InteractiveFly: GeneBrief

Panoramix: Biological Overview | References

Gene name - Panoramix

Synonyms - Silencio/CG9754

Cytological map position - 57D11-57D11

Function - chromatin factor

Keywords - transposon repression - positive regulation of methylation-dependent chromatin silencing - piRNA-guided transcriptional silencing - forms a protein complex with Nxf2, and Nxt1/p15 that provides the key molecular connection between Piwi, the nascent target RNA, and the cellular heterochromatin machinery

Symbol - Panx

FlyBase ID: FBgn0034617

Genetic map position - chr2R:21,310,368-21,312,354 '

Cellular location - nuclear

NCBI link: Gene, Nucleotide, Protein
Panx orthologs: Biolitmine
Recent literature
Eastwood, E. L., Jara, K. A., Bornelov, S., Munafo, M., Frantzis, V., Kneuss, E., Barbar, E. J., Czech, B. and Hannon, G. J. (2021). Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila. Elife 10. PubMed ID: 33538693
In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. This study reports that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. This study showrf that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.
Zhang, G., Yu, T., Parhad, S. S., Ho, S., Weng, Z. and Theurkauf, W. E. (2021). piRNA-independent transposon silencing by the Drosophila THO complex. Dev Cell 56(18): 2623-2635.e2625. PubMed ID: 34547226
piRNAs guide Piwi/Panoramix-dependent H3K9me3 chromatin modification and transposon silencing during Drosophila germline development. The THO RNA export complex is composed of Hpr1, Tho2, and Thoc5-7. Null thoc7 mutations, which displace Thoc5 and Thoc6 from a Tho2-Hpr1 subcomplex, reduce expression of a subset of germline piRNAs and increase transposon expression, suggesting that THO silences transposons by promoting piRNA biogenesis. This study shows that the thoc7-null mutant combination increases transposon transcription but does not reduce anti-sense piRNAs targeting half of the transcriptionally activated transposon families. These mutations also fail to reduce piRNA-guided H3K9me3 chromatin modification or block Panoramix-dependent silencing of a reporter transgene, and unspliced transposon transcripts co-precipitate with THO through a Piwi- and Panoramix-independent mechanism. Mutations in piwi also dominantly enhance germline defects associated with thoc7-null alleles. THO thus functions in a piRNA-independent transposon-silencing pathway, which acts cooperatively with Piwi to support germline development.
Andreev, V. I., Yu, C., Wang, J., Schnabl, J., Tirian, L., Gehre, M., Handler, D., Duchek, P., Novatchkova, M., Baumgartner, L., Meixner, K., Sienski, G., Patel, D. J. and Brennecke, J. (2022). Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery. Nat Struct Mol Biol 29(2): 130-142. PubMed ID: 35173350
Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. This study explained the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. This study found that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.

The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. This study identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. These results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing (Murano, 2019).

Transposable elements (TEs) act as endogenous mobile mutagens to alter the sequence and structure of the genome, thereby changing the transcriptome and chromatin structure. TEs are often deleterious to the host by, for example, disrupting a gene, but may also be adaptive and drive genome evolution. TEs comprise nearly half of the human genome and approximately 30% of the genome of Drosophila melanogaster; thus, the successively arising families of TEs are the main drivers of genome expansion. In metazoans, TEs are silenced by the piRNA pathway, in which Piwi proteins are guided by Piwi-interacting RNAs (piRNAs) to their targets. The piRNA pathway is also essential for germline development and fertility in animals (Murano, 2019).

In the Drosophila ovary, two cytoplasmic PIWI paralogs, AGO3 and Aubergine (Aub), engage an amplification loop termed the 'ping-pong cycle' to cleave both TE transcripts and long piRNA precursor transcripts arising from piRNA clusters, which comprise a large number as well as various types of fragmented TEs, leading to the post-transcriptional silencing of TEs and production of piRNAs. These piRNAs can in turn trigger the production of phased piRNAs from piRNA precursors, which generates a dazzling variety of piRNAs and is coupled to the activity of a third Piwi protein. Phased piRNAs are also produced in ovarian somatic cells by a ping-suggests the independent mechanism, which are in turn also loaded onto Piwi. This loading initiates the transport of Piwi into the nucleus where it drives the transcriptional silencing of target TEs, by inducing specific histone modification and/or facilitating the folding of chromatin into a higher order structure (Murano, 2019).

