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Mutation Research 714 (2011) 95– 104

Contents lists available at ScienceDirect

Mutation Research/Fundamental and MolecularMechanisms of Mutagenesis

jo ur n al hom ep a ge: www.elsev ier .com/ locate /molmutC om mun i ty a ddress : www.elsev ier .com/ locate /mutres

eview

iRNAs, transposon silencing, and germline genome integrity

ulio Castanedaa,b,1, Pavol Genzora,b,1, Alex Bortvina,b,∗

Biology Department, Johns Hopkins University, Baltimore, MD 21218, United StatesDepartment of Embryology, Carnegie Institution for Science, 3520 San Martin Drive, Baltimore, MD 21218, United States

r t i c l e i n f o

rticle history:eceived 16 December 2010ccepted 4 May 2011vailable online 11 May 2011

eywords:iRNAransposable elementsEINE-1pigenetic

a b s t r a c t

Integrity of the germline genome is essential for the production of viable gametes and successful repro-duction. In mammals, the generation of gametes involves extensive epigenetic changes (DNA methylationand histone modification) in conjunction with changes in chromosome structure to ensure flawless pro-gression through meiotic recombination and packaging of the genome into mature gametes. Althoughepigenetic reprogramming is essential for mammalian reproduction, reprogramming also provides a per-missive window for exploitation by transposable elements (TEs), autonomously replicating endogenouselements. Expression and propagation of TEs during the reprogramming period can result in insertionalmutagenesis that compromises genome integrity leading to reproductive problems and sporadic inher-ited diseases in offspring. Recent work has identified the germ cell associated PIWI Interacting RNA(piRNA) pathway in conjunction with the DNA methylation and histone modification machinery in silenc-

NA methylationistone modificationsermlineouseechanism

ing TEs. In this review we will highlight these recent advances in piRNA mediated regulation of TEs inthe mouse germline, as well as mention the repercussions of failure to properly regulate TEs.

© 2011 Elsevier B.V. All rights reserved.

nmtaelstrom

ontents

1. Mammalian germline specification and reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 962. Eukaryotic genomes and the transposable element challenge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973. LINE-1 transposable elements as mutagens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.1. ORF1p and ORF2p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984. Epigenetic reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

4.1. DNA methylation and L1 expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984.2. Histone modifications and TE silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

5. The piRNA pathway-mechanistic overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985.1. The discovery of piRNAs in Drosophila and mouse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985.2. The primary piRNA pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 985.3. The secondary/“ping-pong” pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

6. The piRNA pathway in the male mouse germline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996.1. The pi-Body: the presumed site of the primary piRNA pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 996.2. The piP-Body: the presumed site of the secondary piRNA pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

7. Summary and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Department of Embryology, Carnegie Institution for Scienceel.: +1 410 246 3034; fax: +1 410 243 6311.

E-mail address: bortvin@ciwemb.edu (A. Bortvin).1 These authors contributed equally to this work.

027-5107/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.mrfmmm.2011.05.002

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

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, 3520 San Martin Drive, Baltimore, MD 21218, United States.

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. Mammalian germline specification and reprogramming

Following fertilization, male and female haploid pronuclei fuseo form the diploid nucleus of the mammalian zygote. After sev-ral rounds of cleavage, the blastocyst is generated (at embryonicay 3.5, E3.5) where a group of interior cells, called the inner cellass, will give rise to the embryo proper. The identity of these early

mbryonic cells is thought to be determined and established basedn their position and inductive cues they receive from neighboringells. Prior to gastrulation (E6.5), a small subset of cells at the junc-ion between the embryo and extra-embryonic tissue (the posteriorpiblast) is induced by ligands of the Bone Morphogenic ProteinBMP) family to initiate a germ-cell developmental cascade (Fig. 1A)1–4]. After gastrulation, these germ cell precursors (often referredo as primordial germ cells or PGCs) reside at the most posterioregion of the epiblast and then migrate to the genital ridge, theuture gonad. During germ cell migration, PGCs begin to undergopigenetic changes that include loss of transcriptionally repres-ive marks such as DNA methyl-cytosine [5,6]. When germ cellseach and colonize the genital ridge (E10.5), the rate of epigenetichanges increases rapidly and in the case of DNA de-methylation isompleted within days [7]. This process (called “epigenetic repro-ramming” or simply “reprogramming”) is thought to allow PGCs toeacquire a pluripotent state [8] and to establish a “blank” genomeor which to “paint” the sex-specific imprints of the embryo [9–13].

