x\ Rhino

InteractiveFly: GeneBrief

rhino: Biological Overview | References

Gene name - rhino

Synonyms -

Cytological map position - 54D2-54D2

Function - chromatin protein

Keywords - oogenesis, nuage organization, transposon silencing; ping-pong amplification of Piwi-interacting RNAs, germline homolog of the heterochromatin protein 1

Symbol - rhi

FlyBase ID: FBgn0004400

Genetic map position - 2R:13,515,994..13,521,018 [-

Classification - Chromatin organization modifier (chromo) domain

Cellular location - nuclear

NCBI link: EntrezGene
rhi orthologs: Biolitmine
Recent literature
Hermant, C., Boivin, A., Teysset, L., Delmarre, V., Asif-Laidin, A., Beek, M. V., Antoniewski, C. and Ronsseray, S. (2015). Paramutation in Drosophila requires both nuclear and cytoplasmic actors of the piRNA pathway and induces cis-spreading of piRNA production. Genetics [Epub ahead of print]. PubMed ID: 26482790
Transposable element (TE) activity is repressed in the germline in animals by PIWI-Interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. In Drosophila, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. This paper reports that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21nt RNAs which are produced by the paramutated cluster in addition to 23-28nt piRNAs are not necessary for paramutation to occur. Production of these 21nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus which produces piRNAs along the whole length of the transgenes. Finally, it was observed that maternally-inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between non-fully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.
Parhad, S. S., Tu, S., Weng, Z. and Theurkauf, W. E. (2017). Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery. Dev Cell [Epub ahead of print]. PubMed ID: 28919205
Reproductive isolation defines species divergence and is linked to adaptive evolution of hybrid incompatibility genes. Hybrids between Drosophila melanogaster and Drosophila simulans are sterile, and phenocopy mutations in the PIWI interacting RNA (piRNA) pathway, which silences transposons and shows pervasive adaptive evolution, and Drosophila rhino and deadlock encode rapidly evolving components of a complex that binds to piRNA clusters. This study shows that Rhino and Deadlock interact and co-localize in simulans and melanogaster, but simulans Rhino does not bind melanogaster Deadlock, due to substitutions in the rapidly evolving Shadow domain. Significantly, a chimera expressing the simulans Shadow domain in a melanogaster Rhino backbone fails to support piRNA production, disrupts binding to piRNA clusters, and leads to ectopic localization to bulk heterochromatin. Fusing melanogaster Deadlock to simulans Rhino, by contrast, restores localization to clusters. Deadlock binding thus directs Rhino to piRNA clusters, and Rhino-Deadlock co-evolution has produced cross-species incompatibilities, which may contribute to reproductive isolation.
Akulenko, N., Ryazansky, S., Morgunova, V., Komarov, P. A., Olovnikov, I., Vaury, C., Jensen, S. and Kalmykova, A. (2018). Transcriptional and chromatin changes accompanying de novo formation of transgenic piRNA clusters. RNA [Epub ahead of print]. PubMed ID: 29358235
Expression of transposable elements in the germline is controlled by Piwi-interacting (pi) RNAs produced by genomic loci termed piRNA clusters and associated with Rhino, a Heterochromatin Protein 1 (HP1) homolog. Previously, it was shown that transgenes containing a fragment of the I retrotransposon form de novo piRNA clusters in the Drosophila germline providing suppression of I-element activity. It was noted that identical transgenes located in different genomic sites vary considerably in piRNA production, and they were classified as "strong" and "weak" piRNA clusters. This study investigated what chromatin and transcriptional changes occur at the transgene insertion sites after their conversion into piRNA clusters. The formation of a transgenic piRNA cluster was found to be accompanied by activation of transcription from both genomic strands that likely initiates at multiple random sites. The chromatin of all transgene-associated piRNA clusters contain high levels of trimethylated lysine 9 of histone H3 (H3K9me3) and HP1a, whereas Rhino binding is considerably higher at the strong clusters. None of these chromatin marks was revealed at the "empty" sites before transgene insertion. Finally, this study showed that in the nucleus of polyploid nurse cells, the formation of a piRNA cluster at a given transgenic genomic copy works according to an "all- or- nothing" model: either there is high Rhino enrichment or there is no association with Rhino at all. As a result, genomic copies of a weak piRNA transgenic cluster show a mosaic association with Rhino foci, while the majority of strong transgene copies associate with Rhino and are hence involved in piRNA production.
Yu, B., Lin, Y. A., Parhad, S. S., Jin, Z., Ma, J., Theurkauf, W. E., Zhang, Z. Z. and Huang, Y. (2018). Structural insights into Rhino-Deadlock complex for germline piRNA cluster specification. EMBO Rep. PubMed ID: 29858487
PIWI-interacting RNAs (piRNAs) silence transposons in germ cells to maintain genome stability and animal fertility. Rhino, a rapidly evolving heterochromatin protein 1 (HP1) family protein, binds Deadlock in a species-specific manner and so defines the piRNA-producing loci in the Drosophila genome. This study determined the crystal structures of Rhino-Deadlock complex in Drosophila melanogaster and simulans. In both species, one Rhino binds the N-terminal helix-hairpin-helix motif of one Deadlock protein through a novel interface formed by the beta-sheet in the Rhino chromoshadow domain. Disrupting the interface leads to infertility and transposon hyperactivation in flies. These structural and functional experiments indicate that electrostatic repulsion at the interaction interface causes cross-species incompatibility between the sibling species. By determining the molecular architecture of this piRNA-producing machinery, a novel HP1-partner interacting mode was discovered that is crucial to piRNA biogenesis and transposon silencing. This study thus explains the cross-species incompatibility of two sibling species at the molecular level.
Wei, X., Eickbush, D. G., Speece, I. and Larracuente, A. M. (2021). Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline. Elife 10. PubMed ID: 34259629
Large blocks of tandemly repeated DNAs-satellite DNAs (satDNAs)-play important roles in heterochromatin formation and chromosome segregation. Little is known about how satDNAs are regulated, however their misregulation is associated with genomic instability and human diseases. The Drosophila melanogaster germline was used as a model to study the regulation of satDNA transcription and chromatin. This study shows that complex satDNAs (>100-bp repeat units) are transcribed into long noncoding RNAs and processed into piRNAs (PIWI interacting RNAs). This satDNA piRNA production depends on the Rhino-Deadlock-Cutoff complex and the transcription factor Moonshiner-a previously-described non-canonical pathway that licenses heterochromatin-dependent transcription of dual-strand piRNA clusters. This study shows that this pathway is important for establishing heterochromatin at satDNAs. Therefore, satDNAs are regulated by piRNAs originating from their own genomic loci. This novel mechanism of satDNA regulation provides insight into the role of piRNA pathways in heterochromatin formation and genome stability.
Miller, D. E., Dorador, A. P, ..., Hawley, R. S. and Blumenstiel, J. P. (2023). Off-target piRNA gene silencing in Drosophila melanogaster rescued by a transposable element insertion. PLoS Genet 19(2): e1010598. PubMed ID: 36809339
Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. This study describes how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. This local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. It was further shown how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.
Ho, S., Rice, N. P., Yu, T., Weng, Z. and Theurkauf, W. E. (2023). Aub, Vasa and Armi localization to phase separated nuage is dispensable for piRNA biogenesis and transposon silencing in Drosophila. bioRxiv. PubMed ID: 37546958
From nematodes to placental mammals, key components of the germline transposon silencing piRNAs pathway localize to phase separated perinuclear granules. In Drosophila, the PIWI protein Aub, DEAD box protein Vasa and helicase Armi localize to nuage granules and are required for ping-pong piRNA amplification and phased piRNA processing. Drosophila piRNA mutants lead to genome instability and mnk double mutants, this study showed that Chk2 activation disrupts nuage localization of Aub and Vasa, and that the HP1 homolog Rhino, which drives piRNA precursor transcription, is required for Aub, Vasa, and Armi localization to nuage. However, these studies also show that ping-pong amplification and phased piRNA biogenesis are independent of nuage localization of Vasa, Aub and Armi. Dispersed cytoplasmic proteins thus appear to mediate these essential piRNA pathway functions.
Jin, Z., Yu, B. and Huang, Y. (2023). Structural insights into the chromodomain of Oxpecker in complex with histone H3 lysine 9 trimethylation reveal a transposon silencing mechanism by heterodimerization. Biochem Biophys Res Commun 652: 95-102. PubMed ID: 36841100
Oxpecker, the homolog of Rhino/HP1D, exclusively expressed in Drosophila ovaries, belongs to the Heterochromatin Protein 1 family, as does Rhino. Rhi recognizes piRNA clusters enriched with the heterochromatin marker H3K9me3 via its N-terminal chromodomain and recruits Deadlock via its C-terminal chromoshadow domain, further recruits Moonshiner, a paralog of the TATA box-binding protein-related factor 2 large subunits, to promote transcription of piRNA precursors, thereby protecting the genome. Despite Oxp possessing only the chromodomain, its loss leads to the upregulation of transposons in the female germline. This study ∂solved the crystal structure of the Oxp chromodomain in complex with the histone H3K9me3 peptide. As the Oxp chromodomain dimerizes, two H3K9me3 peptides bind to the Oxp chromodomain in an antiparallel manner. ITC experiments and site-directed mutagenesis experiments showed that E44 determines Oxp's five-fold stronger binding ability to H3K9me3 than that of Rhi. In addition, it was found that Oxp and Rhi can form a heterodimer, which may shed light on the molecular mechanism by which Oxp regulates transposon silencing in the absence of CSD.

Piwi-interacting RNAs (piRNAs) silence transposons and maintain genome integrity during germline development. In Drosophila, transposon-rich heterochromatic clusters encode piRNAs either on both genomic strands (dual-strand clusters) or predominantly one genomic strand (uni-strand clusters). Primary piRNAs derived from these clusters are proposed to drive a ping-pong amplification cycle catalyzed by proteins that localize to the perinuclear nuage. This study shows that the HP1 homolog Rhino is required for nuage organization, transposon silencing, and ping-pong amplification of piRNAs. rhi mutations virtually eliminate piRNAs from the dual-strand clusters and block production of putative precursor RNAs from both strands of the major 42AB dual-strand cluster, but not of transcripts or piRNAs from the uni-strand clusters. Furthermore, Rhino protein associates with the 42AB dual-strand cluster, but does not bind to uni-strand cluster 2 or flamenco. Rhino thus appears to promote transcription of dual-strand clusters, leading to production of piRNAs that drive the ping-pong amplification cycle (Klattenhoff, 2009).

Mutations in the Drosophila piwi-interacting RNA (piRNA) pathway disrupt transposon silencing, cause DNA break accumulation during female germline development, and lead to defects in posterior and dorsoventral axis specification (Brennecke, 2007; Chambeyron, 2008; Klattenhoff, 2007; Vagin, 2006). The axis specification defects associated with piRNA pathway mutations are dramatically suppressed by mutations in mnk and mei-41, which encode Chk2 and ATR kinase homologs that function in DNA damage signaling. The developmental defects linked to piRNA pathway mutations thus appear to be secondary to DNA damage, which may result from transposon mobilization. PIWI proteins bind piRNAs, and mutations in genes encoding mouse and Zebrafish piwi homologs lead to transposon overexpression and germline-specific apoptosis, which could be triggered by DNA damage. The piRNA pathway may therefore have a conserved function in transposon silencing and maintenance of germline genome integrity (Klattenhoff, 2009).

Drosophila piRNAs appear to be derived from transposon rich clusters, most of which are localized in pericentromeric and subtelomeric heterochromatin (Brennecke, 2007). The majority of clusters produce piRNAs from both genomic strands (dual-strand clusters). However, two major clusters on the X chromosome produce piRNAs predominantly from one genomic strand (uni-strand clusters) (Brennecke, 2007; Brennecke, 2008). One of these uni-strand clusters maps to flamenco, a locus required for transposon silencing in the somatic follicle cells. The flamenco cluster contains fragments of a number of transposons, including Zam, idefix, and gypsy, and flamenco mutations disrupt silencing of these transposons (Desset, 2008; Mevel-Ninio, 2007; Prud'homme, 1995). In addition, transgenes carrying fragments of transposons in this cluster show flamenco-dependent silencing (Sarot, 2004). These findings suggest that piRNAs encoded by flamenco trans-silence complementary transposons located outside this cluster (Brennecke, 2007) (Klattenhoff, 2009).

The mechanism of trans-silencing by piRNA is not well understood. piRNA-PIWI protein complexes catalyze homology-dependent target cleavage, suggesting that target transposon mRNAs are cotranscriptionally or posttranscriptionally degraded (Gunawardane, 2007; Saito, 2006). However, several Drosophila piRNA pathway mutations have been reported to modify position effect variegation (PEV), which is linked to spreading of transcriptionally silent heterochromatin from pericentric and telomeric regions. Piwi protein also binds to heterochromatin in somatic cells, and interacts with Heterochromatin protein-1 (HP1) in yeast two-hybrid and immunoprecipitation assays (Brower-Toland, 2007). piRNA-Piwi protein complexes could therefore silence target transposons by directing assembly of heterochromatin-like domains. In fission yeast, which do not have piRNAs, small interfering RNAs (siRNAs) and Argonaute 1 (Ago1) appear to recognize nascent transcripts at the centromere, triggering both transcript destruction and HP1 recruitment and assembly of centromeric heterochromatin (Buhler, 2006; Verdel, 2005). A similar combination of homology dependent cleavage and heterochromatin assembly could drive piRNA based silencing in the Drosophila germline (Klattenhoff, 2009).

The mechanism of piRNAs biogenesis also remains to be fully elucidated. Dicer endonucleases cleave double-stranded precursors to produce miRNAs and siRNAs, but piRNA production is Dicer independent. A subset of sense and antisense piRNAs overlap by 10 base pairs and show a strong bias toward an A at position 10 of the sense strand and a complementary U at the 5' end of the antisense strand, suggesting that positions 1 and 10 base pair. As Argonautes cleave their targets between positions 10 and 11 of the guide strand, these finding suggest that piRNAs are produced by a 'ping-pong' amplification cycle in which antisense strand piRNAs bound to Argonaute proteins cleave complementary RNAs to produce the 5' end of sense piRNAs, which in turn direct a reciprocal reaction that generates the 5' end of antisense strand piRNAs (Brennecke, 2007; Gunawardane, 2007). However, most piRNAs cannot be assigned to ping-pong pairs, some clusters produce piRNAs from only one strand (Brennecke, 2007), and the mechanism of 3' end generation has not been determined. It is also unclear how ping-pong amplification is initiated, since the cycle depends on pre-existing primary piRNAs (Klattenhoff, 2009).

This study shows that Rhino (Rhi), a member of the Heterochromatin Protein 1 (HP1) subfamily of chromo box proteins, is required for transposon silencing, production of piRNAs by dual-strand heterochromatic clusters, and efficient ping-pong amplification. Significantly, Rhi protein associates with the 42AB dual-strand cluster and is required for production of longer RNAs from both strands of this cluster. Rhi thus appears to promote expression of trigger RNAs that are processed to from primary piRNAs that drive ping-pong amplification and transposon silencing. Protein coding genes carrying transposons and transposon fragments within introns escape silencing, suggesting that piRNA silencing is imposed after RNA processing. Furthermore, rhi mutations disrupt nuage, a perinuclear structure that is enriched in piRNA pathway components. It is therefore speculated that the nuage functions as a perinuclear surveillance machine that scans RNAs exiting the nucleus and destroys transcripts with piRNA complementarity (Klattenhoff, 2009).