Lack of the nuclear Piwi activity results in de-repression of TEs, which is concomitant 6 with decreases in both H3K9me3 repressive epigenetic marks and RNA polymerase II 7 (Pol II) occupancy on target TE loci This suggests a scenario of how the nuclear piRNA pathway works: Piwi and its bound piRNAs scan for target TEs by complementary base-pairing with their nascent transcripts. Upon targeting, Piwi recruits chromatin factors including H3K9me3 methyltransferases such as Eggless (also called dSetDB1) to initiate heterochromatin formation. The H3K9me3 modification is then bound by HP1a to maintain and propagate epigenetic silencing. However, thus far, direct association of the Piwi-piRNA complex with target transcripts and/or H3K9me3 methyltransferases has not been demonstrated (Murano, 2019).

Furthermore, depletion of Maelstrom (Mael), a Piwi cofactor in the nuclear piRNA pathway, does not decrease H3K9me3 levels at Piwi-targeted TE loci, suggesting that the repressive histone mark per se is not the final silencing mark for transcriptional gene silencing mediated by Piwi-piRNA complexes. Recently, linker histone H1 was identified as a component of a nuclear Piwi complex; depletion of H1 de-represses Piwi-targeted TEs and their surrounding genes without affecting the density of H3K9me3 marks and HP1a at target TE loci at target TE loci. Instead, it was demonstrated that the chromatin accessibility at Piwi-targeted TE loci is modulated by H1. These findings suggested a model in which Piwi recruits H1 and forces it to stay on target TE loci to induce chromatin compaction, thereby repressing target TE transcription and that the nuclear piRNA pathway adopts collaborated actions of H1 and H3K9me3 mark to maintain silencing of the TE state by modulating the chromatin state (Murano, 2019).

In addition to Mael and H1, biochemical and genetic analyses have also identified a number of putative Piwi cofactors including DmGTSF1/Asterix (Arx) and Panoramix (Panx)/Silencio. These findings together suggest that multiple pathways leading to Piwi-mediated TE silencing may exist, and raise the question of whether these pathways are independently initiated or have a common initial event (Murano, 2019).

To investigate this issue, Panx, which interacts with Piwi and promotes the deposition of H3K9me3 marks on target TE chromatin by H3K9me3 histone methyltransferase, Eggless (Sienski, 2015; Yu, 2015), was reinvestigated. This study biochemically isolated Nxf2, nuclear RNA export factor (NXF) variant, as a component of Panx-associated complexes. Nxf2 further associates with p15 (also called Nxt1), a co-adaptor for nuclear RNA export. The NXF family comprises four members in Drosophila, among which Nxf1 is an essential mRNA nucleocytoplasmic export factor. However, other members (Nxf2-4) are gonad-specific and their functions are not known, although they share common domain structures with Nxf1. Detailed analysis of Nxf2 function in the nuclear piRNA pathway revealed that the interactions between Panx, Nxf2, and p15 are necessary to maintain the protein stability of Nxf2 and Panx. Moreover, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter gene prior to heterochromatin formation, and H3K9me3 marks and H1 are required at later time points. Notably, the RNA binding domain of Nxf2 is essential for recruitment of the complex to target TEs. CLIP experiments demonstrated that both Piwi and Nxf2 directly interact with Piwi-targeted TE transcripts and that the association of Nxf2 with target TE transcripts is Piwi-dependent. These results suggest that Nxf2 enforces the association of Piwi-Panx-Nxf2-p15 (PPNP) complexes with the nascent transcripts of target TEs and triggers co-transcriptional repression in the nuclear silencing pathway (Murano, 2019).