Unlike this equal epigenetic erasure that occurs contempora-eously in both sexes [14,15], the timing of meiotic entry and the

e-establishment of DNA methylation between males and femaless dimorphic (Fig. 1B) [15]. In females, the entire germ cell poolnters meiosis before DNA re-methylation during embryonic devel-pment (E13.5), arrests at prophase I, and establishes primordial

ig. 1. Germline specification and reprogramming. (A) After fertilization, cellular divisiomplantation, the ICM will give rise to the epiblast (light grey) from which the embryo proerm cells (PGCs – green). After gastrulation, PGCs end up at the posterior of the embrypigenetic reprogramming and meiotic entry in male and female germ cells. PGCs in the

e-establish DNA methylation (dark blue) before meiotic entry after birth. Female germ cells, hypo 5meC – de-methylated PGCs, G – gonocyte, PSP – prospermatogonia, Sp – Spegrowing follicle.

earch 714 (2011) 95– 104

follicles. At puberty and at each subsequent estrous cycle, a subsetof primordial follicles are recruited for maturation where growthand re-methylation occurs [16]. The completion of the first roundof meiosis (MI) occurs during ovulation while the second round(MII) is only completed upon fertilization of the ovum. In males,DNA methylation in germ cells (termed prospermatogonia at thistime) begins to be re-established during embryonic development(E14.5) and is completed by post natal day 2 (P2). Meiosis in malesinitiates from spermatogonia, the stem cell pool established byprospermatogonia, after birth in waves spaced several days apart(10 days in the mouse) and is completed within one to two weeks,depending on the species (∼12 days in the mouse) [17]. The accu-rate progression through meiosis in males is highly contingent onthe re-establishment of methyl-cytosine marks that were erasedin PGCs [18–20]. Female meiosis is also dependent on DNA methy-lation [21]; however, meiosis in females can be completed in theabsence of the DNA methylation machinery required for male meio-sis [18–20]. Thus, DNA methylation is essential to the mammaliangermline in both sexes, however, to varying degrees.

A major reason, although not exclusive, for the re-establishmentof DNA methylation is that it is the primary mode of silencingTEs in mammals. TEs are selfish DNA elements highly abundant inmammalian genomes, which cause DNA damage via transposition[22–24]. Deposition and maintenance of methyl-cytosine is medi-ated by the DNA methyltransferases (DNMT), which are requiredto silence expression of TEs [25]. In addition to the DNMTs, workwithin the last several years has shown that the piRNA pathway is

essential for de novo DNA methylation of TEs [26,27]. Members ofthis pathway associate with germline enriched piRNAs (26–31 ntslong), allowing for recognition and silencing of TEs. Why is epige-netic silencing of TEs, especially in the context of the germline, so

n produces the blastocyst containing the inner cell mass (ICM – light grey). Afterper forms. BMP signals from extra-embryonic tissue (dark-grey) induce primordialo and migrate to the genital ridge (red), (extra-embryonic tissue not shown). (B)

gonad undergo DNA demethylation (indicated by lighter shading). Male germ cellsells re-establish DNA methylation (dark pink) after birth. PGCs – primordial germrmatogonia, PMS – pre-meiotic S-phase, M – meiocyte, PF – primordial follicle, GF

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mportant in mammalian germline development? What are the keyathways (their constituents and molecular mechanisms) involved

n silencing of TEs, and what are the repercussions of their absence?hese and other questions will be discussed in the followingections.

. Eukaryotic genomes and the transposable elementhallenge

Metazoan genomes are generally large, containing DNA mea-uring in the thousands of megabases; however, the fraction thatad been thought to be functionally important (coding sequencesnd their regulatory elements) comprises less than two percentf the genome depending on the organism [23,28]. The Humanenome Project revealed many surprises about the organizationnd content of the human genome, one of which is the large pres-nce of repetitive elements. Repetitive elements within the humanenome include transposable elements (TEs), mobile pieces of DNAhat were initially discovered in maize by Barbara McClintock [29].Es make up roughly ∼45% of the human genome and can beivided into DNA and RNA TEs [23]. DNA based transposons prop-gate by a “cut and paste” mechanism using a transposase enzymeor their excision and insertion [30]. RNA TEs replicate via an RNAntermediate and comprise up to 42% of the human genome. TheseEs consist of Long Terminal Repeat (LTR) and non-LTR TEs. Struc-urally, LTR TEs resemble and appear to be forbearers of retroviruses

hat gained the capacity for horizontal transfer through acquisitionf an Envelope gene [31]. The non-LTRs TEs represent the biologi-ally most relevant class since they are the only active transposonsn humans and comprise the majority of TEs in the human genome

ig. 2. Transposon regulation in the germline. (A) Transposable elements are transcribed dytoplasm (b), encoded proteins (ORF1p and ORF2p) are translated allowing for the assembhe nucleus and integrated into new genomic locations (d) via target-primed reverse trannd secondary (2◦) piRNAs are proposed to be generated in the pi-Body and piP-Body, resomplex) to silence transposon expression. The exact mechanism of piRNA-guided DNAathway are shown.