Drosophila piRNA pathway mutations lead to germline DNA damage and disrupt axis specification through activation of Chk2 and ATR kinases, which function in DNA damage signaling. Mutations in the rhi locus lead to very similar patterning defects (Volpe, 2001). The mei-41 and mnk genes encode ATR and Chk2, respectively. To determine whether the axis specification defects associated with rhi result from damage signaling, double mutants were generated with mnk and mei-41 and axis specification was quantified by scoring for assembly of dorsal appendages, which are egg shell structures that form in response to dorsal signaling during oocyte development. Only 17% of embryos from rhiKG/rhi2 females had two wild-type appendages. However, 80% of embryos from mnk;rhiKG/rhi2 double-mutant females had two appendages. In addition, 33% of embryos from mei-41;rhiKG/rhi2 double-mutant females had two appendages. Consistent with these observations, rhi mutations disrupt dorsal localization of Gurken and posterior localization of Vasa in the oocyte, and localization of both proteins is restored in mnk; rhiKG/rhi2 double mutants (Klattenhoff, 2009).

Both ATM and ATR kinases have been reported to activate Chk2. Mutations in the Drosophila atm gene are lethal, but caffeine inhibits ATM and to a lesser extent ATR. Strikingly, 88% of embryos from rhi mutant mothers fed caffeine had wild-type dorsal appendages. Similarly, only 2% of embryos from armi mutant females had two dorsal appendages, compared with 11% after caffeine treatment. In addition, 56% of embryos from mei41D3/mei41D3; armi72.1/armi1 females had wild-type appendages, but 83 of embryos from mei41D3/mei41D3; armi72.1/armi1 double mutants fed with caffeine had two appendages. Caffeine combined with mei-41 mutations thus leads to levels of suppression that are similar to mnk single mutations, suggesting that ATM and ATR redundantly activate Chk2 in armi and rhi mutants (Klattenhoff, 2009).

The mei-W68 locus encodes the Drosophila Spo11 homolog, which is required for meiotic double-strand break formation (McKim, 1998). However, mei-W68 mutations fail to suppress the dorsal appendage defects associated with rhi, indicating that DNA damage signaling in rhi mutants is not due to defects in meiotic break repair (Klattenhoff, 2009).

The phosphorylated form of the Drosophila histone H2AX (γ-H2Av) accumulates near DNA double-strand break sites. In wild-type ovaries, γ-H2Av foci are generally restricted to region 2 of the germarium, where meiotic double-strand breaks are formed. As the cysts mature and pass through region 3 of the germarium, γ-H2Av labeling is reduced. Stage 2 egg chambers, which bud from the germarium, show only low levels of γ-H2Av labeling. In rhi mutants, prominent γ-H2Av foci are present in germline cells of the germarium, and these foci persist and increase in intensity as cysts mature and bud to form stage 2 egg chambers. rhi mutations thus appear to trigger germline-specific DNA breaks and damage signaling through ATM, ATR, and Chk2 (Klattenhoff, 2009).

The piRNA pathway is required for transposon silencing in the Drosophila female germline (Vagin, 2006) but has also been implicated in heterochromatic gene silencing in somatic cells (Brower-Toland, 2007; Pal-Bhadra, 2002; Pal-Bhadra, 2004). Therefore both transposon and protein-coding gene expression were assayed using whole-genome tiling arrays. In both rhi and armi mutants, most transposon families show a relatively modest 1.5- to 2-fold increase in expression, which is not statistically significant. However, a subset of transposon families are dramatically overexpressed in both rhi and armi mutants. For example, HeT-A expression increased 70-fold in rhino and 117-fold in armi. In total, 15 of 17 transposon families that are significantly overexpressed in rhi are also overexpressed in armi. 11 families are overexpressed in armi mutants, but not in rhi. Rhino thus appears to silence a subset of the transposons silenced by Armi. This could reflect a role for Armi in transposon silencing in both somatic follicle cells and the germline (Klattenhoff, 2007), while Rhi appears to be restricted to the germline (Klattenhoff, 2009).

Both rhi and armi mutations increased expression of long terminal repeat (LTR) elements, non-LTR retrotransposons, and inverted repeat (IR) elements (Vagin, 2006). Similar patterns of transposon overexpression are observed in aub and ago3 mutants, which disrupt piRNA biogenesis. Mutations in established piRNA pathway genes and in the rhino locus thus disrupt transposon silencing, independent of transposition mechanism (Klattenhoff, 2009).

To define the subcellular distribution of Rhi, a GFP-rhi transgene was generated and anti-Rhi antibodies were raised and used to localize the protein in vivo and immunolabel whole-mount egg chambers. Both methods revealed germline-specific nuclear foci that are present throughout oogenesis. In addition, germline-specific expression of the GFP-Rhi fusion protein rescued fertility and axial patterning in rhi mutations. Rhi thus appears to function specifically within the germline cells of the ovary (Klattenhoff, 2009).

To determine whether Rhi foci are associated with centromeres, for Rhi and CID, the Drosophila homolog of the centromere-specific, histone H3-like CENP-A, were labeled. Rhi accumulated in regions adjacent to most CID foci in germline nuclei, consistent with localization to pericentromeric heterochromatin. However, many Rhi foci were not obviously linked to CID. Some of these foci could be linked to telomeres or other chromatin domains. Resolving this question will require higher-resolution molecular approaches (Klattenhoff, 2009).

To determine whether Rhi localization depends on the piRNA pathway, egg chambers mutant for aub and armi were immunolabeled. Rhino localization to nuclear foci was not disrupted by either mutation. In striking contrast, rhi mutations disrupt localization of Aub and Ago3 to nuage, a perinuclear structure implicated in RNA processing. Vasa is a core component of nuage, and perinuclear localization of Vasa was also lost in rhi mutants. Piwi localizes to nuclei in both germline cells and the somatic follicle cells. In wild-type ovaries, Piwi is most abundant in germline nuclei during early stages of oogenesis. In rhi mutants, nuclear localization of Piwi is reduced during these early stages. However, in later-stage egg chambers, which make up the bulk of the ovary, Piwi localization in rhi is similar to wild-type controls. These findings suggest rhi functions upstream of Ago3 and Aub, but may have a less critical role in Piwi-dependent processes (Klattenhoff, 2009).

To determine whether Rhi is required for piRNA expression, small RNAs were sequenced from control and rhi mutant ovaries. Unlike miRNAs, piRNAs carry 2' methoxy, 3' hydroxy termini that render them resistant to oxidation and stabilize these RNAs in vivo (Vagin, 2006). To enrich for piRNAs and increase effective sequencing depth, RNA samples were oxidized prior to library construction and sequencing and the data were normalized to surviving noncoding RNA fragments. These studies indicate that rhi mutations reduce total piRNA abundance by approximately 80%. Northern blotting for specific piRNAs and miRNAs support these findings. Defects in 3' modification destabilize piRNAs and would lead to preferential loss of piRNAs in oxidized samples. Therefore unoxidized RNAs were deep sequenced and piRNA abundance was normalized to miRNAs. These studies confirm that rhi mutations reduce piRNA abundance by 80%, and indicate that this reduction does not result from a defect in end modification (Klattenhoff, 2009).

The majority of Drosophila piRNAs are derived from transposons and other repeated elements. This study analyzed the impact of rhi mutations on piRNA expression from 95 families with at least 500 matching reads in control samples (Li, 2009). rhi mutations lead to a 50% or greater reduction in antisense piRNA abundance for 83% of these transposon families, and a 98% reduction in antisense piRNAs for approximately 30% of these elements. For 66 of 95 families, both sense and antisense piRNAs are reduced. For example, rhi mutations nearly eliminate sense and antisense piRNAs from the telomeric transposon HeT-A. Eight transposon families continue to express at least 50% of wild-type sense strand piRNAs but show an 80% or greater reduction in antisense piRNAs. The jockey element falls into this class. Mutations in rhi reduce sense strand piRNAs linked to jockey by only 10%, but antisense strand piRNAs are reduced by 95%. For all of the transposon families that show reduced antisense piRNAs, including those that retain sense strand piRNAs, there is a clear reduction in opposite strand piRNAs that overlap by 10 nt, consistent with defects in ping-pong amplification. A comparison of the p values for the 10 nt overlap bias across all transposon families confirms that the loss of ping-pong pairs in rhi is very highly significant. The loss of species that overlap by 10nt is also clear from an analysis of total piRNAs. The rhi mutations thus lead to a near collapse of the ping-pong cycle amplification cycle (Klattenhoff, 2009).

Only 10 of 95 transposon families continue to express antisense piRNAs at or above 75% of wild-type levels in rhi mutants (blood, mdg-1, Tabor, Stalker, Stalker 2, Stalker3, Stalker4, 412, 297, gypsy 5. Eight of these families (blood, mdg-1, Tabor, Stalker, Stalker 2, Stalker3, Stalker4, 412) also show an increase in sense strand piRNAs. The sense strand piRNAs generally map to the same regions as peaks of antisense piRNAs. This pattern could indicate that antisense strand piRNA direct production of the sense strand piRNAs. Alternatively, specific regions within full-length elements or fragments of elements that lie within specific clusters may be preferentially utilized during piRNA production. The available data cannot distinguish between these alternatives (Klattenhoff, 2009).

An analysis of piRNAs encoded by the ten transposon families that show Rhi-independent piRNA production revealed three patterns with respect to overlapping sense and antisense species. The overlapping piRNAs encoded by Stalker3 did not show a statistically significant 10 nt overlap bias in either wild-type or rhi mutants, indicating that their production is independent of ping-pong amplification. However, six families showed a statistically significant 10 nt overlap peak in both wild-type and rhi mutants, indicating that at least some of the piRNAs are produced by a ping-pong cycle that is independent of Rhi (Tabor, Stalker, Stalker 2, Stalker4, 412, 297). The final class of elements includes blood, mdg1, and gypsy5, which show a statistically significant ping-pong peak in wild-type, but loose the 10 nt overlap bias in rhi mutants. For this class, Rhi thus appears to promote production of only a subset of piRNAs through ping-pong amplification. Intriguingly, rhi leads to a 10-fold increase in blood expression, suggesting the minor ping-pong pool of piRNAs may be critical to transposon silencing (Klattenhoff, 2009).

Therefore, although piRNAs encoded by transposon-rich heterochromatic clusters have been proposed to initiate a ping-pong cycle that amplifies the piRNA pool and mediates transposon silencing, the mechanisms of piRNA biogenesis and silencing are not well understood, and it is unclear how the piRNA clusters are differentiated from other chromatin domains. This study shows that the HP1 homolog Rhino is required for production of piRNAs from dual-strand clusters and associates with the major 42AB cluster by ChIP. Significantly, putative piRNA precursor RNAs were identify from both strands of the 42AB cluster, and it was shown that Rhino is required for production of these RNAs. These findings lead to a proposal that Rhi binding promotes transcription of dual-strand clusters, and that the resulting RNAs are processed to form primary piRNAs that drive the ping-pong amplification cycle and transposon silencing (Klattenhoff, 2009).

While Rhino protein appears to be restricted to germline nuclei, rhi mutations disrupt perinuclear localization of Ago3 and Aub, which catalyze the ping-pong amplification cycle (Li, 2009). Mutations in krimper, which encodes a component of the perinuclear nuage, also disrupt transposon silencing and piRNA production (Lim, 2007). piRNA silencing and nuage assembly thus appear to be codependent processes. These observations, along with the finding that protein coding genes carrying piRNA homology within introns escape silencing by the piRNA pathway, suggest that transcripts are scanned for piRNA homology within the nuage, after splicing and nuclear export. Mature protein coding mRNAs thus pass through the nuage and are translated because piRNA homology has been removed by splicing. By contrast, mature transposon transcripts carry piRNA complementarity are recognized by the perinuclear ping-pong machine, leading to destruction. Interestingly, mutations in the mouse maelstrom gene disrupt nuage and lead to male sterility and significant overexpression of LINE-1 elements (Soper, 2008). Nuage may therefore have a conserved function in transposon RNA surveillance and silencing (Klattenhoff, 2009).

In S. pombe, siRNAs bound to Ago1 appear to recruit HP1 to centromeres through interactions with nascent transcripts, thus triggering heterochromatin assembly and transcriptional silencing. The current data indicate that the HP1 homolog Rhino is required for transposon silencing, but this process appears to be mechanistically distinct from centromeric heterochromatin silencing in yeast. For example, localization of the Rhino HP1 homolog to nuclear foci is independent of piRNA production, and Rhino binding appears to promote transcription of heterchromatic clusters. This in turn generates piRNAs that may direct silencing through posttranscriptional target cleavage. However, piRNAs bound to PIWI proteins have been implicated in heterochromatin assembly in somatic cells, and this process could be related evolutionarily to heterochromatin assembly in fission yeast (Klattenhoff, 2009).

Intriguingly, rhi is a rapidly evolving gene, and all three Rhi protein domains (chromo, chromo shadow, and hinge) show evidence of strong positive selection (Vermaak, 2005). On the basis of these observations, Vermaak (2005) proposed that rhino is involved in a genetic conflict within the germline. The observations reported in this study suggest that the conflict between transposon propagation and maintenance of germline DNA integrity drives rhi evolution, and that the heterochromatic dual-strand clusters have a key role in this battle. Rhino appears to define heterochromatic domains that produce transposon silencing piRNAs. Rhino could therefore have evolved to bind transposon integration proteins, which would promote transposition into clusters and production of trans-silencing piRNAs. In this model, the transposon integration machinery would evolve to escape Rhino binding and silencing. The rapid pace of rhino evolution makes identification of homologs in other species difficult (Vermaak, 2005), but the conserved role for piRNAs in germline development suggests that HP1 variants may have critical roles in the conflict between selfish elements and genome integrity in other species, including humans (Klattenhoff, 2009).

Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection

Transposons and other selfish DNA elements can be found in all phyla, and mobilization of these elements can compromise genome integrity. The piRNA (PIWI-interacting RNA) pathway silences transposons in the germline, but it is unclear if this pathway has additional functions during development. This study shows that mutations in the Drosophila piRNA pathway genes, armi, aub, ago3, and rhino, lead to extensive fragmentation of the zygotic genome during the cleavage stage of embryonic divisions. Additionally, aub and armi show defects in telomere resolution during meiosis and the cleavage divisions; and mutations in ligase-IV, which disrupt non-homologous end joining, suppress these fusions. By contrast, lig-IV mutations enhance chromosome fragmentation. Chromatin immunoprecipitation studies show that aub and armi mutations disrupt telomere binding of HOAP, which is a component of the telomere protection complex, and reduce expression of a subpopulation of 19- to 22-nt telomere-specific piRNAs. Mutations in rhi and ago3, by contrast, do not block HOAP binding or production of these piRNAs. These findings uncover genetically separable functions for the Drosophila piRNA pathway. The aub, armi, rhi, and ago3 genes silence transposons and maintain chromosome integrity during cleavage-stage embryonic divisions. However, the aub and armi genes have an additional function in assembly of the telomere protection complex (Khurana, 2010).