A model is proposed in which the engagement of the PPNP complex with nascent TE transcripts initiates co-transcriptional silencing prior to heterochromatin formation. Although the heterochromatin factors are not required for the initial step of the silencing, they play a significant role in maintenance of the repressed status in the later stages of TE silencing. Because the depletion of Piwi activity rapidly results in the de-repression of TEs, this also suggests that silencing initiation and heterochromatin formation undergo continuous cycling to enforce a silenced state of TEs (Murano, 2019).

Four groups have obtained the similar findings regarding the essential role of Nxf2 in the Piwi-piRNA pathway rather than in nuclear RNA export. This studies have identified a new complex of Panx-Nxf2-P15, which associates with Piwi. Although two studies did not observe significant amount of Piwi peptides in their shotgun MS analysis of Panx immunoprecipitants, they showed the interaction between Panx-Piwi in immunoprecipitation followed by Western blot analysis. The inconsistency could be due to the immunoprecipitation conditions and/or antibodies used. Additionally, the UBA domain of Nxf2 associates with Panx, and the NTF2 domain associates with p15. The first LRR domain with RNA binding activity is needed for the regulation of TEs, and direct association of TE transcripts with Nxf2. Among these four studies, this is the only one which was able to observe the initial steps of the silencing. This is because this analysis was carried out using cultured cell line OSCs, and different timepoints were taken after expression of Nxf2. The present study is the only one to consider time points as early as 48 h. The regulation observed at 96 h after transfection, representing later stages of TE silencing, was consistent with the observation by one of the four studies. An important issue to be clarified is the mechanism by which the PPNP complex initiates the silencing of TEs (Murano, 2019 and references therein).

In C. elegans, NRDE-2 (nuclear RNAi defective-2) associates with the Argonaute protein NRDE-3 in the nucleus and is recruited by the NRDE-3/siRNA complex to nascent transcripts. This complex inhibits Pol II during the elongation and directs the accumulation of Pol II at genomic loci targeted by RNAi. This study has investigated whether λN-Nxf2 directs the accumulation of Pol II at boxB sites in the co-transcriptional silencing. Although the time course of Pol II occupancy upon λN-Nxf2 was examined by ChIP-qPCR at the boxB sites, no accumulation of Pol II was seen along the reporter gene (Murano, 2019).

Recently, it was reported that Pol II-associated proteins, PAF1 and RTF1, antagonize Piwi-directed silencing, which are factors involved in the modulation of promoter release, elongation, and termination of Pol II. The Panx-Nxf2-P15 complex might interfere with Pol II via the PAF1 complex, considering that fission yeast PAF1 represses AGO1/siRNA-directed silencing. Although this study examined the effect of PAF1 and RTF1 on the co-transcriptional silencing, PAF1 and RTF1 do not appear to be involved in the co-transcriptional silencing mediated by λN-Nxf2. These results suggest that Pol II regulation by the PPNP complex differs from that of the small RNA-mediated Pol II regulation model proposed previously. It may be necessary to identify key factors involved in this regulation, possibly by performing proteome analysis of the unknown factors tethered to the reporter recruitment system (Murano, 2019).

A decrease was found in the active histone H3K4me3 marks on the reporter gene even at an earlier time point (48 hpt) at which the level of H3K9me3 marks remained low. Two reports about H3K4 methylation in Piwi-mediated silencing have been published. The level of H3K4 methylation on TEs is increased upon Piwi-KD. The expression of artificial piRNAs that target a reporter locus induced transcriptional silencing associated with a decrease in the active H3K4-methylation marks. In addition, the depletion of LSD1, which removes H3K4-methylation marks from promoters, had significant effects on the ability of Panx to repress the reporter gene. Therefore, this study performed LSD1-KD and examined the effect of LSD1 on co-transcriptional silencing by the enforced tethering of Nxf2. However, the depletion of LSD1 had a limited impact on the silencing, whereas the level of mdg1 was increased 27-fold upon LSD1-KD. These findings together suggest that the decrease observed in H3K4me3 levels at 48 hpt in the tethering assay may not have been due to LSD1 (Murano, 2019).