earch 714 (2011) 95– 104 97

(∼34%) [23,28,32,33]. This class can be subdivided into autonomous(TEs capable of transposition) and non-autonomous (TEs depen-dent on autonomous elements for transposition) that comprise 21%and 13% of the genome, respectively. Long interspersed nuclearelements (LINE-1 or L1) are the autonomous elements, whilethe non-autonomous include short interspersed nuclear elements(SINEs). Both autonomous and non-autonomous transposons areof serious concern for germline development as indicated by theactivation of these elements during the reprogramming window.The spotlight belongs especially to L1 elements, whose ability toretrotranspose themselves and mobilize other non-autonomouselements has been linked to reproductive disorders and other dis-eases [34–36].

3. LINE-1 transposable elements as mutagens

L1 expression can be detected in the germ line, during embry-onic development, in neuronal tissue, and cell lines derived fromvarious cancers [37–43]. The L1 life cycle begins with transcrip-tion of the element by RNA polymerase II. The L1 mRNA encodestwo proteins [Open Reading Frame 1 and 2 proteins (ORF1p andORF2p) described in the following section] that facilitate L1 mRNAreverse transcription and integration at novel locations in thegenome (Fig. 2A). Detailed mechanisms of L1 insertion into thegenome (termed target-primed reverse transcription, TPRT) havebeen reviewed previously [44–46], however, it should be empha-

sized that the complete mechanism of L1 insertion is not fullyunderstood. Insertion into new genomic locations is detrimentalto genome integrity as it produces DNA breaks and has the poten-tial to disrupt gene coding regions [47]. For example, L1 mediated

uring the period of genome reprogramming (a). TE mRNA is then transported to thely of the ribonucleoprotein particles (RNPs) (c). RNPs are then transported back into

scription (TPRT) which consequently results in genomic instability. (B) Primary (1◦)pectively. These piRNAs facilitate de novo DNA methylation (by DNMT3a/DNMT3L

methylation remains to be elucidated, however, several implicated genes in this

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utagenesis described 20 years ago, was shown to be the causativegent of hemophilia A [48]. In addition, an increase association of1 and its encoded proteins in human diseases (i.e. cancers) [36,49]uggests TEs as the etiological basis for these diseases and man-ates a better understanding of proteins encoded by L1 elementsnd of their regulation.

.1. ORF1p and ORF2p

The first open reading frame of the L1 dicistronic mRNA encodesRF1p [50]. ORF1p is a roughly 40 kDa RNA binding protein that

orms a trimer in solution and binds L1 mRNA via its noncanoni-al RNA-recognition motif [51–57]. ORF1p preferentially binds itsoding mRNA thereby facilitating cis-TPRT (Fig. 2A) [53,55,58]. Theinding of ORF1p to L1 RNA is independent of ORF2p and is impor-ant for the formation of ribonucleoprotein complexes thoughto chaperone L1 mRNA back into the nucleus [59–61]. The sec-nd open reading frame, ORF2p, encodes a 150 kDa protein withndonuclease (EN) and reverse transcriptase (RT) activities [62,63].oth ORF1p and ORF2p are required for retrotransposition of L1.esides facilitating L1 transposition, ORF2p is required for non-L1lement transposition such as SVA or SINEs in trans by a simi-ar TPRT mechanism [64]. Retrotransposition assays in HeLa cellsave shown that EN and RT activities of ORF2p alone are suffi-ient for SINE retrotransposition, but the efficiency of this process isncreased in the presence of ORF1p [65]. Expression of L1 mRNA andts encoded proteins poses significant threats to the genome. Theamifications are most serious in the context of the germline, sinceesulting defects can be fixed and inherited by successive genera-ions thereby expanding the active TE population. In order to keephe TE threat at bay, epigenetic control mechanisms, described in

ore detail below, have been adapted and refined over evolution-ry time to ensure the silencing of these parasites.