Drosophila piRNAs have been implicated in transposon silencing and maintenance of genome integrity during female germline development. However, piRNA pathway mutations lead to complex developmental phenotypes, and piRNAs have been implicated in control of gene expression. Furthermore, the majority of piRNAs in other systems, including mouse testes, are not derived from repeated elements. The full extent of piRNA functions thus remains to be explored (Khurana, 2010).

Mutations in the majority of Drosophila piRNA pathway genes disrupt asymmetric localization of RNAs along the axes of the oocyte, and lead to maternal effect embryonic lethality. The axis specification defects linked to several of piRNA pathway mutations are dramatically suppressed by a null mutation in mnk, which encodes a Checkpoint kinase 2 (Chk2) homolog required for DNA damage signaling, indicating that the loss of asymmetric RNA localization is downstream of DNA damage. Oocyte patterning defects generally lead to embryonic lethality, but the mnk allele that suppresses the axis specification defects associated with piRNA mutations does not suppress embryonic lethality. piRNAs thus have an essential function during embryogenesis that is independent of Chk2 activation and DNA damage signaling. To gain insight into potential new functions for the piRNA pathway, the embryonic lethality associated with four piRNA pathway mutations was characterized. These studies reveal a novel function for a subset of piRNA genes in assembly of the telomere protection complex, and suggest that this process is directed by a subpopulation of 19-22 nt piRNAs (Khurana, 2010). The armi and aub genes encode a putative RNA helicase and a piRNA binding PIWI Argonaute protein, and recent studies suggest that they have distinct functions in piRNA biogenesis. Mutations in aub dramatically reduce piRNA species that overlap by 10 nt, which is characteristic of ping-pong amplification, while armi mutations reduce total piRNA production but enhance the ping-pong signature. Mutations in aub and armi lead to maternal-effect embryonic lethality, however, suggesting that these genes share an essential function. To gain insight into the lethality associated with these mutations, DNA break accumulation during oogenesis was analyzed. Germline-specific DNA breaks normally form during early oogenesis, as meiosis is initiated. In several piRNA mutants, however, DNA breaks persist, which could compromise the female pronucleus and thus lead to genetic instability in the early zygote. DNA breaks trigger phosphorylation of histone H2Av, producing γ-H2Av foci near the break sites. In wild-type ovaries, γ-H2Av foci begin to accumulate in region 2 of the germarium, as meiotic breaks are formed. These foci are significantly reduced in stage 2 egg chambers, which have completed meiotic repair and budded from the germarium. Later in oogenesis, γ-H2Av foci accumulate in the nurse cell nuclei, which undergo endoreduplication. However, these foci remain undetectable in the oocyte. In ovaries mutant for aub or armi, γ-H2Av foci appear in germarium region 2, but persist in nurse cells and the oocyte through stage 4. By stage 5, however, γ-H2Av foci are undetectable in 50% of armi and aub mutant oocytes, and are significantly reduced in the remaining oocytes. Both armi and aub mutations thus increase DNA damage during early oogenesis, but most of the damage in the oocyte appears to be repaired as oogenesis proceeds (Khurana, 2010).

As wild type oocytes mature and initiate meiotic spindle assembly, the major chromosomes form a single mass at the spindle equator and the non-exchange 4th chromosomes move toward the poles. In OregonR, distinct 4th chromosomes were observed in 79% of stage 13 oocytes. In stage 13 aub and armi mutants, by contrast, distinct 4th chromosomes were observed in only 11% and 18% of stage 13 oocytes, respectively. However, a single primary mass of chromatin was always observed. These observations are consistent with γ-H2Av data suggesting that DNA breaks formed during early oogenesis are often repaired as the oocyte matures. In addition, both aub and armi mutations appear to inhibit separation of the small 4th chromosomes, although it is also possible that this small chromosome is fragmented and thus difficult to detect cytologically (Khurana, 2010).

Drosophila oocytes are activated as they pass through the oviduct, which triggers completion of the meiotic divisions. The first meiotic division is completed in the oviduct, but meiosis II can be observed in freshly laid eggs and is characterized by four well-separated meiotic products on tandem spindles. In aub and armi mutant embryos, the meiotic chromatin was either stretched across the paired meiotic spindles, or fragmented and spread over both spindles. No wild type meiotic figures were observed. Breaks thus appear to persist in some stage 14 oocytes, although this does not disrupt the karyosome organization during earlier stages. However, other oocytes appear to have intact chromosomes that fail to resolve during the meiotic divisions (Khurana, 2010).

Chromatin fragmentation could result from replication of broken chromosomes inherited from the female, or from post-fertilization fragmentation of the zygotic genome. To directly assay zygotic genome integrity, mutant females were mated to wild type males and dual-label FISH was used to monitor physically separate regions of the Y chromosome. In male embryos derived from wild type females, the two Y chromosome probes always co-segregated through anaphase and telophase. Mutant embryos showing chromatin fragmentation, by contrast, contained chromatin clusters that did not label for either Y chromosome probe, or that labeled for only one of the two probes. In mutant embryos that proceeded through cleavage stage mitotic cycles, the majority of segregating chromatids retained both Y chromosome markers, indicating that chromosome continuity had been maintained. Chromatids with only one of two markers were observed, however, indicating that breaks had separated regions on a Y chromosome arm from the centromere. The axial patterning defects associated with piRNA mutations are suppressed by mutations in mnk, but mnk did not suppress either the chromatin fragmentation or segregation defects linked to aub and armi. Mutations in aub and armi thus destabilize the genome of the zygote and disrupt chromosome resolution during the cleavage divisions through processes that are independent of DNA damage signaling (Khurana, 2010).

Mutations in the armi and aub genes disrupt piRNA production and transposon silencing, but have also been reported to inhibit homology dependent target cleavage by siRNAs. In addition, null mutations in argonaute2 (ago2), which block siRNA based silencing, have been reported to disrupt mitosis during the syncytial blastoderm stage. These observations raise the possibility that chromatin fragmentation and fusion in aub and armi mutants result from defects in the siRNA pathway. Therefore, cleavage was analyzed in embryos from females homozygous for null mutations in ago2 and dcr2, which block siRNA production and silencing. Consistent with previous studies, it was found that embryos from ago2 and dcr2 mutant females are viable. However, neither chromosome fragmentation nor a statistically significant increase in anaphase bridge formation was found relative to wild type controls. The loquacious (loqs) gene encodes a Dicer-1 binding protein required for miRNA production, and it was found that embryos from loqs mutant females also proceed through normal cleavage stage divisions. Chromosome segregation and maintenance of zygotic genome integrity during early embryogenesis thus appear to be independent of the siRNA and miRNA pathways, but require at least two components of the piRNA pathway (Khurana, 2010).

In S. pombe, mutations in ago1, dcr1 and rdp1 disrupt kinetochore assembly and thus lead to lagging mitotic chromosomes due to defects in centromere movement to the spindle poles. To determine if Drosophila piRNA mutations disrupt kinetochore assembly, dual label FISH was performed for centromeric dodeca-satellite sequences and the telomere-specific transposon HeT-A. In aub and armi mutants, centromeric sequences segregated to the spindle poles in essentially every anaphase figure, but telomere specific sequences were consistently present at the chromatin bridges. These observations indicate that armi and aub are not required for kinetochore assembly, but are needed for telomere resolution (Khurana, 2010).

Telomeres are protected from recognition as DNA double strand breaks by the telomere-protection complex (TPC), and defects in telomere protection thus lead to covalent ligation of chromosome ends by the non-homologous end-joining (NHEJ) pathway. DNA Ligase IV is required for NHEJ, and ligase IV mutations suppress fusions that result from covalent joining of unprotected chromosome ends. To determine if chromosome fusions in aub and armi are due to NHEJ, ligIV;aub and ligIV;armi double mutant females were generated and chromosome segregation was analyzed in the resulting embryos. In aub single mutant embryos, 50% of anaphase figures show bridges, but anaphase bridges are present in only 15% of ligIV;aub double mutants. By contrast, the fraction of embryos showing chromosome fragmentation increases in ligIV;aub double mutants. Chromosome fragmentation also increased in ligIV;armi mutant embryos, and as a result morphologically normal anaphase figures could not be observed. These findings strongly suggest that lagging chromosomes result from covalent ligation of chromosome ends by the NHEJ pathway, while chromatin fragmentation results from DNA breaks that are repaired by NHEJ. Mutations in armi and aub lead to significant over-expression of transposable elements, including DNA elements that are mobilized by a 'cut and paste' mechanism that directly produces double strand breaks. In addition, NHEJ pathway has been implicated in repair of gapped retroviral integration intermediates. Chromosome fragmentation may therefore result from transposon over-expression and mobilization, which induces breaks that overwhelm the NHEJ pathway. Telomere fusions, by contrast, appear to result from defects in telomere protection, which lead to chromosome end recognition by the NHEJ pathway (Khurana, 2010).

The Drosophila TPC includes HOAP and Modigliani (Moi), which may function only at chromosome ends, and HP1a and the MRN complex, which have additional roles in heterochromatic silencing and DNA repair. To directly assay for TPC recruitment, chromatin immunoprecipitation (ChIP) was used to measure HP1a and HOAP binding to the telomere specific transposon HeT-A. In wild type ovaries, HOAP and HP1a bind to multiple regions of HeT-A. In armi and aub mutants, by contrast, HOAP and HP1a binding to the Het-A 5'-UTR and ORF are significantly reduced. The 5' end of Het-A is oriented toward the chromosome end, and is therefore likely to lie at the telomere. Ovarian tissue consists of germ cells with a surrounding layer of somatic cells, which complicates interpretation of these biochemical studies. However, ChIP on 0-3 hour old embryos from aub and mnk,aub mutant females revealed significant reduction in HOAP binding at the HeT-A 5'-UTR. The aub and armi genes thus appear to be required for TPC recruitment, consistent with ligation of chromosome ends in mutant embryos (Khurana, 2010).

To determine if other piRNA pathway mutations disrupt telomere protection, the cleavage stage embryonic divisions was analyzed in ago3 and rhi mutants. The ago3 locus encodes a PIWI clade protein that primarily binds sense strand piRNAs, and rhi encodes a rapidly evolving HP1 homologue required for production of precursor RNAs from a subset of piRNA clusters. Essentially all of the rhi and ago3 mutant embryos showed chromatin fragmentation, as observed in the majority of aub and armi mutants. Therefore TPC assembly was analyzed in ovarian chromatin using ChIP for HOAP and HP1a. Surprisingly, neither ago3 nor rhi mutations disrupt HOAP or HP1a binding to Het-A, and rhi mutants show greater than wild type levels of HOAP binding to Het-A. By contrast, these rhi alleles reduce total piRNA production by 10 fold. The ago3 mutations appear to be null, and the rhi mutations are strong hypomorphc alleles. Assembly of the TPC in the ago3 and rhi mutants is therefore unlikely to be mediated by residual protein. Instead, these findings strongly suggest that aub and armi have a function in telomere protection that is not shared by ago3 or rhi (Khurana, 2010).

In Drosophila, chromosome breaks can be converted to stable telomeres, called terminal deletions, which accumulate additional copies of the telomeric elements HeT-A and TART. When terminal deletions are passaged in animals heterozygous for aub or the piRNA pathway gene spnE, the number of terminal TART repeats increase. The defects in TPC assembly in aub and armi could therefore be triggered by increased HeT-A and TART copy number, which could titrate TPC components. Therefore telomeric transposon copy number was assayed in aub and armi mutants, which show defects in TPC assembly, and in rhi and ago3 mutants, which do not. Telomeric transposon copy number and mitotic chromosome segregation was also analyzed in a wild-type variant, Gaiano, that has been reported to carry additional HeT-A repeats. Consistent with previous reports, it was found that Gaiano has 10 to 15 fold more HeT-A copies than OregonR controls. Despite the increase in telomere length, this stock is viable and fertile, and no telomere fusions or lagging chromosomes were observed during the cleavage stage embryonic divisions. In addition, it was found that aub mutants that show defects in TPC assembly do not accumulate additional copies of HeT-A or TART, while rhi and ago3 mutants that are wild type for TPC binding show an increase in telomere-specific transposon copy number. Assembly of the TPC is therefore independent of telomere specific transposon copy number (Khurana, 2010).

piRNAs are proposed to direct PIWI clade proteins to targets through sequence specific interactions. The current observations raised the possibility that armi and aub promote production of piRNAs that direct the telomere protection complex to transposons that make up chromosome ends. Published small RNA deep sequencing data was analyzed for species derived from a fourth chromosome cluster, defined by a high density of uniquely mapping piRNAs, containing multiple repeats of the telomeric transposons. This bioinformatic analysis showed that 70-80% of telomere specific piRNAs match this cluster. Length histograms for small RNAs from wt, rhi, ago3, aub and armi mutant ovaries map to this cluster. Significantly, aub and armi mutations lead to a preferential loss of shorter piRNAs mapping to the minus genomic strand. Loss of these shorter RNAs highlights the peak at 21 nt, which is retained in all of the mutants and likely represent endogenous siRNAs. The telomeric elements (HeT-A and TART) are almost exclusively on the minus genomic strand in this cluster, and the RNAs that are lost in aub and armi thus correspond to the sense strand of the target elements. Ovaries mutant for ago3 and rhi, by contrast, retain these shorter sense strand RNAs (Khurana, 2010).

The relative abundance of typical 23-29nt long piRNAs and the shorter 19-22nt species were quantified, excluding the 21nt endo-siRNA peak. All four mutations significantly reduce 23 to 29 nt piRNAs, although rhi mutants retain approximately 50% of wild type minus strand species. Loss of these piRNAs is consistent with over-expression of transposons matching this cluster in all four mutants. By contrast, the shorter minus strand RNAs are reduced by 3 to 10 fold in armi and aub, but are expressed at 80% to 95% of wild type levels in ago3 and rhi. In addition, short piRNA species from the telomeric cluster co-immunoprecipitate with Piwi protein, which localizes to the nucleus and is a likely effector of chromatin functions for the piRNA pathway. Binding of this subpopulation of piRNAs by Piwi is retained in ago3 mutants, which assemble the TPC, but significantly reduced in armi mutants, which block assembly of the TPC (Khurana, 2010).

Taken together, these observations suggest that the piRNA pathway has two genetically distinct functions during oogenesis and early embryogenesis. The pathway prevents DNA damage during oogenesis and maintains the integrity of the zygotic genome during the embryonic cleavage divisions, which likely reflects the established role for piRNAs in transposon silencing. This function requires aub, armi, rhi and ago3, which are also required for wild type piRNA production. In addition, these studies reveal a novel function for the piRNA genes aub and armi in telomere protection, whch may be mediated by a novel class of short RNAs that bind to Piwi. Consistent with this hypothesis, it has been reported that germline clones of piwi null alleles do not significantly disrupt oogenesis, but lead to maternal effect embryonic lethality and severe chromosome segregation defects during the cleavage division. A subpopulation of Piwi-bound piRNAs may therefore direct assembly of the TPC (Khurana, 2010).

