InteractiveFly: GeneBrief

rhino: Biological Overview | References

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 rhi^KG/rhi² females had two wild-type appendages. However, 80% of embryos from mnk;rhi^KG/rhi² double-mutant females had two appendages. In addition, 33% of embryos from mei-41;rhi^KG/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; rhi^KG/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 mei^41D3/mei^41D3; armi^72.1/armi¹ females had wild-type appendages, but 83 of embryos from mei^41D3/mei^41D3; armi^72.1/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 4^th chromosomes move toward the poles. In OregonR, distinct 4^th 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 4^th 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).

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

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

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

Brower-Toland, B., et al. (2007). Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21: 2300-2311. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 18253480

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 Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 17428915

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 Citation: 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 Citation: 17277359

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 Citation: 14752161

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

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 Citation: 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 Citation: 18694567

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

Verdel, A. and Moazed, D. (2005). RNAi-directed assembly of heterochromatin in fission yeast. FEBS Lett. 579: 5872-5878. PubMed Citation: 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 Citation: 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 Citation: 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 Citation: 11729157

date revised: 20 January 2010

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