. Epigenetic reprogramming

.1. DNA methylation and L1 expression

Erasure of methyl-cytosine marks during germ cell devel-pment (starting at E10.5 in mouse PGCs) is crucial for thestablishment of sex-specific imprinting [25], but also provides anpportunistic environment for L1 propagation as methylation ofhe upstream regions of L1 is essential to maintain their silencedtate [66–68]. In the male germline, this mark is re-establishedy the de novo methyltransferases DNMT3a and 3L and main-ained by DNMT1 [19,20,69–74]. Deletion of DNMT3a or 3L resultsn the activation of TEs in the male germline resulting in apo-tosis of spermatocytes and sterility. Interestingly, in male germells lacking DNMT3a or 3L the methylation patterns of satelliteNA remain unperturbed suggesting this complex specifically tar-ets TEs [19,72]. The female germline can develop mature oocytesnd ova without DNMT3a, 3b, or 3L but the resulting ova arencapable of supporting embryonic development [18–20]. Mech-nistically, DNMT3L binds to and stimulates DNMT3a which thenransfers a methyl group from S-adenosyl-l-methionine to the fiftharbon on cytosine [73]. The DNA substrate preference for this com-lex has been suggested to be transposon regions enriched in CGinucleotides [75,76], however, the exact mechanism of how theNMT3 complex is targeted to specific regions of the genome (i.e.Es) is not clear. One possibility is that the DNMT3 complex isuided to genomic sites through complementarity to a small RNA

s is the case with Arabidopsis [77,78]; however, Ross et al. havealled this idea into question using an in vitro approach [79]. It haseen shown that DNMT3L interacts with the non-methylated tailf histone 3 (H3) [80,81]. The presence of H3 lysine 4 methylation

earch 714 (2011) 95– 104

(H3K4) abolishes this interaction and prevents de novo DNA methy-lation [80,81]. These results suggest H3K4 methylation could be thetargeting mechanism for the DNMTs to regulate to TEs.

4.2. Histone modifications and TE silencing

Recent work has shown other histone tail modificationsinvolved in regulating TEs in addition to H3K4 methylation.Acetylation of histone tails were implicated in human embryoniccarcinoma cell lines where the silencing of a L1 transgene was alle-viated by the addition of the histone deacetylase inhibitors [82].Biotinylation of H4K12 and H2AK9 was shown to be essential forsilencing TEs in human and mouse cell lines and Drosophila [83,84].In addition, increased levels in acetylated H3 and H3S10 phospho-rylation upregulate expression of specific transposable elements[85,86]. Work in Arabidopsis implicated deubiquitination of H2Band H3K9 demethylation with TE expression [87]. These resultslead to further questions about whether there is one or a com-bination of histone modifications specific to TEs. Indeed, on agenome-wide level 38 histone modifications show enrichment atTE regions, further suggesting more complex regulation besidesH3K4 methylation [66]. However, detailed analysis of histone mod-ifications of TEs in animal models have not been performed thusfar. The modification of histone tails could be the targeting mech-anism for the DNMT3 enzyme complex, however, this begets thequestion as to how the histone modifiers themselves are targetedto TEs within the genome. Recent work, which we will describebelow, has shown that the additional targeting mechanism for his-tone modifiers could include the piRNA pathway, a class of smallRNAs predominately expressed in the germline, thought to facili-tate genomic TE recognition.

5. The piRNA pathway-mechanistic overview

5.1. The discovery of piRNAs in Drosophila and mouse

The existence of piRNAs were hinted at in Drosophila [88,89]but were named repeat-associated RNAs (rasiRNAs) [90]. Thebreakthrough in understanding piRNA biology, however, was notuntil next generation sequencing became available. In 2006, fourgroups reported the discovery of RNAs bound to the rodenthomologs of PIWI proteins [91–94], demonstrating that these werelonger (26–31 nt) than miRNAs and siRNAs, were encoded in clus-ters throughout the genome, predominately correspond to TEsequences, and are specific to testes. All four studies differentiatedthese novel small RNAs (named piRNAs for their association withPIWI proteins) from miRNAs and siRNAs since piRNAs mapped toregions that did not produce either a dsRNA or hairpin intermediateand appeared to originate from transcripts several kilobases long[91–94]. Shortly thereafter, Drosophila rasiRNAs from testes werecharacterized as piRNAs and shown to be generated by a distinctpathway from miRNAs and siRNAs [95].

5.2. The primary piRNA pathway

Characterization of piRNAs since 2006 has shown that piRNAsare generated via two distinct molecular mechanisms, the primaryand secondary/amplification (or “ping-pong”) pathways [96–98].Considering the large tandem arrays of piRNAs in the genome,primary piRNAs are thought to be transcribed as long ssRNA tran-scripts [27,96]. However, whether a multi-kb RNA transcript isproduced and gets processed into mature piRNAs remains to be

determined. The molecular players at the top of the primary path-way have only recently been described using Drosophila genetics.Four groups independently showed that an RNA helicase (Armitage,Armi), a Tudor domain protein (Yb), and an putative exonuclease

J. Castaneda et al. / Mutation Research 714 (2011) 95– 104 99

Table 1Mouse embryonic piRNA pathway components. Genes implicated in the mouse embryonic piRNA pathway, their Drosophila homologs, encoded domains, and deducedfunction based on mutant phenotype. 1◦ – primary, 2◦ – secondary.