The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline

In a broad range of organisms, Piwi-interacting RNAs (piRNAs) have emerged as core components of a surveillance system that protects the genome by silencing transposable and repetitive elements. A vast proportion of piRNAs is produced from discrete genomic loci, termed piRNA clusters, which are generally embedded in heterochromatic regions. The molecular mechanisms and the factors that govern their expression are largely unknown. This study shows that Cutoff (Cuff), a Drosophila protein related to the yeast transcription termination factor Rai1, is essential for piRNA production in germline tissues. Cuff accumulates at centromeric/pericentromeric positions in germ-cell nuclei and strongly colocalizes with the major heterochromatic domains. Remarkably, Cuff is enriched at the dual-strand piRNA cluster 1/42AB and is likely to be involved in regulation of transcript levels of similar loci dispersed in the genome. Consistent with this observation, Cuff physically interacts with the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi). These results reveal a link between Cuff activity, heterochromatin assembly and piRNA cluster expression, which is critical for stem-cell and germ-cell development in Drosophila (Pane, 2011).

A significant fraction of eukaryotic genomes is made up of repetitive sequences, such as transposable elements (TEs) and tandem repeats, whose deregulation has been linked to DNA damage and sterility. Germline tissues, which are responsible for transferring the genetic information to the progeny, appear to be the most sensitive targets of TE deregulation. A specialized RNAi pathway, centered on small non-coding RNAs known as Piwi-interacting RNAs (piRNAs), guarantees the repression of transposable and repetitive elements and ensures the maintenance of genomic stability during germ-cell division and differentiation. piRNAs are 23-30-nt-long non-coding RNAs and mainly correspond to sequences in transposable and repetitive elements dispersed in the genome. In Drosophila, the production of these molecules relies on the activity of the Argonaute family members Piwi, Aubergine (Aub) and Argonaute-3 (Ago3). Deep-sequencing analyses of the small RNAs associated with these proteins revealed that Piwi and Aub complexes are enriched in antisense piRNAs, while Ago3 is mostly bound by sense piRNAs. Furthermore, a subset of piRNAs displays a 10-nt overlap at their 5' end. These observations were captured in a model, whereby antisense piRNAs bound to Aub/Piwi can pair with transposon transcripts and catalyse the production of sense piRNA molecules through cleavage of the transposon transcripts. The latter are then loaded into an Ago3 complex, which in turn triggers the production of antisense piRNAs by cleaving cluster-derived antisense transcripts. This cycle is the core of the so-called 'Ping-pong' model, a feed-forward loop that amplifies the piRNA population and is thought to reinforce the repression of active transposons dispersed in the genome. piRNA processing requires additional activities, including the eIF-4A-like translation factor Vasa (Vas), the Heterochromatin Protein 1 (HP1) variant Rhino (Rhi), the helicases Spindle-E and Armitage, the putative nucleases Maelstrom, Zucchini and Squash and the Tudor-domain proteins Tejas and Krimper. The precise biochemical function of many of these proteins is, however, still largely unclear (Pane, 2011).

In Drosophila, primary sources of piRNAs are discrete genomic regions termed piRNA clusters. These genomic loci are composed of repetitive sequences, TEs and inactive transposon remnants. The transcripts produced from these loci are processed by cytoplasmic activities to produce the mature piRNAs. The major sources of piRNAs in Drosophila are the cluster 1/42AB located on chromosome 2R in pericentromeric position and clusters 2 and flamenco/COM (flam) located on chromosome X. Cluster 1/42AB and the majority of the piRNA clusters in Drosophila are transcribed from both DNA strands (dual-strand clusters). Conversely, a few loci, including cluster 2 and flam, produce piRNAs only from one genomic DNA strand (uni-strand clusters). To date, flam represents the best-characterized piRNA cluster. The flam cluster harbours fragments of the Zam, gypsy and idefix retro-transposons and is required for the silencing of these retroviral elements in the follicle cells of the Drosophila ovaries. Interestingly, the vast majority of the transposon remnants that populate the flam locus are oriented towards the centromere of the X chromosome. Genetic and molecular analyses strongly suggest that the transcription of this cluster occurs from an external promoter on the opposite strand and is likely to produce a long transcript encompassing the entire locus. The structure of the cluster and the strand bias of the transcriptional events thus generate mostly antisense piRNAs, which do not engage a ping-pong amplification loop. piRNA clusters in zebrafish, mouse and human also display a clear strand bias similar to flam, whereby piRNAs map only to one DNA strand. The mechanism that regulates the expression of the piRNA clusters and the proteins involved in this nuclear process remain largely elusive. It has recently been shown that a germline-specific HP1 variant encoded by the rhino (rhi) gene is essential for the regulation of the dual-strand clusters. Accordingly, mutations in rhi abolish the expression of these loci, which results in a general depletion of the corresponding piRNA population. The activity of Rhi seems to be limited to the dual-strand clusters, since mutations in this protein do not affect the production of piRNAs from the uni-strand clusters. Surprisingly, the expression of the piRNA loci appears to also rely on the activity of the Piwi proteins Aub and Piwi. In mutants of these factors, heterochromatic states spread over the clusters causing their transcriptional downregulation. These findings, therefore, implicate the production of piRNAs in the maintenance of active transcription from the piRNA clusters (Pane, 2011).

It has been reported that the protein product of the cutoff (cuff) gene is essential for transposon silencing and germline development. Mutations in cuff impair the establishment of dorsal–ventral polarity during oogenesis and cause a significant loss of germline cells, possibly due to defects in stem-cell maintenance and/or division. Cuff shows similarity to the yeast transcription termination factor Rai1. In yeast, Rai1 is found in a complex with the 5'- 3' exoribonuclease Rat1/Xrn2 and promotes the termination of transcriptional events initiated by the PolI and PolII RNA polymerases. Different from Rai1, however, Cuff does not display a Dom3Z domain, which is critical for Rai1 activity. Given the observation that an additional Rai1 homologue exists in Drosophila which contains the Rai1 catalytic domain, it is hypothesized that Cuff might be a germline-specific Rai1 variant, which exerts a novel function in germline cells (Pane, 2011).

This study shows that Cuff localizes to discrete foci in germline nuclei and accumulates in proximity of and within heterochromatic domains. Cuff is essential for the expression of the piRNA clusters embedded in heterochromatic regions, including the major dual-strand cluster 1/42AB, but not for the flam locus, which is primarily expressed in the soma. In agreement with these observations, it was found that Cuff colocalizes and physically interacts with Rhi. It is proposed that these proteins assemble into a complex, which regulates the production of piRNAs from the dual-strand clusters at a transcriptional level. Mutations in Cuff cause a severe disruption of the nuage, which in turn might contribute to the depletion of piRNAs not only from the dual-strand, but also from some uni-strand clusters, including the major cluster 2. These results uncover a role for Cuff in piRNA cluster regulation, nuage assembly and piRNA production and highlight a critical function for the piRNA pathway in stem- and germ-cell development during Drosophila oogenesis (Pane, 2011).

In Drosophila, piRNAs are mostly produced from discrete genomic loci, termed piRNA clusters, which are composed of repetitive sequences and inactive transposon remnants . The major piRNA clusters, including the dual-strand cluster 1/42AB and the uni-strand cluster 2 and flam, are located at the border between euchromatic and heterochromatic regions or within the major heterochromatic domains. Heterochromatic states have been generally associated with transcriptionally repressed genomic loci. The expression of the piRNA clusters, therefore, needs to be tightly controlled to ensure piRNA production, while the heterochromatin is being assembled. This study reports the functional characterization of Cuff, a Drosophila protein with similarity to the yeast transcription termination factor Rai1, and this protein is shown to control piRNA cluster expression and piRNA production during Drosophila germline development. Cuff is critical for the correct levels of transcripts produced from the major dual-strand cluster 1/42AB in the pericentromere of chromosome II. piRNAs originating from this locus and from virtually all the dual-strand clusters dispersed in the genome are substantially reduced or depleted in the cuff mutant. Consistent with a germline-specific function of Cuff, mutations in this protein do not impact the flam locus, which is known to be active only in the somatic follicle cells. The data suggest that Cuff regulates the piRNA master loci presumably acting in a complex with the HP1 variant Rhi. Both Cuff and Rhi are enriched at the cluster 1/42AB, while they do not interact with sequences in the cluster 2 and flam. These observations strongly suggest that a Cuff/Rhi complex regulates the transcription of the dual-strand piRNA clusters. The activity of this complex appears to be crucial for the expression of the dual-strand clusters and might be critical to overcome the complexity of these genomic loci, where bidirectional transcription of the different elements can potentially hinder the production of the proper piRNA population. Different from Rhi, cuff also affects the production of piRNAs from some uni-strand clusters, including the major cluster 2. ChIP assays, however, failed to reveal a significant enrichment of Cuff at this locus. It is, therefore, likely that the loss of piRNAs from cluster 2 is not directly caused by mutations in the Cuff protein, but it might rather ensue from the disruption of the nuage observed in the cuff ovaries and the mislocalization of factors required for piRNA production. Nevertheless, the comparison of cuff and rhi piRNA libraries revealed some interesting differences between the two mutants. For instance, mutations in cuff appear to have a more prominent impact on the sense piRNA population compared with the rhi mutant, thus suggesting that these proteins might display partially different functions in piRNA cluster expression and piRNA production. Intriguingly, the expression levels of the piRNA clusters in the cuff mutant do not precisely mirror the profiles of the corresponding piRNA population. One would predict that the depletion of a specific piRNA set would ensue from a general downregulation of the corresponding cluster-derived transcripts. While some regions within cluster 1/42AB are clearly downregulated in the cuff mutant, other regions are not affected and only one, among those analysed, appears to be upregulated. These data point to a role for Cuff in the transcriptional control of dual-strand cluster expression, whereby this protein may be required to activate/permit the transcription of these loci. In the absence of Cuff, cluster-derived piRNA precursor transcripts do not accumulate, thus leading to a general collapse of the piRNA population. It is noteworthy that some regions in the cluster 1/42AB are not affected in the cuff mutant. This observation suggests that the dual-strand clusters might not produce single transcripts spanning the entire locus, as it is the case for the flam locus, but they might be rather transcribed by multiple internal and external promoters (Pane, 2011).

It has been recently reported that mutations in the Argonaute proteins Piwi and Aub reduce the expression levels of genomic regions located within or next to the piRNA clusters. In these mutants, HP1 spreading triggers a significant repression of the piRNA loci, which led to the proposal that in Drosophila, piRNAs ensure the transcription of the clusters by counteracting the spreading of heterochromatic states. Remarkably, the recruitment of Cuff and Rhi to nuclear foci early in oogenesis seems to parallel the assembly of the major heterochromatic blocks. The levels of Cuff, Rhi and HP1 are low in the stem cells and in the early mitotic cyst, while these proteins appear to accumulate in the late mitotic cyst, where they extensively colocalize in specific regions of the nuclei. It is, therefore, tempting to speculate that Cuff and Rhino might protect the clusters from the repressive effects associated with the assembly of the major heterochromatic blocks, which takes place early in oogenesis while the germ cells undergo the mitotic divisions (Pane, 2011).

Mutations in cuff negate the production of both sense and antisense piRNAs corresponding to 46 transposon families. For a significant number of transposons only piRNAs matching one DNA strand seem to be affected in cuff, while piRNAs corresponding to a few transposon families are apparently unaltered. It is noteworthy that the HeT-A and TART retro-transposons, which are involved in the maintenance of telomere integrity, display contrasting piRNA profiles despite the fact that the transcript levels of both these elements are upregulated in cuff ovaries. Virtually, all the piRNAs corresponding to HeT-A sequences are depleted in the cuff mutant, which might account for the severe deregulation of this retro-transposon in the absence of Cuff. Conversely, only a limited number of piRNAs matching TART sequences could be identified in wild-type ovaries, while a surprising variety of reads corresponding to this element are produced in cuff mutant ovaries. Similar to the piRNA clusters, also the different classes of transposons appear to undergo differential regulation, and it will be a challenge for the future to uncover the underlying rules (Pane, 2011).

The introduction of a sensor transgene based on the Zam retro-transposon in the germline of cuff, rhi, vas and aub flies led to further insight into the temporal requirement of these proteins during Drosophila oogenesis. Zam is an LTR retro-transposon, which is specifically repressed in the ovarian follicle cells by the piRNAs produced from the flam locus. While the sensor transgene is consistently repressed in wild-type germ cells, it is significantly deregulated in cuff, rhi, vas and aub germline. The silencing of the reporter appears to be very sensitive to mutations in cuff and rhi, which produce a strong GFP signal in stem and germ cells as well as in fully formed egg chambers. Mutations in vas and aub instead seem to have a milder impact on the sensor construct, whereby a detectable GFP signal is mostly observed in the dividing or late mitotic cysts. The mechanism by which the sensor construct is silenced in wild-type ovaries is unlikely to mirror the piRNA-dependent repression of the Zam retro-transposon in somatic cells, since mutations in cuff do not impact the production of piRNAs from the flam locus. Similarly, flam piRNA levels are unaltered in rhi, aub and vas mutant backgrounds. These observations reveal that Zam sequences can be targeted for silencing also in germline tissues in a flam-independent fashion. Interestingly, no piRNAs corresponding to the env fragment were detected either in wild-type or in the cuff mutant libraries. While it is not possible to exclude that the expression of the sensor transgene induced by the Act5C-Gal4 driver in the wild-type germline might trigger the production of piRNAs directed against the reporter, it is possible that the silencing of the pGFP-ZenvAS transgene might not proceed through a piRNA-based mechanism. Since nuclear Piwi is dramatically lost in the germaria of all these mutants, it is also unlikely that the strong deregulation of the reporter observed in cuff and rhi mutant stem and germ cells is primarily caused by Piwi mislocalization. Instead, these results suggest that Cuff and Rhi might act upstream of Piwi to regulate the expression of the piRNA clusters and ensure piRNA production early in oogenesis. The mature piRNAs are then loaded into Piwi and serve to drive the nuclear accumulation of this protein. It is conceivable that the activity of Cuff and Rhi protects the piRNA clusters from the repressing effects associated with heterochromatin formation. In later stages, Vas, Aub and most likely other piRNA pathway components might further contribute to the maintenance of active transcription at these loci (Pane, 2011).

Mutations in cuff cause a progressive loss of germ cells, including stem cells, over time. In addition, previous studies have implicated germline Piwi in the control of germ-cell division rate, while somatic Piwi is required for stem-cell self-renewal. The current data strongly suggest that stem- and germ-cell loss in the cuff mutant is caused by a failure to express the piRNA master loci. In the absence of primary piRNAs, the entire piRNA machinery is dispersed and Piwi fails to accumulate in the stem- and germ-cell nuclei, thus further contributing to the cuff phenotype (Pane, 2011).

piRNA-based mechanisms appear to be conserved across the phyla and homologues of the Piwi-clade Argonaute proteins are present in organisms as distant as flies and mouse. Similarly, members of the Rai1 superfamily can be found both in unicellular organisms and in higher Eukaryotes. Therefore, it is conceivable that specific Rai1 and HP1 variants might exist in other organisms and, similar to Drosophila, their interaction might ensure the maintenance of genome integrity during stem- and germ-cell division (Pane, 2011).

Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing

Small noncoding RNAs that associate with Piwi proteins, called piRNAs, serve as guides for repression of diverse transposable elements in germ cells of metazoa. In Drosophila, the genomic regions that give rise to piRNAs, the so-called piRNA clusters, are transcribed to generate long precursor molecules that are processed into mature piRNAs. How genomic regions that give rise to piRNA precursor transcripts are differentiated from the rest of the genome and how these transcripts are specifically channeled into the piRNA biogenesis pathway are not known. This study found that transgenerationally inherited piRNAs provide the critical trigger for piRNA production from homologous genomic regions in the next generation by two different mechanisms. First, inherited piRNAs enhance processing of homologous transcripts into mature piRNAs by initiating the ping-pong cycle in the cytoplasm. Second, inherited piRNAs induce installment of the histone 3 Lys9 trimethylation (H3K9me3) mark on genomic piRNA cluster sequences. The heterochromatin protein 1 (HP1) homolog Rhino binds to the H3K9me3 mark through its chromodomain and is enriched over piRNA clusters. Rhino recruits the piRNA biogenesis factor Cutoff to piRNA clusters and is required for efficient transcription of piRNA precursors. It is proposed that transgenerationally inherited piRNAs act as an epigenetic memory for identification of substrates for piRNA biogenesis on two levels: by inducing a permissive chromatin environment for piRNA precursor synthesis and by enhancing processing of these precursors (Le Thomas, 2014).

A previous study and the current results reveal an essential role for a maternally transmitted transgenerationally inherited cytoplasmic factor in the generation of piRNAs. de Vanssay (2012) showed that a maternal factor supplied to the progeny by females expressing piRNAs from the T1 locus activates piRNA generation from the homologous inactive BX2 locus. Furthermore, maintaining the activity of T1 in the subsequent generation also requires the maternal factor. This observation was extended to other systems, and it was shown that generation of piRNAs from a single-copy transgene inserted into a telomeric piRNA cluster also depends on a maternally transmitted cytoplasmic factor. This maternal factor also activates piRNA generation from a single-copy euchromatic sequence, which simultaneously becomes the target of repression and the source of new piRNAs. Finally, the activity of endogenous clusters in D. melanogaster also seems to require a maternally inherited factor: An analysis of previously published piRNA profiles in interspecies hybrids between D. melanogaster females and Drosophila simulans males showed that only D. melanogaster piRNA clusters generate piRNAs, while D. simulans piRNA clusters are inactive (Le Thomas, 2014).

What is the nature of the maternally supplied epigenetic factor that triggers piRNA generation in the progeny? Multiple lines of evidence point to piRNAs themselves as the carriers of this epigenetic signal. First, as initially shown by de Vanssay (2012), the epigenetic signal produced by the T1 locus does not require inheritance of the locus itself, indicating that the signal has a nonchromosomal nature. This eliminates the possibility that the signal is any kind of chromatin mark linked to the active locus. Second, the process of BX2 activation genetically depends on piRNA pathway genes but is independent of Dicer, which is required for siRNA biogenesis (de Vanssay, 2012). Third, both piRNAs and Piwi proteins are inherited from the maternal germline to the early embryos, while piRNAs are not transmitted through the sperm. Finally, piRNAs can be sequence-specific guides to identify and activate homologous loci. Importantly, recent studies have shown that piRNAs and the nuclear Piwi protein trigger installation of the H3K9me3 mark on homologous targets, providing a possible mechanism by which inherited piRNAs could lead to chromatin changes. Together, these results strongly support the role of inherited piRNAs as a transgenerationally transmitted epigenetic signal that activates piRNA generation from homologous loci in the progeny (Le Thomas, 2014).

How can transgenerationally inherited piRNAs activate piRNA generation from homologous loci? The results imply two mechanisms that cooperate and work at different steps of piRNA biogenesis. In the cytoplasm, transgenerationally inherited piRNAs activate processing of complementary transcripts by the ping-pong amplification loop, as evidenced by a dramatic increase in piRNAs generated by the ping-pong processing upon MD of cognate piRNAs (Le Thomas, 2014).

In the ping-pong processing, initial piRNAs guide generation of secondary piRNAs from complementary sequences. Previously, it was proposed that the ping-pong cycle requires two types of piRNA precursors: cluster transcripts and transcripts from active transposons provided in trans. The current results indicate that the ping-pong cycle can be activated by inherited piRNAs derived from the very same locus, provided that it is bidirectionally transcribed. Importantly, with the exception of one locus, all major piRNA clusters in the D. melanogaster germline are transcribed from both genomic strands, providing an abundant source of complementary transcripts to be used by the ping-pong process (Le Thomas, 2014).

The major players in ping-pong processing in Drosophila are two Piwi proteins, AUB and AGO3, while the third Piwi protein, PIWI itself, is not involved in this process. AUB and AGO3 colocalize in cytoplasmic nuage granules, where the ping-pong processing is believed to take place. Therefore, the effect of inherited piRNAs on ping-pong processing impacts a late step of piRNA biogenesis after piRNA precursor transcripts are exported to the cytoplasm (Le Thomas, 2014).

Although enhancing the ping-pong processing is clearly an important mechanism by which transgenerationally inherited piRNAs boost piRNA biogenesis, it cannot explain all of the observations, suggesting the existence of another mechanism. This study found that maternal piRNAs are also required for the biogenesis of PIWI-associated piRNAs, although those are not generated by ping-pong processing. Using several genetic systems, it was shown that inheritance of piRNAs leads to an increase of the H3K9me3 mark on regions homologous to the piRNAs. Importantly, acquisition of the H3K9me3 mark by genomic regions that did not previously produce piRNAs triggered piRNA generation in two transgenic systems. In contrast, the absence of inherited piRNAs led to a decreased H3K9me3 signal on homologous regions and a concomitant decrease of the corresponding piRNAs. These results suggest that modification of the chromatin structure of homologous genomic regions is the other mechanism by which transgenerationally inherited piRNAs turn on piRNA biogenesis in the progeny. Counterintuitively, it was found that enrichment of the H3K9me3 mark, which is generally assumed to be repressive, strongly correlates with enhanced piRNA biogenesis. In agreement with these results, a previous study showed that biogenesis of piRNAs from double-stranded clusters requires Eggless/SETDB1, one of the methyltransferases responsible for installation of the H3K9me3 mark (Le Thomas, 2014).

Analysis of several transgenic piRNA clusters revealed differences in the impact of inherited piRNAs on the level of the H3K9me3 mark. The inherited piRNAs seem indispensable to maintain high H3K9me3 signal on the transgenic T1 and BX2* loci. However, the absence of maternal piRNAs leads to a relatively mild decrease in H3K9me3 on the telomeric piRNA cluster in the P1152 strain. These results indicate that natural piRNA clusters are able to maintain a certain level of the H3K9me3 mark in a piRNA-independent fashion. This is not unexpected, as natural piRNA clusters are located close to heterochromatin, which is known to have a high level of H3K9me3 signal. In contrast, the T1 and BX2 transgenes are inserted in a euchromatic site that is normally lacking this mark. Overall, the data strongly support an essential role of the H3K9me3 mark in piRNA generation. They further reveal that enrichment of this mark on regions that generate piRNAs at least partially depends on the inheritance of homologous piRNAs from the previous generation. Finally, acquisition of the H3K9me3 mark by a naive locus as a result of exposure to homologous piRNAs strongly correlates with the initiation of de novo primary piRNA biogenesis from such a locus. The exact mechanism for piRNA-dependent deposition of the H3K9me3 mark on piRNA regions remains to be elucidated; however, recent studies suggest that it might occur through recognition of nascent transcripts by the nuclear PIWI/piRNA complex, which is known to be deposited by the mother into the developing egg and has been shown to install H3K9me3 on its genomic targets (Le Thomas, 2014).

The proposal that inherited piRNAs trigger piRNA biogenesis by changing the chromatin structure of homologous sequences raises the question of how the piRNAs distinguish a genuine transposon, a target that needs to be silenced, from a piRNA cluster that needs to be activated. Surprisingly, the results indicate that targeting by piRNAs leads to simultaneous repression of the target and activation of piRNA biogenesis from the same sequence. It was found that targeting of a unique sequence by piRNAs triggers accumulation of the H3K9me3 mark, a decrease in target expression, and activation of piRNA biogenesis. Importantly, the target-derived piRNAs are not generated by the ping-pong mechanism (which would be a trivial explanation), as they are present in complex with PIWI, which does not participate in ping-pong processing. The similarity between transposon targets and piRNA-producing regions is supported by recent work that demonstrated that new transposon insertions in euchromatin start to generate piRNAs; i.e., they are becoming de novo piRNA clusters. Indeed, careful consideration suggests that the requirement to 'silence' a genuine transposon target versus 'activate' a piRNA cluster is a false dichotomy: If nascent transcripts generated from piRNA target loci are channeled into the piRNA processing machinery instead of the standard mRNA processing pathway, the transcript will be effectively silenced, since no full-length mRNA will accumulate. The idea that the target of piRNA repression becomes a source of new piRNAs makes the distinction between piRNA clusters (source of piRNAs) and targets obsolete. Furthermore, the results expose a case in which the same genomic region is 'silenced' and 'activated' at the same time, depending on the exact output the researcher is looking at (generation of full-length mRNA or piRNAs). Similar phenomena might be more widespread than previously suspected, as studies in yeast suggest a very similar model in which centromeric repeats are 'silenced' and generate siRNAs at the same time (Le Thomas, 2014).

How can the high level of the allegedly repressive H3K9me3 mark enhance piRNA biogenesis? The results show that the H3K9me3 mark provides a platform for the binding of Rhino, a chromodomain protein that shows specific enrichment over piRNA clusters. As high levels of the H3K9me3 mark are also present in other genomic regions, it is possible that recognition of H3K9me3 is not sufficient for Rhino’s stable binding and that it interacts with other proteins to achieve its localization on chromatin of double-stranded clusters. Rhino forms a complex with Cutoff, a protein that is also required for piRNA biogenesis. The H3K9me3 mark, Rhino, and Cutoff colocalize at double-stranded piRNA clusters, and Cutoff is de-localized from nuclear foci in rhino mutants, suggesting that it is recruited to piRNA clusters through its interaction with Rhino. Taken together, these results suggest that Rhino and Cutoff, which were previously shown to be indispensable for piRNA generation from double-stranded piRNA clusters, are recruited to cluster chromatin through the H3K9me3 mark (Le Thomas, 2014).

The exact molecular mechanism by which the Rhino/Cutoff complex activates piRNA biogenesis in the nucleus remains to be elucidated; however, two not necessarily mutually exclusive hypotheses can be proposed. First, Cutoff might bind and target nascent transcripts generated from piRNA clusters to the piRNA processing machinery instead of the normal pre-mRNA processing. In support of this idea, this study found that the association of Cutoff with chromatin is RNA-dependent. It has been shown previously that inserting intron-containing heterologous gene sequences into piRNA clusters results in abundant piRNAs from both the exonic and intronic sequences, indicating that normal splicing is perturbed. According to the second hypothesis, the Rhino/Cutoff complex might enhance transcription of piRNA clusters, hence providing more precursors for piRNA biogenesis. Indeed, the run-on experiment showed that Rhino is required for efficient transcription of dual-stranded piRNA clusters. Furthermore, in agreement with an effect on transcription, this study found that, in the cutoff mutant, both siRNAs and piRNAs as well as long RNAs are eliminated from double-stranded piRNA clusters, arguing against a role of Cutoff exclusive to piRNA processing (Le Thomas, 2014).

The counterintuitive idea that the H3K9me3 mark might enhance rather than suppress transcription through binding of nonconventional epigenetic 'readers' has interesting parallels in yeast. In Schizosaccharomyces pombe, H3K9 methylation induces binding of Swi6/HP1, which then recruits the Jumonji protein Epe1 that promotes nucleosome turnover, resulting in increased transcription of heterochromatic repeats and generation of siRNAs. One possible mechanism by which Cutoff might enhance cluster transcription is by suppressing RNA polymerase II (Pol II) termination. Indeed, transgenic insertions that contain polyA cleavage/termination signals into piRNA clusters generate piRNAs downstream from the polyA signal, indicating that not only splicing but transcription termination is also suppressed in piRNA clusters. Ignoring transcription termination signals is likely an important feature of piRNA clusters, as otherwise, multiple signals within transposon sequences present in the clusters would terminate transcription and disrupt piRNA generation (Le Thomas, 2014).

Overall, these data revealed that regions that produce piRNAs in Drosophila germ cells are defined by the epigenetic process of the transgenerational inheritance of cognate small RNAs. It was found that inherited piRNAs trigger piRNA generation in the progeny by two mechanisms that seem to work simultaneously and cooperate to shape the final piRNA population. In the nucleus, inherited piRNAs mark genomic regions that will give rise to new piRNAs and enhance early steps of piRNA biogenesis. In the cytoplasm, inherited piRNAs further trigger the post-transcriptional processing of cluster transcripts through the ping-pong amplification loop (Le Thomas, 2014).

Transcription termination by nuclear RNA polymerases

Argonaute proteins of the PIWI clade are central to transposon silencing in animal gonads. Their target specificity is defined by 23-30 nt PIWI interacting RNAs (piRNAs), which mostly originate from discrete genomic loci termed piRNA clusters. This study shows that a complex composed of Rhino, Deadlock, and Cutoff (RDC) defines dual-strand piRNA clusters genome-wide in Drosophila ovaries. The RDC is anchored to H3K9me3-marked chromatin in part via Rhino's chromodomain. Depletion of Piwi results in loss of the RDC and small RNAs at a subset of piRNA clusters, demonstrating a feedback loop between Piwi and piRNA source loci. Intriguingly, profiles of RNA polymerase II occupancy, nascent transcription, and steady-state RNA levels reveal that the RDC licenses noncanonical transcription of dual-strand piRNA clusters. Likely, this process involves 5' end protection of nascent RNAs and suppression of transcription termination. These data provide key insight into the regulation and evolution of piRNA clusters (Mohn, 2014).

The data indicate that the Rhi-Del-Cuff complex acts as the central licensing factor for dual-strand piRNA cluster transcription in Drosophila. This discussion focuses on two aspects that emerge from this work. (1) The molecular function of the RDC, and (2) the role of Piwi in specifying piRNA source loci and resulting implications for maternally deposited Piwi (Mohn, 2014).

Rhi, Del, and Cuff form an interdependent protein complex that occupies hundreds of mostly heterochromatic loci in ovarian germline cells. Loss of any individual RDC factor results in loss of transcription at dual-strand piRNA source loci and in downstream distortions of piRNA populations and TE silencing (Mohn, 2014).

At the molecular level, Rhi appears to be an adaptor that anchors Del and Cuff to H3K9 methylated chromatin via its chromodomain. While required, it is unlikely that H3K9me3 is sufficient for RDC recruitment as only a subset of H3K9me3-enriched domains are occupied by the RDC. Further, Su(var)205 is highly expressed in germline cells and occupies H3K9me3 domains genome-wide, suggesting that additional factors or chromatin marks exist that allow Rhi to compete against Su(var)205 (Mohn, 2014).

