters are determined will be great interest.
One important observation is that bidirectional piRNA
clusters are associated with Rhino, which is required forthe production of piRNA precursors (Klattenhoff et al.,2009). In particular, it will be of great interest toaddress what factors Rhino recognizes and how Rhino
affects piRNA precursor transcription. Given thatpiRNA clusters contribute to the euchromatic TE content
in the genome, understanding the mechanisms that
determine piRNA clusters will shed light on the molecular basis of how higher eukaryotes obtained and amplified
DNA elements during evolution.
I thank Haruhiko Siomi for helpful comments. This work was
supported by the Funding Program for Next Generation WorldLeading Researchers (LS109).

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