Mouse gene Drosophila homolog Protein domains Function Mouse mutant phenotype References

Mili Aubergine PAZ, PIWI Binds 1◦ piRNAs, generates 2◦ piRNAs,required for piP-Body formation

Loss of most piRNAs, failure to methylateTEs, upregulation of TEs at meiosis

[26,27,87,108,114]

Miwi2 Ago3 PAZ, PIWI Binds 2◦ piRNAs, possible effector ofRNA-directed DNA methylation

Loss of 2◦ piRNAs, failure to methylate TEs,upregulation of TEs at meiosis

[26,27,108,115]

Mov10L1 Armitage DEXDc-helicase,AAA ATpase

Generates 1◦ piRNAs and/or loads piRNAsonto Mili

Loss of all piRNAs, failure to methylate TEs,upregulation of TEs at meiosis

[108,109]

MVH Vasa DEADc-helicase,HELICc

Required for “ping-pong” pathway andloading Miwi2 with piRNAs

Reduced levels of piRNAs (20% of WT),mislocalization of many piRNAcomponents, upregulation of TEs at meiosis

[124,125]

Tdrd1 CG14303 MYND domain, 4Tudor domains

Ensures proper piRNA loading onto Mili,required for piP-Body formation

Increase in exon-derived piRNAs onto Mili,failure to methylate TEs, upregulation ofTEs at meiosis

[111,112]

Tdrd9 Spn-EHls DEXDc-helicase,HELICc, HA2, 1Tudor domain

Ensures production of 2◦ piRNAs Reduction of 2◦ piRNAs, increase of 1◦

piRNAs, failure to methylate TEs,upregulation of L1 at meiosis

[107]

Tdrd12 Yb 1 Tudor domain Not examined None reported N/APld6 Zucchini PLDc Not examined None reported N/AMael Mael HMG-like box Required for proper timing of 1◦ piRNA

production, required for piP-Bodyformation

Loss of all piRNAs at E16.5, recovery ofpiRNAs by P2, failure to maintain TEmethylation at meiosis

[104,105]

dy forA comprote

◦ ◦

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5

ootaAgAaaopAo1piNsiekp

GASZ CG2183? 4 ankyrin repeats,sterile alpha motif

Required for pi-Boexpression of piRNfacilitates protein–

Zucchini, Zuc) are required for the generation of primary piRNAshat are loaded onto Piwi protein [99–103]. A mechanistic model forrimary piRNA production based on these papers suggests that Ybay act as a scaffold (analogous to murine Tudor domain proteins)

ringing together all the players into a cytoplasmic body (the Ybody) [104]. Armi and Zuc, in the Yb-body, process pre-piRNA tran-cripts into mature piRNAs that are then loaded onto Piwi, allowingiwi to enter the nucleus and mediate silencing [99–102]. However,he molecular mechanism of downstream TE silencing in Drosophilas unclear. It is possible that piRNAs recognize RNA transcripts andarget them for degradation since the Yb body sits adjacent to pro-essing bodies (sites of RNA degradation) [104,105]. Alternatively,ilencing may include piRNA mediated recognition of DNA targetshat then mediate epigenetic changes of these genomic targets toeep them off.

.3. The secondary/“ping-pong” pathway

The main effectors of the “ping-pong” pathway are the twother Drosophila encoded PIWI proteins, Aubergine (Aub) and Arg-naute3 (Ago3). Initiation of piRNA mediated silencing begins withranscripts from piRNA loci (antisense with regard to TEs), thatre processed to functional size RNAs and bound by Aub [96]. Theub–piRNA complex directs the cleavage of sense TE transcripts toenerate degradation products that are processed and loaded ontogo3 [96]. The Ago3–piRNA complex can then direct the cleavage ofntisense transcripts to produce sense degradations products thatlso get processed and loaded onto Aub, thus completing the loop,r so-called “ping-pong” cycle. The main signature of the secondaryathway is the complementarity between the first 10 nt of Aub andgo3 piRNAs, where the uridine (U) predominates as the first basef Aub piRNAs, and adenine (A) as tenth of Ago3 piRNAs [96]. ThisU-10A “ping-pong” signature is absent from the primary piRNAathway in Drosophila [97,98,106]. As a result of back-and-forth

nteractions of Aub and Ago3, zygotic or maternally deposited piR-As can be amplified and their targets silenced [96,107]. The down-

tream mechanism TE silencing by the piRNA pathway in Drosophila

s thought to include recruitment of the RNA degradation machin-ry and a yet to be described epigenetic silencing mechanism thateeps TEs in a silent state [108,109]. In the case of the mouse piRNAathway, the major downstream epigenetic mechanism implicated

mation andponents, possibly

in interactions

Reduction of 1 and 2 piRNAs,mislocalization of MILI and Tdrd1, reducedexpression of piRNA pathway components

[106,110]

in repression of TEs is the establishment of DNA methylation, whichis absent in Drosophila. Major murine counterparts of the DrosophilapiRNA pathway components are described in detail below.