Rhi's chromo-shadow domain interacts with Del, which-due to lack of any recognizable domains-might act as a flexible linker between Rhi and Cuff. Intriguingly, Cuff is related to the Rai1/Dom3Z family of proteins. This is highly interesting in light of the transcriptional patterns at piRNA source loci: the data strongly point toward a central role of the RDC in preventing termination of RNA Pol II, either from flanking transcription units or from atypical promoters. Termination of Pol II transcription occurs in a poorly understood process but is typically linked to endonucleolytic cleavage of the nascent RNA at poly(A) signals by the cleavage and poly-adenylation specificity factor (CPSF) complex (reviewed in Richard, 2009). While RNA Pol II continues transcription, the cleaved and 5' phosphorylated end of the nascent RNA is targeted by the Rat1/Xrn2 5'-to-3' exonuclease. Upon reaching the elongating Pol II, Xrn2 has been postulated to elicit transcription termination . A central step in preventing termination would therefore be the protection of the nascent RNA's 5' end against degradation. It is speculated that Cuff is centrally involved in this process as it resembles a catalytically inactive Dom3Z/Rai1 paralog. Cuff's binding to the nascent RNA 5' end would prevent RNA degradation and would uncouple transcription from the CBC. This in turn would result in noncanonical Pol II transcription, consistent with RDC licensed transcription ignoring poly(A) sites and splice sites (Mohn, 2014).

While the Pol II scavenging model predicts that Cuff stabilizes 5' phosphorylated transcripts, it is noted that Cuff likely also acts on nascent RNA 5' ends without prior CPSF-mediated cleavage, such as would be the case at the promoters of cluster 38C or the Hsp70A genes. It is speculated that Cuff competes with the capping enzyme or with the CBC for the nascent RNA 5' end. Furthermore, it is also conceivable that Cuff protects 5' ends of spurious noncanonical initiation events within RD-SL. In particular, at large heterochromatic piRNA-SL such as 42AB, which encompass nearly 250 kb, internal initiation events would ensure steady piRNA generation over the entire length (Mohn, 2014).

Taken together, dual-strand piRNA source loci are not hardwired transcription units but are dynamic features of heterochromatin that depend on their heterochromatic nature and the transcriptional status of nearby loci. Considering this, dual-stranded transcription of RD-SL appears to be a consequence of, rather than a requirement for, RDC binding (Mohn, 2014).

The data indicate that Piwi is involved in the definition of piRNA source loci, likely by guiding H3K9 methylation that is required for RDC recruitment. This was particularly evident for stand-alone euchromatic TE insertions. In contrast, most RD-SL within pericentromeric heterochromatin and the major dual-strand piRNA clusters were not or only mildly affected upon Piwi depletion. This resembles findings from S. pombe where concepts of heterochromatin initiation and maintenance have been established and where loci also depend differentially on the RITS complex. Probably, more robust maintenance pathways for chromatin patterns exist within heterochromatin versus euchromatin. Of note, mutations in the Drosophila H3K9 methyl-transferase SETDB1 result in widespread defects in piRNA cluster transcription (Mohn, 2014).

The fact that Piwi can impact RDC recruitment indicates that evolution of piRNA source loci is much more dynamic than previously anticipated. Any TE can act as a piRNA source as long as it inserts at a favorable position and is transcribed to provide a piRNA target. Piwi therefore has a dual function in the germline: First, it guides transcriptional silencing of TE promoters. Second, it permits low-level transcription of TE sequences and specification of emerging transcripts as piRNA precursors via the RDC (Mohn, 2014).

Piwi-piRNA complexes are maternally deposited into the developing egg and are abundant at the posterior pole where future primordial germ cells will form. Given its role in RDC guidance, maternal Piwi might in fact be the central factor to specify piRNA source loci during early development. Subsequently, heterochromatin-and thus RDC occupancy-at most sites is probably maintained independent of Piwi (Mohn, 2014).

Hybrid dysgenesis and paramutation, two epigenetic TE repression phenomena, have been linked to maternally deposited piRNA populations in Drosophila. The findings that Piwi can specify genomic piRNA sources are probably central for the further understanding of the molecular events underlying these phenomena (Mohn, 2014).

The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing

piRNAs guide an adaptive genome defense system that silences transposons during germline development. The Drosophila HP1 homolog Rhino is required for germline piRNA production. Rhino is shown to bind specifically to the heterochromatic clusters that produce piRNA precursors; binding directly correlates with piRNA production. Rhino colocalizes to germline nuclear foci with Rai1/DXO-related protein Cutoff (Cuff) and the DEAD box protein UAP56, which are also required for germline piRNA production. RNA sequencing indicates that most cluster transcripts are not spliced and that rhino, cuff, and uap56 mutations increase expression of spliced cluster transcripts over 100-fold. LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger piRNA production from a trans combination of sense and antisense reporters. It is therefore proposed that Rhino anchors a nuclear complex that suppresses cluster transcript splicing and it is speculated that stalled splicing differentiates piRNA precursors from mRNAs (Zhang, 2014).

The piRNA pathway has an evolutionarily conserved role in transposon control during germline development and is essential for transmission of the inherited genetic complement. In the Drosophila ovary, unique piRNAs are concentrated in 'clusters' composed of complex arrays of nested transposon fragments that are generally localized to pericentromeric or subtelomeric heterochromatin. These loci fall into two classes, based on strand bias. Clusters that produce piRNAs from both genomic strands (dual-strand clusters) are dominant in the germline, while clusters that are expressed on only one genomic strand (uni-strand clusters) produce most of the piRNAs in somatic follicle cells that surround the germline. Primary piRNAs from dual-strand clusters, bound to Piwi proteins, appear to drive a ping-pong cycle that amplifies the silencing RNA pool. Primary piRNAs that initiate the amplification cycle, by definition, are produced by a ping-pong-independent mechanism. Similarly, ping-pong amplification is not required for production of piRNAs from uni-strand clusters. These observations suggest a simple model in which primary piRNAs from uni-strand and dual-strand clusters are produced by a common mechanism, and dual-strand clusters are equivalent to convergently transcribed uni-strand clusters. However, uni-strand cluster piRNA production is independent of rhi, uap56, and cuff, which are essential for production of piRNAs that map uniquely to dual-strand clusters. In addition, this study showed that Rhi-dependent piRNA production from an ectopic locus requires a combination of transgenes expressing complementary transcripts. Primary piRNA production by dual-strand and uni-strand clusters thus appear to proceed by distinct mechanisms (Zhang, 2014).

These findings also suggest that piRNA production by dual-strand clusters requires complementary precursors. The role of complementary RNAs in the germline piRNA biogenesis pathway, however, remains to be determined (Zhang, 2014).

piRNA pathway mutations increase expression of a subset of transposons by over 200-fold, but do not alter germline gene expression. This remarkable specificity is almost certainly essential to gamete production, but how piRNA precursors are differentiated from mRNAs is not understood. The vast majority of protein coding premRNAs are efficiently spliced, exported from the nucleus, and translated in the cytoplasm. By contrast, splicing is suppressed at a transgene inserted into the Drosohila X-TAS piRNA cluster, and transcriptome wide studies indicate that rapidly evolving HP1 homolog Rhi, the Rai1-related protein Cuff, and the DEAD box protein UAP56 suppress slicing at resident consensus donor and acceptor sites in germline clusters. This is most clearly illustrated at the sox102F locus, which produces efficiently spliced pre-mRNAs in the soma but is the source of piRNAs from unspliced primary transcripts in the germline. Significantly, accumulation of both unspliced transcripts and piRNAs requires rhi, cuff, and uap56, and tethering a LacI::Rhi fusion to a intron-containing reporter transgene suppresses splicing and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. It is therefore proposed that Rhi functions with Cuff and UAP56 to suppress cluster transcript splicing and that the stalled splicing intermediates are the precursors for primary piRNAs (Zhang, 2014).

Cuff is a homolog of Rai1/DXO, which binds and degrades mRNAs carrying incomplete cap structures. This would appear to support a role for Cuff in destabilizing spliced cluster transcripts, but the residues required for the 5' to 3' exonuclease activity of Rai1/DXO are not conserved in Cuff. Murine DXO has been cocrystallized with an uncapped RNA, and with a cap analog. The crystal structures reveal the protein residues that interact with the RNA backbone, and indicate that the cap is bound in a pocket in the interior of the protein. The sequences of Drosophila Cuff was aligned with the murine DXO. Twenty one percent of the positions are identical, and conserved amino acids are present throughout the entire alignment. Overall protein fold of Cuff is therefore highly likely to resemble DXO. Therefore the Modeler and I-TASSER algorithms were used to build a homology model of Cuff based on the Murine DXO structures. Essentially, all of the RNA-binding interactions are preserved in the homology model of Cuff. It is therefore proposed that Cuff is not a catalytically active nuclease, but binds to uncapped RNA ends as they emerge from Pol2 or to newly capped cluster transcripts (Zhang, 2014).

Cap binding by the nuclear Cap Binding Complex (CBC) is required for splicing and polyadenylation. It is therefore proposed that Cuff binds to Rhi and cotranscriptionally associates with cluster transcripts, preventing capping or recognition of capped RNAs by the CBC, which blocks splicing. Previous work has shown that UAP56 immunoprecipitation significantly enriches for cluster transcripts, not for mRNAs, and that RNAs mapping to the major 42AB cluster are the most highly enriched species in the immunoprecipitated pool. The point mutation in uap56 that specifically blocks piRNA biogenesis disrupts a salt bridge predicted to stabilize the ATP and RNA bound form of the protein. These observations suggest that stable cluster transcript binding by UAP56 is required for piRNA biogenesis. Mutations in the yeast cap-binding complex lead to arrest at an early step in the splicing pathway, with the U2 snRNP bound to primary transcripts. UAP56 was identified as a binding partner of the U2 snRNP protein U2AF65 (Zhang, 2014).

Cuff binding to capped cluster transcripts may therefore prevent cap recognition by the CBC, arresting splicing with UAP56 stably bound. This aberrant stable complex could differentiate piRNA precursors from pre-mRNAs. While this model is highly speculative, it makes several clear predictions and should therefore serve as a useful starting point to additional studies. Adaptation to transposon invasion by the piRNA pathway appears to be initiated by insertion of the invading element into a cluster. This speculate model, with the observation that Rhi can spread from anchor sites, suggest an adaption model in which Rhi spreads into active transposons that insert into clusters, leading to Cuff binding to capped transcripts from transposon promoters. This would block processing and promote production of new piRNAs, thus coordinately silence the inserted element and produce the transsilence species that control dispersed active elements (Zhang, 2014).

Studies in the pathogenic yeast Crypotoccous provide evidence for a direct link between stalled splicing and transposon silencing by the siRNA pathway. It has been shown that splicing factors associate with the Crypotoccous siRNA biogenesis machinery and siRNAs are produced from unspliced transposon transcripts. In addition, intron removal reduces siRNA production, and splice site mutations that reduce splicing efficiency increase siRNA production. Furthermore, recent genome-wide screens have implicated splicing factors in transposon silencing, and the splicing and small RNA-silencing pathways appears to be coevolving. These findings, with the the current studies, suggest that stalled splicing generates a conserved molecular signature for potentially deleterious RNAs, which directs these transcripts to small silencing RNA biogenesis pathways. Retrotransposons and retroviruses encode essential spliced transcripts, but splicing must be suppressed to produce full-length genomic RNAs. This novel feature of the retroviral life cycle may have driven evolution of silencing systems that use stalled splicing as a hallmark of pathogenic RNAs (Zhang, 2014).

Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster

In Drosophila, 23-30 nt long PIWI-interacting RNAs (piRNAs) direct the protein Piwi to silence germline transposon transcription. Most germline piRNAs derive from dual-strand piRNA clusters, heterochromatic transposon graveyards that are transcribed from both genomic strands. These piRNA sources are marked by the heterochromatin protein 1 homolog Rhino (Rhi), which facilitates their promoter-independent transcription, suppresses splicing, and inhibits transcriptional termination. This study reports that the protein Maelstrom (Mael) represses canonical, promoter-dependent transcription in dual-strand clusters, allowing Rhi to initiate piRNA precursor transcription. Mael also represses promoter-dependent transcription at sites outside clusters. At some loci, Mael repression requires the piRNA pathway, while at others, piRNAs play no role. It is proposed that by repressing canonical transcription of individual transposon mRNAs, Mael helps Rhi drive non-canonical transcription of piRNA precursors without generating mRNAs encoding transposon proteins (Chang, 2018).

Fly piRNA clusters must solve a gene expression paradox. They record the ancient and contemporary exposure of the animal to transposon invasion, and this information must be copied into RNA in order to generate protective, anti-transposon piRNAs. However, recent transposon insertions retain the ability to produce mRNA encoding proteins required for their transposition. In flies, dual-strand piRNA clusters solve this paradox by using Rhi to initiate non-canonical transcription of unspliced RNA from both genomic strands, generating piRNA precursors, while repressing promoter-initiated, canonical transcription. These data suggest that Mael is required for this second process, allowing dual-strand piRNA clusters to safely generate piRNA precursor transcripts without risking production of transposon mRNAs. Within dual-strand clusters, Mael is likely guided to its targets by the piRNA pathway. However, the current analyses also predict that for some loci, Mael functions to repress canonical transcription in heterochromatin separately from the piRNA pathway, probably via one or more proteins that direct Mael to specific genomic sites (Chang, 2018).

In maelM391/r20 ovaries, piRNAs mapping to dual-strand clusters decrease, despite a concomitant increase in canonical transcription from these same loci. The data suggest that in maelM391 mutants, Rhi-mediated non-canonical transcription, cluster transcript export, and ping-pong amplification become uncoupled. Perhaps, canonical transcription and Rhi-mediated non-canonical transcription compete for Pol II. Instead of fueling piRNA production, the canonical transcripts from dual-strand piRNA clusters produced in the absence of Mael are translated into protein. Mael therefore contributes to dual-strand cluster piRNA production by tipping the balance toward non-canonical transcription (Chang, 2018).

The data indicate that in addition to relying on the piRNA pathway, Mael can also be guided to its targets by piRNA-independent mechanisms. Moreover, piRNA-dependent repression by Mael may be widespread outside of flies: although Drosophila melanogaster piwi is found only in the gonads, piwi is expressed broadly in the soma of most arthropods. In fact, 12 arthropods with somatic piRNAs also express mael in the soma, while 3 arthropods with no detectable piRNAs outside the gonads have low or undetectable somatic mael mRNA (Chang, 2018).

In male mice, loss of MAEL also leads to loss of piRNAs, germline transposon derepression, and sterility. As in flies, loss of MAEL in mice does not trigger loss of heterochromatin: DNA methylation of L1 elements is unchanged (Chang, 2018).

Because Mael is conserved from protists to humans, it is hypothesized that in different organisms Mael may be co-opted by different pathways to repress transcription of various targets. Prior studies suggest a model for how Mael confers repression. In protists, the MAEL domain was predicted to degrade RNA and may directly destroy nascent transcripts. In insects, the MAEL domain interacts with single-stranded RNA; it is speculated that fly Mael may have retained a role in destabilizing RNA. In this view, Mael may promote premature termination or degradation of nascent transcripts. In addition, because fly Mael has a partial HMG domain, it may also directly bind to DNA and repress transcription by preventing canonical core transcription factors from binding to promoters. Another possibility is that fly Mael may play a role in establishing or maintaining chromatin modifications not monitored in this study. Consistent with all of these possible mechanisms, non-canonical transcription mediated by Rhi is expected to be unaffected by Mael because the Rhi allows transcriptional initiation in dual-strand clusters without need for promoters and prevents degradation of unspliced piRNA precursor transcripts (Chang, 2018).