Nxf2 is the core factor of the PPNP complex, and the current results suggest that the nuclear export factor variant has been co-opted to repress TEs in the Piwi-piRNA pathway. It is speculated that the co-option of Nxf2 into TE silencing is not coincidental but may reflect previous TE adaptation to exploit cellular mRNA transport pathways promoting the export of TE transcripts and replication/transposition. Why is it necessary for Piwi to use the RNA binding activity of Nxf2 to silence their targets? Piwi, as a member of the Argonaute family, is thought to search for targets through random interactions between Piwi-piRNA and transcripts in the ocean of the transcriptome, many of which likely show partial complementarity with Piwi-loaded piRNAs. To distinguish 'scanning Piwi' and 'target-engaged Piwi', the silencing pathway may need an engaged-in system to repress only targets and avoid unnecessary silencing of other cellular transcripts. It is possible that Nxf2 plays a role in supporting the association of Piwi that has found its bona fide targets. Panx preferentially associates with Piwi, which loads piRNAs targeting TEs, suggesting that there may be a system for Panx-Nxf2 to form the PPNP complex, specifically with Piwi that is engaged with its target TEs. CLIP analysis shows that Nxf2 cannot associate with target TEs without the activity of Piwi, indicating that the Panx-Nxf2-P15 complex itself does not recognize target transcripts, but rather recognizes target-engaged Piwi. In line with this, while piRNA-targeted Piwi can repress the reporter gene, λN-tethered Piwi cannot. This may be due to the Panx-Nxf2-p15 complex recognizing only piRNA-directed target-engaged Piwi, but not scanning or λN-tethered Piwi. Further studies will be necessary to confirm this speculation and elucidate the precise mechanism by which the Panx-Nxf2-P15 complex selectively recognizes only the 'target-engaged Piwi' and forms a PPNP complex to induce silencing of its targets (Murano, 2019).

The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. This study has identified SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2-Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1-Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. These data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly (Batki, 2019).

The discovery of SFiNX, a nuclear protein complex consisting of Panoramix, Nxf2, and Nxt1/p15, provides the key molecular connection between Piwi, the nascent target RNA, and the cellular heterochromatin machinery. In the absence of SFiNX, piRNA-loaded Piwi is incapable of inducing co-transcriptional silencing. Conversely, experimental recruitment of SFiNX to a nascent RNA independently of Piwi results in potent silencing and local heterochromatin formation. The data indicate that within SFiNX, Panoramix, but not Nxf2, provides the molecular link to the downstream cellular heterochromatin effectors. Based on genetic experiments, the histone methyl-transferase SetDB1/Eggless, the histone-demethylase Lsd1/Su(var)3-3, and the heterochromatin binding protein HP1/Su(var)205 are required for piRNA-guided co-transcriptional silencing (Sienski, 2015; Yu, 2015). None of these factors were found enriched in a SFiNX co-IP mass-spectrometry experiments. This suggests a transient or regulated molecular interaction between Panoramix and the cellular heterochromatin machinery. In this respect, the recent finding that the SUMOylation machinery is critically involved in piRNA-guided co-transcriptional silencing (Ninova, 2019) is of considerable interest (Batki, 2019).