6. The piRNA pathway in the male mouse germline

When mouse PGCs enter the genital ridge (E10.5), DNA methylmarks on cytosine are erased [7], relieving the suppression of TEs.The especially prominent expression of L1 TEs is corroborated bythe presence of ORF1p at E15.5 and E14.5 in females and males,respectively [110,111]. It is essential that DNA methylation of theseTEs be re-established in embryonic male germ cells; failure to do soresults in TE transcript accumulation, transposition-induced DNAdamage, defects in homologous chromosome synapsis, meioticarrest, and sterility [19,72,112]. Interestingly, in the male, certainpiRNA pathway components [the PIWI proteins MILI & MIWI2,Maelstrom (MAEL), GASZ, Mouse Vasa Homolog (MVH), TDRD1,and TDRD9] begin to accumulate before ORF1p is detected as ifanticipating the release of L1 inhibition [27,111,113–115]. Unlikein Drosophila, there is a substantial presence of both primary and“ping-pong” cycles in male mouse embryonic germ cells whichcomplicates the molecular dissection of this pathway [27,111];nevertheless, many studies of mouse mutants have elucidated cer-tain aspects of the piRNA pathway in the male germline (Table 1).

6.1. The pi-Body: the presumed site of the primary piRNApathway

The upstream components of the primary pathway includeMOV10L1, GASZ, MILI, TDRD1, and MAEL. The cytoplasmic orga-nization of the male embryonic pathway is such that most ofthe upstream components (MOV10L1, GASZ, MILI, and TDRD1)reside in the piRNA-Body or pi-Body (previously known as theintermitochondrial cement) while MAEL (along with MIWI2, andTDRD9) resides in the adjacent cytoplasmic structure termed thepiRNA-Processing Body or piP-Body (Fig. 2B) [111,113,114]. Ofthese primary components, only MAEL has been localized to the

nucleus by immunofluorescence [111]. MOV10L1 (a homolog ofArmi) is required for the generation of primary piRNAs, which inthe mouse are sense with respect to TEs [116,117]. In Mov10l1mutants, total small RNA deep sequencing at P10 fails to detect

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iRNAs and immunoprecipitation of MIWI2 and MILI at P0 (beforeeiosis initiates) shows that these proteins are devoid of piRNAs

s well [117]. MOV10L1, like Drosophila Armi, is therefore requiredor the primary piRNA pathway. piRNA defects in mutants for the

ouse homologues of Yb (TDRD12) and Zuc (PLD6) are yet to beeported. GASZ, a protein with many protein–protein interactionotifs [118], was found to be required for correct localization ofILI and for expression of many piRNA pathway proteins [113].

he mislocalization of MILI in Gasz mutants suggests GASZ maylay the role of Drosophila Yb (which localizes PIWI) in the mouseossibly with TDRD12 (Yb). In the Gasz mutant, piRNAs are dras-ically reduced but not absent at P7, P10, and P14 based on totalmall RNA sequencing. Germ cells deficient for TDRD1 show a dis-roportionate accumulation of sense, exonic-derived piRNAs (aspposed to piRNAs from TEs) in purified MILI complexes by P15,hich suggests that TDRD1 helps direct proper piRNA selection

nd loading onto MILI [119,120]. However, the localization of MILIo the pi-Body is not affected in Tdrd1 mutants.

Mael mutants show an interesting phenotype in embryonic anderinatal male germ cells. At E16.5, MAEL deficient germ cells cor-ectly localize MILI and TDRD1, however they show a completebsence of piRNAs suggesting that MAEL functions in the primaryiRNA processing pathway unlike Drosophila Mael [101,111,112].erhaps MAEL aids in the initial production of piRNAs as it is thenly primary piRNA component localized to the nucleus, however,hy MAEL also localizes to a distinct cytoplasmic body from the

ther primary components is unexplained. Unexpectedly in theael mutant, at P2 piRNA profiles recover to near wild-type lev-

ls. piRNA recovery may result from spurious degradation productsenerated by the RNA degradation machinery leading to the pro-uction of small RNAs that encounter MILI, which then initiates theping-pong” cycle [121]. Since total small RNA profiles from embry-nic testes in Mov10l1, Gasz, and Tdrd1 mutants were not analyzed,his piRNA recovery seen in Mael mutants may not be unique.