Adaptive evolution targets a piRNA precursor transcription network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription (Parhad, 2020).

Transposable elements (TEs) are major genome components that can induce mutations and facilitate ectopic recombination, but transposons have also been co-opted for normal cellular functions, and transposon mobilization has rewired transcriptional networks to drive evolution. Species survival may therefore depend on a balance of transposon silencing and activation. The Piwi interacting RNA (piRNA) pathway transcriptionally and post-transcriptionally silences transposons in the germline. However, how this pathway is regulated is not completely understood (Parhad, 2020).

In Drosophila, piRNAs are produced from heterochromatic clusters composed of complex arrays of nested transposon fragments, which appear to provide genetic memory of past genome invaders. Adaptation to new genome invaders is proposed to involve transposition into a cluster, which leads to sequence incorporation into precursors that are processed into trans-silencing anti-sense piRNAs. However, a subset of isolated transposon insertions also produce sense and anti-sense piRNAs, providing an independent adaptation mechanism and epigenetic memory of genome invaders. Expression of piRNAs from these loci is disrupted by piwi mutations, but Piwi-bound piRNAs map to all insertions, not just the subset that function in piRNA biogenesis. The mechanism that defines these 'mini-cluster' thus remains to be determined (Parhad, 2020).

In Drosophila, germline piRNA clusters and transposon mini-clusters are bound by the RDC complex, which is composed of the HP1 homolog Rhino (Rhi), which co-localizes with the linker protein Deadlock (Del) and the Rai1 homolog Cutoff (Cuff). The three components of the RDC are co-dependent for localization to clusters and essential to germline piRNA production. Rhi is composed of chromo, hinge, and shadow domains. The chromo domain binds to H3K9me3-modified histones, and the shadow domain binds Del, which recruits Moonshiner (Moon) and TATA box binding protein-related factor 2 (TRF2), promoting 'non-canonical' transcription from both genomic strands (Parhad, 2020).

The third RDC component, Cuff, was identified in a screen for female sterile mutations and found to encode a homolog of the decapping exonuclease Rai1 required for transposon silencing and piRNA biogenesis. Critical residues in the catalytic pocket of Rai1 are not conserved in Cuff, but sidechains that bind the RNA backbone are retained, suggesting that Cuff may be an RNA 5' end-binding protein. Intriguingly, germline piRNAs in Drosophila are preferentially produced from unspliced transcripts, and cuff mutations significantly increase piRNA precursor splicing, and 5' cap binding by the nuclear cap binding complex (CBC) promotes splicing. Together, these findings suggest that that Cuff competes with the CBC for binding to capped cluster transcripts, suppressing splicing and promoting piRNA biogenesis. However, tethering Cuff to a reporter transcript increases read-through transcription, consistent with suppression of transcription termination. The molecular function for Cuff in piRNA biogenesis thus remains enigmatic (Parhad, 2020).

All three RDC genes are rapidly evolving under positive selection, suggesting that adaptive evolution of the complex is driven by a genetic conflict with the transposons the piRNA pathway silences, but other mechanisms are possible. Previous work found that rapid evolution has modified the Rhi-Del interface, producing orthologs that function as mutant alleles when moved across species. Analysis of these cross-species incompatibilities defined an interaction between the Rhi shadow domain and Del that prevents ectopic assembly of piRNA cluster chromatin. Crosses between Drosophila melanogaster and Drosophila simulans, which are sibling species, lead to hybrid sterility and are important model for genetic control of reproductive isolation. Significantly, sterile hybrids between these species phenocopy piRNA pathway mutations. Adaptive evolution of piRNA pathway genes may therefore contribute to reproductive isolation and speciation (Parhad, 2020).

These findings also suggest that cross-species analysis of rapidly evolving genes may provide a powerful genetic approach to structure-function analysis. This study applies this approach to cuff. These studies indicate that adaptive evolution has targeted direct or indirect interactions among Cuff, the Del-TRF2 non-canonical transcriptional complex, and the transcriptional co-repressor C-terminal binding protein (CtBP). CtBP was first identified as a host binding partner of Adenovirus E1A and subsequently implicated in diverse developmental pathways and cancer. This study shows that Drosophila CtBP suppresses canonical transcription from promoters in transposon terminal repeats and from promoters flanking two major germline piRNA clusters. Significantly, in both contexts, activation of canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution has therefore targeted interactions between Cuff and two transcription regulators, which coordinately control germline piRNA expression (Parhad, 2020).

Adaptive evolution is a hallmark of genes engaged in a genetic conflict, which typically leads to co-evolution of host-pathogen gene pairs that encode interacting proteins. However, pathogens can also produce mimics that target interactions within host defense systems, raising the possibility that adaptation can also remodel interaction between host proteins. Supporting the possibility, adaptive evolution has remodeled an interface between the Rhi and Del, which are core components of the host transposon defense machinery. These adaptive changes prevent gene function across closely relates species and define an interaction that is required to restrict the RDC to piRNA clusters, which defines the specificity of the transposon silencing machinery. These findings suggest that adaptive evolution targets important functional domains, which can be functionally analyzed using cross-species complementation. This approach is applied to the third RDC component, cuff and shows that adaptive evolution targets interactions between this Rai1 homolog and proteins that coordinate canonical and non-canonical piRNA cluster transcription and piRNA biogenesis (Parhad, 2020).

Transposon silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, and Cuff co-localizes with Rhi and Del at these piRNA source loci. Rhi binds to H3K9me3 marks and recruits Del. Del, in turn, binds Moon, which recruits TRF2 to initiate non-canonical transcription from both genomic strands. In contrast, the current data suggest that Cuff coordinates canonical and non-canonical cluster expression. D. simulans cuff ortholog fails to rescue D. melanogaster cuff mutations and leads to dominant sterility when overexpressed in wild-type flies. Significantly, these phenotypes are associated with stable binding to Del, TRF2, and CtBP. As noted above, Del and TRF2 function in non-canonical transcription of piRNA clusters. CtBP is a conserved transcriptional co-repressor, first identified as a host factor that binds to Adenovirus E1a, and subsequently shown to function in numerous developmental pathways. CtBP does not directly interact with DNA, but binds sequence specific transcription factors and recruits histone-modifying enzymes. This study shows that CtBP-kd activates canonical promoters linked to piRNA source loci. Adaptive evolution has therefore remodeled interactions between Cuff and factors that control both canonical and non-canonical transcription of piRNA precursors loci (Parhad, 2020).

Dominant phenotypes can result from mutations that produce new interactions or functions (neomorphic mutations) and assembly of complexes that are not formed by wild-type proteins. However, the current findings, with previous studies, suggest that substitutions in sim-Cuff stabilize normally transient complexes with both TRF2 and CtBP. In D. melanogaster, Cuff and Del do not co-precipitate, but the proteins co-localize to nuclear foci, interact in two-hybrid assays, and are co-dependent for association with piRNA clusters (Mohn, 2014). Del, in turn, co-precipitates with TRF2 and Moon, and all three proteins are required for non-canonical cluster transcription (Andersen, 2017), but TRF2 does not normally accumulate at clusters. In contrast, overexpression of sim-Cuff drives TRF2 co-localization with the RDC. Similarly, ChIP-seq shows that Cuff and Del localize to canonical promoters that are suppressed by CtBP, but CtBP does not accumulate at these promoters. Substitutions in the sim-Cuff ortholog thus appear to stabilize normally transient associations with Del and TRF2 and with CtBP (Parhad, 2020).

The majority of Drosophila germline clusters are transcribed from internal non-canonical initiation sites and do not have flanking canonical promoters. CtBP-kd does not significantly alter long RNA or piRNA expression from these loci. However, canonical promoters flank the right side of the 42AB cluster and both ends of the 38C cluster, and CtBP-kd increases transcription from these canonical promoters, which is associated with reduced transcription and piRNA production from downstream regions]. It has not been possible directly assay non-canonical transcription at most transposon insertions that produce piRNAs, as the inserted sequences are repeated, but CtBP-kd increases canonical transcription of transposons and is linked to collapse of piRNAs mapping to sequences flanking these insertions. In addition, deletion of the promoters flanking 42AB and 38C leads to spreading of piRNA production into flanking domains (Andersen, 2017). Together, these findings indicate that canonical transcription directly or indirectly represses non-canonical transcription and piRNA production (Parhad, 2020).

On the basis of these findings, it is proposed that Cuff coordinates canonical and non-canonical piRNA precursor transcription. By stabilizing Rhi, Del, Moon and TRF2, Cuff promotes non-canonical transcription. By contrast, Cuff appears to function with CtBP to control canonical transcription. Rescue of cuff mutants with sim-Cuff, which shows enhanced binding to CtBP, is phenocopied by CtBP-kd: both lead to increased canonical transcription. Formation of stable complexes with sim-Cuff thus appears to inhibit CtBP, activating canonical transcription and reducing downstream non-canonical transcription. Normally, the interaction between Cuff and CtBP is weak and free CtBP suppresses canonical promoters, while Cuff functions with Del-TRF2 to drive of non-canonical transcription. It is speculated that this balance may be altered in response to stress or environmental signals, which can activate transposons. Intriguingly, CtBP is also an NADH/NAD binding protein, suggesting that the balance between canonical and non-canonical piRNA precursor transcriptions may be regulated in response to metabolic state (Parhad, 2020).

The RDC proteins Moon and TRF2 are required for piRNA precursor transcription, and all of these factors are rapidly evolving. By contrast, CtBP is conserved from flies to humans, and a putative human oncogene. The data presented here, with earlier analysis of Rhi and Del (Parhad, 2017), indicate that rapid evolution has modified multiple interactions between rapidly evolving proteins in the piRNA biogenesis, and association of these proteins with a highly conserved transcriptional co-repressor. Rapidly evolving genes with specialized functions are frequently the most accessible to phenotype-based forward genetic approaches in model systems, and linking these specialized genes to conserved pathways can be a challenge. The studies reported in this study indicate that cross-species studies can help define these links, bridging the gap between genetically tractable model organisms and human biology (Parhad, 2020).

Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription

The piRNA pathway is a highly conserved mechanism to repress transposon activation in the germline in Drosophila and mammals. This pathway starts from transcribing piRNA clusters to generate long piRNA precursors. The majority of piRNA clusters lack conventional promoters, and utilize heterochromatin- and HP1D/Rhino-dependent noncanonical mechanisms for transcription. However, information regarding the transcriptional regulation of piRNA clusters is limited. This study reports that the Drosophila acetyltransferase Enok, which can activate transcription by acetylating H3K23, is critical for piRNA production from 54% of piRNA clusters including 42AB, the major piRNA source. Surprisingly, it was found that Enok not only promotes rhino expression by acetylating H3K23, but also directly enhances transcription of piRNA clusters by facilitating Rhino recruitment. Taken together, this study provides novel insights into the regulation of noncanonical transcription at piRNA clusters and transposon silencing (Tsai, 2021).

In a wide range of organisms, repressing the activation of transposon insertions is essential for maintenance of genome stability. Small RNA-mediated heterochromatin formation plays important roles in silencing transposons in eukaryotic genomes. Mammals and Drosophila utilize the PIWI-interacting RNA (piRNA) pathway to achieve transcriptional and post-transcriptional silencing of transposons in the germline. In Drosophila, the piRNA pathway starts from transcription of the 142 piRNA clusters, usually ranging from 50 to a few hundred kilobases and containing multiple copies of truncated or full-length transposons, which produce long piRNA precursors. The long RNA precursors would then be processed through slicer- and Zucchini (Zuc)-dependent mechanisms into mature 23–29 nt piRNAs that get loaded to the Piwi protein. In addition, another two PIWI-clade Argonaute proteins, Ago3 and Aubergine (Aub), function in the ping-pong cycle to specifically amplify piRNAs against active transposons. Guided by complementary piRNAs, Ago3 and Aub can mediate degradation of transposon transcripts, and the Piwi-piRNA complex can also direct heterochromatin formation at the loci of transposons and piRNA clusters by recruiting epigenetic factors, resulting in effective repression of transposons both transcriptionally and post-transcriptionally (Tsai, 2021).

The Drosophila piRNA clusters can be divided into two classes: uni-strand, which produces piRNAs mainly from one genomic strand, and dual-strand, which produces piRNAs from both genomic strands. Transcription of the uni-strand clusters is proposed to be similar to the canonical transcription of protein-coding genes, as they contain clear promoter structures with enriched H3K4me2 and peaks of RNA polymerase II (Pol II). In contrast, dual-strand clusters lack clear Pol II promoter regions and are enriched for the heterochromatic H3K9me3 mark. Therefore, these clusters undergo noncanonical transcription that utilizes multiple internal initiation sites via heterochromatin- and Rhino (Rhi)-dependent mechanisms (Tsai, 2021).

Rhi is the germline-specific heterochromatin protein 1D (HP1D), and it associates with Deadlock (Del) and Cutoff (Cuff) to form the RDC complex. The RDC complex is recruited to dual-strand clusters by the interaction between H3K9me3 and the chromodomain of Rhi. At dual-strand clusters, the RDC complex licenses and promotes their transcription through four mechanisms. First, Del interacts with the germline-specific paralog of transcription factor IIA (TFIIA)-L, Moonshiner (Moon), and in turn recruits TFIIA and the TATA-box binding protein (TBP)-related factor TRF2 for transcription initiation. Second, the RDC complex has been shown to suppress the splicing of piRNA cluster transcripts, which is proposed to facilitate piRNA production. Third, Cuff recruits the transcription-export (TREX) complex to nascent transcripts to promote efficient transcription at piRNA clusters. Fourth, Cuff interferes with recruitment of the cleavage and polyadenylation specificity factor (CPSF) complex, and therefore prevents premature termination during transcription of piRNA precursors. While the positive roles of the RDC complex in noncanonical transcription of piRNA clusters were studied extensively, further transcriptional regulation upstream to the recruitment of this complex to piRNA clusters is still unclear (Tsai, 2021).

The KAT6 acetyltransferases are highly conserved from budding yeast to mammals, and preferentially acetylate histone H3 among the four core histones. The fly KAT6, Enok, has been shown to function as the major acetyltransferase for establishing the H3K23ac mark, which plays activating roles in transcription of genes. H3K23ac has been suggested to destabilize the interaction between H3K27me3 and the chromodomain of Polycomb, and therefore may contribute to transcription activation. In the ovarian germline, Enok is important for the maintenance of germline stem cells, and is required for proper polarization of oocytes by promoting expression of the actin nucleator spir. This study reports a novel role for Enok in the piRNA pathway. Mutating or knocking-down enok in the ovarian germline led to derepression of transposons and reduction in levels of piRNAs produced from a subset of piRNA clusters including the major piRNA source 42AB. Enok binds to and acetylates H3K23 in the 5' region of rhi, and is required for its normal expression levels in the ovary. It was further shown that Enok is also required for proper Rhi recruitment to a subset of piRNA clusters to promote their transcription. Therefore, Enok contributes to proper transposon silencing in the germline by promoting transcription of rhi and piRNA clusters (Tsai, 2021).

This paper reports a novel role for Enok in suppressing the activation of transposons in the germline. Loss of functional Enok in the ovarian germline resulted in activation of 7 transposon families. This amount of activated transposon families in enok mutant ovaries is comparable to the 17 families activated in the rhi mutant. RNA-seq analysis showed a ~75% reduction in the mRNA levels of rhi in enok mutant germline clone ovaries as compared with the WT control. Knocking down enok in the ovarian germline using two different UAS-shRNA-enok constructs also reduced the mRNA levels of rhi as compared with two different control fly lines. In addition, Enok ChIP-seq analysis revealed that Enok is localized to the 5' region of rhi, and the Enok-dependent enrichment of H3K23ac at the 5' end of rhi suggests that the Enok-mediated H3K23ac mark promotes rhi expression, contributing to proper piRNA production. Indeed, enok mutant germline clone ovaries showed decreased levels of piRNAs that mapped to Rhi-dependent source loci (RD-SL). However, not all RD-SL showed decreased piRNA levels in enok mutants. About 20% of the 6426 RD-SL showed reduced piRNA levels in enok mutants. Therefore, the remaining 25% of rhi levels in enok mutant ovaries may be sufficient to support transcription of the RD-SL that were not affected by loss of Enok. More strikingly, knocking down enok in the germline, without affecting the global protein levels of Rhi, reduced Rhi occupancies at Enok-dependent source loci (ED-SL) but not at Enok-independent source loci (EI-SL). This result suggests that Enok regulates Rhi recruitment specifically at ED-SL. The enok and rhi mutants show similar effects on the fold changes in transposon family expression and in antisense piRNAs. However, among the top 24 most highly overexpressed families in rhi, loss of Enok in the germline specifically activates 7 families. This specificity suggests that these 7 families may be more sensitive to reductions in Rhi recruitment to a subset of piRNA source loci. Taken together, Enok may contribute to fine-tuning transcription of piRNA clusters by modulating rhi expression and by regulating Rhi recruitment to Enok-dependent piRNA source loci (Tsai, 2021).

Three genome-wide RNAi screens have been reported before, but two of them were specifically performed in ovarian somatic cells. Knocking down enok in ovarian somatic cells using the tj-Gal4 driver did not activate the soma-dominant transposon, Gtwin, suggesting that Enok may be dispensable for transposon silencing in the soma. In the genome-wide screen in the germline, the enok RNAi construct (KK108400) is a long hairpin RNA. The efficiency of knocking down enok by long hairpin RNAs is lower than by short hairpin RNAs in the germline, even in the presence of additional Dicer-2. It has been shown that knocking down enok weakly activated the blood and Burdock transposons (z-scores of -0.5 and -0.74, respectively). However, this activation effect did not reach the threshold (z-score of -1.5 or lower) applied in the screen. This study used two different short hairpin RNA constructs against enok to deplete Enok in the germline, and therefore it was possible to detect stronger activation of transposons, possibly due to better knockdown efficiencies (Tsai, 2021).

Enok is the major enzyme responsible for the abundant H3K23ac mark. It was previously demonstrated that Enok is localized to the 5' end of its target genes, spir and mael, and promotes their expression by acetylating H3K23. This study further reports rhi and a subset of RD-SL (defined as ED-SL) as novel targets that are transcriptionally regulated by Enok. Intriguingly, while the 5' region of rhi is enriched with Enok and H3K23ac, Enok is not enriched at ED-SL relative to EI-SL. Also, knocking down enok in ovaries reduced the H3K23ac levels at the 5' end of rhi but not at piRNA clusters. These results suggest that Enok facilitates rhi expression by acetylating H3K23, but regulates the transcription of ED-SL through other mechanisms. Notably, knocking down enok in the ovarian germline severely reduced the Rhi occupancy to sites in 42AB, while global protein levels of Rhi and the H3K9me3 levels at 42AB were largely unaffected. Therefore, Enok is likely to promote transcription of ED-SL by regulating Rhi recruitment (Tsai, 2021).

The transcription of dual-strand piRNA clusters utilizes noncanonical heterochromatin-dependent internal initiation. Transcription initiation at these clusters was proposed to take place by the H3K9me3-bound RDC complex recruiting the germline-specific paralog of TFIIA-L. This study shows that Enok is important for both Rhi and Pol II occupancies at a subset of RD-SL (defined as ED-SL), suggesting that Enok can facilitate transcription of these piRNA source loci. As Rhi is highly enriched across the entire 42AB cluster and no Enok peaks were detected within 42AB, Enok is unlikely to regulate the Rhi occupancy at 42AB by directly recruiting it. Also, the Co-IP assay failed to detect interaction between Enok and the overexpressed Rhi in ovaries. Interestingly, the HAT activity of Enok is critical for transcription of 42AB even when rhi is overexpressed. Therefore, it is possible that Enok may play a role in acetylating some factors that are required for Rhi recruitment, or it may have an indirect role in Rhi recruitment by promoting expression of other genes with yet unidentified functions in the piRNA pathway. Notably, while knocking down enok in the germline decreased the RNA levels transcribed from both genomic strands at cl1-A and from the sense strand at cl1-32, RNA levels transcribed from the antisense strand at cl1-32 was not affected by depletion of Enok. Thus, within dual-strand clusters, Enok may regulate the internal initiation in specific regions. Taken together, this study provides novel information regarding noncanonical transcription and transposon silencing in the germline (Tsai, 2021).

Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila

Heterochromatin comprises a significant component of many eukaryotic genomes. In comparison to euchromatin, heterochromatin is gene poor, transposon rich, and late replicating. It serves many important biological roles, from gene silencing to accurate chromosome segregation, yet little is known about the evolutionary constraints that shape heterochromatin. A complementary approach to the traditional one of directly studying heterochromatic DNA sequence is to study the evolution of proteins that bind and define heterochromatin. One of the best markers for heterochromatin is the heterochromatin protein 1 (HP1), which is an essential, nonhistone chromosomal protein. This study investigates the molecular evolution of five HP1 paralogs present in Drosophila melanogaster. Three of these paralogs have ubiquitous expression patterns in adult Drosophila tissues, whereas HP1D/rhino and HP1E are expressed predominantly in ovaries and testes respectively. The HP1 paralogs also have distinct localization preferences in Drosophila cells. Thus, Rhino localizes to the heterochromatic compartment in Drosophila tissue culture cells, but in a pattern distinct from HP1A and lysine-9 dimethylated H3. Using molecular evolution and population genetic analyses, it was found that rhino has been subject to positive selection in all three domains of the protein: the N-terminal chromo domain, the C-terminal chromo-shadow domain, and the hinge region that connects these two modules. Maximum likelihood analysis of rhino sequences from 20 species of Drosophila reveals that a small number of residues of the chromo and shadow domains have been subject to repeated positive selection. The rapid and positive selection of rhino is highly unusual for a gene encoding a chromosomal protein and suggests that rhino is involved in a genetic conflict that affects the germline, belying the notion that heterochromatin is simply a passive recipient of 'junk DNA' in eukaryotic genomes (Vermaak, 2006; Full text of article).

Drosophila rhino encodes a female-specific chromo-domain protein that affects chromosome structure and egg polarity

Rhino is a novel member of the Heterochromatin Protein 1(HP1) subfamily of chromo box proteins. rhino (rhi) is expressed only in females and chiefly in the germline, thus providing a new tool to dissect the role of chromo-domain proteins in development. Mutations in rhi disrupt eggshell and embryonic patterning and arrest nurse cell nuclei during a stage-specific reorganization of their polyploid chromosomes, a mitotic-like state called the 'five-blob' stage. These visible alterations in chromosome structure do not affect polarity by altering transcription of key patterning genes. Expression levels of gurken, oskar, bicoid, and decapentaplegic transcripts are normal, with a slight delay in the appearance of bcd and dpp mRNAs. Mislocalization of grk and osk transcripts, however, suggests a defect in the microtubule reorganization that occurs during the middle stages of oogenesis and determines axial polarity. This defect likely results from aberrant Grk/Egfr signaling at earlier stages, since rhi mutations delay synthesis of Grk protein in germaria and early egg chambers. In addition, Grk protein accumulates in large, actin-caged vesicles near the endoplasmic reticulum of stages 6-10 egg chambers. Two hypotheses are proposed to explain these results. First, Rhi may play dual roles in oogenesis, independently regulating chromosome compaction in nurse cells at the end of the unique endoreplication cycle 5 and repressing transcription of genes that inhibit Grk synthesis. Thus, loss-of-function mutations arrest nurse cell chromosome reorganization at the five-blob stage and delay production or processing of Grk protein, leading to axial patterning defects. Second, Rhi may regulate chromosome compaction in both nurse cells and oocyte. Loss-of-function mutations block nurse cell nuclear transitions at the five-blob stage and activate checkpoint controls in the oocyte that arrest Grk synthesis and/or inhibit cytoskeletal functions. These functions may involve direct binding of Rhi to chromosomes or may involve indirect effects on pathways controlling these processes (Volpe, 2001; Full text of article).


Search PubMed for articles about Drosophila Rhino

Andersen, P. R., Tirian, L., Vunjak, M. and Brennecke, J. (2017). A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549(7670): 54-59. PubMed ID: 28847004

Brennecke, J., et al. (2007). Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128: 1089-1103. PubMed ID: 17346786

Brennecke, J., et al. (2008). An epigenetic role for maternally inherited piRNAs in transposon silencing. Science 322: 1387-1392. PubMed ID: 19039138

Brower-Toland, B., et al. (2007). Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21: 2300-2311. PubMed ID: 17875665

Buhler, Verdel, A. and Moazed, D. (2006). Tethering RITS to a nascent transcript initiates RNAi- and heterochromatin-dependent gene silencing. Cell 125: 873-886. PubMed ID: 16751098

Chambeyron, S., et al. (2008). piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline. Proc. Natl. Acad. Sci. 105: 14964-14969. PubMed ID: 18809914

Desset, S., et al. (2008). In Drosophila melanogaster the COM locus directs the somatic silencing of two retrotransposons through both Piwi-dependent and -independent pathways. PLoS ONE 3: e1526. PubMed ID: 18253480

de Vanssay, A., Bouge, A. L., Boivin, A., Hermant, C., Teysset, L., Delmarre, V., Antoniewski, C. and Ronsseray, S. (2012). Paramutation in Drosophila linked to emergence of a piRNA-producing locus. Nature 490: 112-115. PubMed ID: 22922650

Gunawardane, L. S., et al. (2007). A slicer-mediated mechanism for repeat-associated siRNA 5' end formation in Drosophila. Science 315(5818): 1587-90. PubMed ID: 17322028

Khurana, J. S., Xu, J., Weng, Z. and Theurkauf. W. E. (2010). Distinct functions for the Drosophila piRNA pathway in genome maintenance and telomere protection. PLoS Genet. 6(12): e1001246. PubMed ID: 21179579

Klattenhoff, C., et al. (2007). Drosophila rasiRNA pathway mutations disrupt embryonic axis specification through activation of an ATR/Chk2 DNA damage response. Dev. Cell 12: 45-55. PubMed ID: 17199040

Klattenhoff C., et al. (2009). The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters. Cell 138:1137-49. PubMed ID: 19732946

Li, C., et al. (2009). Collapse of germline piRNAs in the absence of Argonaute3 reveals somatic piRNAs in flies. Cell 137: 509-521. PubMed ID: 19395009

Lim, A. K. and Kai, T. (2007). Unique germ-line organelle, nuage, functions to repress selfish genetic elements in Drosophila melanogaster. Proc. Natl. Acad. Sci. 104: 6714-6719. PubMed ID: 17428915

Le Thomas, A., Stuwe, E., Li, S., Du, J., Marinov, G., Rozhkov, N., Chen, Y. C., Luo, Y., Sachidanandam, R., Toth, K. F., Patel, D., Aravin, A. A. (2014) Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing. Genes Dev 28: 1667-1680. PubMed ID: 25085419

McKim, K. S. and Hayashi-Hagihara, A. (1998). mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved. Genes Dev. 12: 2932-2942. PubMed ID: 9744869

Mevel-Ninio, M., et al. (2007). The flamenco locus controls the gypsy and ZAM retroviruses and is required for Drosophila oogenesis. Genetics 175: 1615-1624. PubMed ID: 17277359

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The Rhino-Deadlock-Cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157: 1364-1379. PubMed ID: 24906153

Pane, A., Jiang, P., Zhao, D. Y., Singh, M. and Schüpbach, T. (2011). The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline. EMBO J. 30(22): 4601-15. PubMed ID: 21952049

Pal-Bhadra, M., Bhadra, U. and Birchler, J. A. (2002). RNAi related mechanisms affect both transcriptional and posttranscriptional transgene silencing in Drosophila, Mol. Cell 9: 315-327

Pal-Bhadra, M. et al. (2004). Heterochromatic silencing and HP1 localization in Drosophila are dependent on the RNAi machinery. Science 303: 669-672. PubMed ID: 14752161

Parhad, S. S., Tu, S., Weng, Z. and Theurkauf, W. E. (2017). Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery. Dev Cell 43(1): 60-70 e65. PubMed ID: 28919205

Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive evolution targets a piRNA precursor transcription network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744

Prud'homme, M., et al. (1995). Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster. Genetics 139: 697-711. PubMed ID: 7713426

Chang, T. H., Mattei, E., Gainetdinov, I., Colpan, C., Weng, Z. and Zamore, P. D. (2018). Maelstrom represses canonical Polymerase II transcription within bi-directional piRNA clusters in Drosophila melanogaster. Mol Cell. PubMed ID: 30527661

Richard, P. and Manley, J. L. (2009). Transcription termination by nuclear RNA polymerases. Genes Dev 23: 1247-1269. PubMed ID: 19487567

Sarot, E., et al. (2004). Evidence for a piwi-dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166: 1313-1321. PubMed ID: 15082550

Saito, K., et al. (2006). Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 20: 2214-2222

Soper, S. F., et al. (2008). Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis. Dev. Cell 15: 285-297. PubMed ID: 18694567

Tsai, S. Y. and Huang, F. (2021). Acetyltransferase Enok regulates transposon silencing and piRNA cluster transcription. PLoS Genet 17(2): e1009349. PubMed ID: 33524038

Vagin, V. V., et al. (2006). A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313 320-324. PubMed ID: 16809489

Verdel, A. and Moazed, D. (2005). RNAi-directed assembly of heterochromatin in fission yeast. FEBS Lett. 579: 5872-5878. PubMed ID: 16223485

Vermaak, D., Henikoff, S. and Malik, H. S. (2005). Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genet. 1(1): 96-108. PubMed ID: 16103923

Vermaak, D., Henikoff, S. and Malik, H. S. (2006). Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genet. 1: 96-108. PubMed ID: 16103923

Volpe, A. M., et al. (2001). Drosophila rhino encodes a female-specific chromo-domain protein that affects chromosome structure and egg polarity. Genetics 159: 1117-1134. PubMed ID: 11729157

Zhang, Z., Wang, J., Schultz, N., Zhang, F., Parhad, S. S., Tu, S., Vreven, T., Zamore, P. D., Weng, Z. and Theurkauf, W. E. (2014). The HP1 homolog Rhino anchors a nuclear complex that suppresses piRNA precursor splicing. Cell 157: 1353-1363. PubMed ID: 24906152

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date revised: 20 October 2023

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