Absence of MILI results in a loss of most piRNAs correspond-ng to TEs [26,27]. The loss of these piRNAs leads to a failureo re-methylate TEs in male embryonic germ cells, which theneads to massive upregulation of L1 in meiosis. Taken together,

OV10L1 and MAEL appear to be responsible for the productionf piRNAs, GASZ for the correct localization of MILI, and TDRD1or proper piRNA loading of MILI, which is essential as MILI is the

ain effector protein of the pathway. Similar to the Mili pheno-ype, absence of MOV10L1, GASZ, and TDRD1 results in activationf L1 during male meiosis as a consequence of their unsuccess-ul DNA methylation, which results in DNA damage, synapsis andairing defects, meiotic arrest, apoptosis, and ultimately sterility26,28,111,113,116,117,122].

.2. The piP-Body: the presumed site of the secondary piRNAathway

Major players of secondary piRNA pathway are piP-Body resi-ent proteins – MIWI2, TDRD9, and MVH (Fig. 2B) [111,114]. Thentisense identity of piRNAs associated with MIWI2 indicates thatIWI2 is downstream in the ping-pong cycle, associating with

iRNAs generated by MILI presumably in the pi-Body [27]. Comple-entarity of MIWI2 antisense piRNAs with L1 transcripts suggests

n mRNA recognition and degradation pathway involving MIWI2’sIWI domain [26,114,123–125]. A direct role of MIWI2 mediatedleavage as the sole mechanism of TE transcript degradation isomplicated by the fact that processing body (P-body) compo-ents also localize with MIWI2 [111]. P-bodies have been shown

n yeast and cell culture studies to be sites of mRNA degradationnd mRNA storage [126,127]. It is possible that MIWI2 recruits P-odies to aid in the degradation of TE transcripts; however, MIWI2an also localize to the nucleus where piRNAs associated with

earch 714 (2011) 95– 104

MIWI2 may recognize TE nascent transcript by complementaritythat then recruit the DNMT proteins [26,27]. TDRD9, which is themouse homolog of Spn-EHls, also has a similar localization patternas MIWI2 suggesting that it aids MIWI2 in generating secondarypiRNAs and helping in an RNA directed DNA methylation (RdDM)process [114,128–131]. Indeed, Tdrd9 mutants show a decrease insecondary piRNAs by total small RNA deep sequencing, however,the localization of MIWI2 is unaffected in the absence of TDRD9[114]. The correct localization of MIWI2 in the Tdrd9 null back-ground suggest that either MIWI2 can localize to the nucleus on itsown or that there is another component that aids in its localization.

MVH (an RNA helicase) localizes to both the pi-Body and thepiP-Body [132,133]. In the Mvh mutant, embryonic germ cellsshow a decrease (20% of normal levels) but not an absence of piR-NAs, with a preferential loss of MIWI2 associated piRNAs. In fact,immunoprecipitation of MIWI2 fails to detect associated piRNAs,suggesting MVH facilitates their loading onto MIWI2. Loss of MIWI2piRNAs and sequence analysis of the remaining piRNAs suggest thatthe primary pathway is intact but the secondary pathway is com-promised. The localization of the primary and secondary pathwaycomponents (MIWI2 and TDRD9) in embryonic germ cells showsthat only TDRD9 is correctly localized in the absence of MVH, whichis at odds with studies implicating other upstream components inTDRD9 localization [114,132]. MILI and TDRD1 mislocalization andconcurrent production of primary piRNAs (albeit at a lower levelto wild type) suggests that the primary pathway does not requireproper pi-Body formation. The inability to load MIWI2 with piRNAs,however, suggests that pi-Body formation and/or piP-Body local-ization of MIWI2 is required for piRNA loading of MIWI2. Since MVHis an RNA helicase and localizes to both the pi-Body and the piP-Body, it is tempting to speculate that MVH shuttles piRNAs betweenthese bodies, however, MVH does not associate with piRNAs [132].

Mutations in all the above-mentioned mammalian piRNA com-ponents (MILI, MIWI2, MAEL, MOV10L1, GASZ, MVH, TDRD1, andTDRD9) lead to a reduction of piRNAs and failure to silence TEs(Table 1). Although all the phenotypes suggest an RdDM mecha-nism, a direct association between several of the above mentionedcomponents (in particular MIWI2) fail to detect the presence of anyDNMT proteins (either 1 or 3), and the reverse is also true (DNMT3Limmunoprecipitation fails to detect piRNA proteins) [80,134,135].These data suggest there is an intermediate step between MIWI2recognition of TEs and recruitment of DNMTs. It seems likely thatthe gap between TE methylation and piRNA mediated silencing isbridged by modification of histone tails, which could function torecruit the DNMTs to specific sites. However this speculation hasnot yet been corroborated by experiments.

7. Summary and perspectives

Proper repression of TE activity is vital for maintaining theintegrity of the genome. In order for the development of gametescompetent for fertilization TEs must be silenced. In the malegermline, silencing of TEs (in particular L1) occurs before meio-sis, and is achieved by the DNMT enzymes in conjunction withthe piRNA pathway. Failure to silence these elements in the maleleads to meiotic arrest and apoptosis due to massive DNA damagecause by unchecked L1 insertion events. In addition to the germline,L1 activity has the potential to compromise genome integrity inall cells as each cell contains thousand of copies of L1 encoded intheir genome. Indeed, L1 activity has been linked to many cancersand correlated with several diseases [35]. The question remains

whether the association of L1 TEs with these cancers is a causativeagent or just a correlation. If this is just correlated, L1 may serve as auseful diagnostic tool. For example, studies in breast cancers havecorrelated nuclear localization of ORF1p with decreased survival

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f patients compared to patients with predominantly cytoplasmicRF1p [37].

While the activity of TEs is largely viewed to have a negative con-equence for genome integrity, there are several lines of evidencehat suggest transposons and transposon encoded genes exert aositive effect. In one case, transposition of the Het-A DNA transpo-on comprises and maintains the telomeres of Drosophila [136,137].n several other cases, it has been the usurpation of TE encodingenes for the benefit of the host. The most well known example inumans is the RAG recombinases, which are thought to have orig-

nated from a DNA transposon [138]. A recent study of a ciliatedrotozoan, Oxytricha trifallax, demonstrated that TE derived trans-osases facilitate large-scale genome rearrangements and removalf transposon sequences in the maturing macronucleus [139–141].ammalian genome-wide analysis suggests that TEs influenceutation and recombination rates in the germ line [142]. In addi-

ion, the TE landscape in various genomes may be indicative of theirole in chromosome biology and evolution, for example there is annrichment of TE elements on sex chromosomes [143]. Further-ore, multiple reports implicate endogenous reverse transcriptase

ORF2p of L1) to be involved in regulation of the embryonic genectivation, modulation of growth, and proliferation of germ cells144–146]. However, more in-depth understanding of TEs, theirncoded proteins, and their effects within their genomic locations required to be able to attribute specific functions to transposons.

TEs have also been useful for the biomedical community. Thebility to randomly integrate into the genome has made TEs a primeubject for developing genetic tools [147]. One of the most wellstablished system is the P-element (a DNA transposon) used toanipulate the Drosophila genome [148,149]. The P-element has

een utilized in numerous genetics screens and has lead to the pro-uction of thousands of genetic lines that have been invaluable inissecting multiple pathways in numerous tissues [150]. In fact,

P-element genetic screen uncovered PIWI (P-element InducedImpy testis) [151]. A more recent and versatile TE is the Sleep-

ng Beauty (SB) transposon, a “resurrected” transposon capable ofopping in numerous genomes [152]. Mobilization of this elementas been shown to result in somatic and embryonic cancers when

nduced [153–155]. The SB TE has also been utilized to generateransgenic rats [156] as well as in gene therapy using animal mod-ls [157]. Modifications of TEs to carry vital genes and to controlheir insertion will be of great interest for future gene therapyrials.

Although much information has been gained from work on TEsnd the piRNA pathway, many questions remain. It is beyond thecope of this review to list all the questions here, however, weould like to highlight some of the most intriguing avenues that

re currently being pursued. In the immediate future, the field ofammalian piRNAs would be advanced by an understanding of

he mechanism that ties the piRNA pathway with methylation ofNA, as neither PIWI proteins nor DNMTs associate together in aomplex [80,134,135]. Concerning the female piRNA pathway, veryittle is known as most piRNA pathway mutants are female fertile112–114,122,133,158]. Does the lack of a female piRNA pheno-ype suggest there is no role for piRNAs in the female germlinend/or that there is a redundant mechanism that ensures the silenc-ng of TEs in primordial follicles [159,160]? Perhaps the differenceetween the male and female germlines in their dependency onhe piRNA pathway may be attributed to the difference in tim-ng of DNA methylation in meiosis. The post-meiotic methylationf the female genomes leads us to question how the primor-ial follicles are protected from TEs during meiosis. On a global

cale, should there be a concern about the increasing use of RTnhibitors used to combat the global AIDS pandemic consideringhe endogenous L1 activity in sperm, embryos and in neurons39–43,144–146]?

earch 714 (2011) 95– 104 101

We have come a long way in understanding the biologyof transposable elements and the mechanisms that regulatethem since the first discovery of these elements in 1950. Withthe increasing availability of next generation sequencing, moresophisticated whole-animal genetic manipulations, and advancinglive-cell imaging techniques we are on the verge of some remark-able discoveries regarding TEs, their role within our genome, andthe potential they hold for our evolution.

Conflict of interest

None.

Funding source

Carnegie Institution for Science.

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