The involvement of Nxf2, a nuclear RNA export variant, in cotranscriptional silencing was surprising. Based on biochemical and structural data, it is proposed that two molecular features of the ancestral NXF protein, the principal mRNA export receptor Nxf1/Tap, facilitated the evolutionary exaptation of Nxf2 into piRNA-guided silencing. First, Nxf2 retained its ability to bind RNA, thereby providing SFiNX a molecular link to the nascent target RNA. Second, crosslink-mass spectrometry data indicate that, similar to Nxf1/Tap, the RNA binding activity of Nxf2 might be gated. It is proposed that this could provide a critical regulatory switch to ensure that SFiNX only associates with transcripts that are specified as targets via the Piwi-piRNA complex. In the case of Nxf1/Tap, various proteins (e.g. SRproteins, THO-complex, UAP56, Aly/Ref) that are recruited during co-transcriptional mRNA maturation, are required to restrict Nxf1/Tap deposition onto export competent mRNAs only. Whether any of these factors is also required for loading Nxf2 onto RNA is currently unclear. It is, however, likely that target-engaged Piwi contributes a critical role in the deposition of SFiNX onto the target RNA, potentially by licensing Nxf2’s RNA binding activity. It is noted, that despite considerable experimental efforts it was not possible to establish a direct molecular link between Piwi and either Nxf2 or Panoramix. Considering that Piwi represses transcription of its targets, the steady state level of a Piwi-SFiNX complex is expected to be small. In support of this, piRNA-independent recruitment of Piwi to a nascent RNA is incapable of target silencing, indicating that the Piwi-SFiNX interaction occurs only once Piwi is bound to a target RNA via a complementary piRNA. An intriguing result from the Nxf2 CLIP experiments is that Nxf2 binds nascent, Piwi-repressed RNAs also far away from piRNA complementary sequences. One possible explanation for this is that Nxf2 or SFiNX, after initial Piwi-mediated recruitment, spread without Piwi along the target RNA. Alternatively, SFiNX might form oligomers in vivo, resulting in multiple RNA binding domains per complex that could tether SFiNX to distant parts of the target transcript. Such an ‘entangling’ of the target RNA at the transcription locus might serve an additional critical role, namely to keep the target RNA at chromatin, thereby providing sufficient time for the recruited effectors to modify chromatin at the locus (Batki, 2019).

Although no direct ortholog of Nxf2 is identifiable in vertebrates, the finding that an NXF variant is involved in transposon silencing in Drosophila could point to a more general scheme. The Nxf1/Tap ancestor diversified through several independent evolutionary radiations into numerous NXF variants in different animal lineages. In flies, the three NXF variants exhibit gonad-specific expression with Nxf2 and Nxf3 being expressed predominantly in ovaries, and Nxf4 being testis-specific. Besides Nxf2 (this study), Drosophila Nxf3 is also an essential piRNA pathway component as it is required for the nuclear export of un-processed piRNA cluster transcripts in the germline (ElMaghraby, 2019). In mice and humans, several NXF variants are preferentially expressed in testes. Intriguingly, Nxf2 mutant mice are male sterile, a phenotype shared with many piRNA pathway mutants. Considering this, it is speculated that also in vertebrates the host-transposon conflict has been a key driver of NXF protein family evolution through frequent duplication and exaptation events. This study highlights that some of these variants might have evolved novel functions, not directly related to RNA export biology (Batki, 2019).

Pandas complex adapted for piRNA-guided transposon silencing

The repression of transposons by the Piwi-interacting RNA (piRNA) pathway is essential to protect animal germ cells. In Drosophila ovaries, Panoramix (Panx) enforces transcriptional silencing by binding to the target-engaged Piwi-piRNA complex, although the precise mechanisms by which this occurs remain elusive. This study shows that Panx functions together with a germline specific paralogue of a nuclear export factor, dNxf2, and its cofactor dNxt1 (p15), as a ternary complex to suppress transposon expression. Structural and functional analyses demonstrate that dNxf2 binds Panx via its UBA domain, which plays an important role in transposon silencing. Unexpectedly, dNxf2 interacts directly with dNxf1 (TAP), a general nuclear export factor. As a result, dNxf2 prevents dNxf1 from binding to the FG repeats of the nuclear pore complex, a process required for proper RNA export. Transient tethering of dNxf2 to nascent transcripts leads to their nuclear retention. Therefore, it is proposed that dNxf2 may function as a Pandas (Panoramix-dNxf2 dependent TAP/p15 silencing) complex, which counteracts the canonical RNA exporting machinery and restricts transposons to the nuclear peripheries. These findings may have broader implications for understanding how RNA metabolism modulates epigenetic gene silencing and heterochromatin formation (Zhao, 2019).

piRNA-guided co-transcriptional silencing coopts nuclear export factors

The PIWI-interacting RNA (piRNA) pathway is a small RNA-based immune system that controls the expression of transposons and maintains genome integrity in animal gonads. In Drosophila, piRNA-guided silencing is achieved, in part, via co-transcriptional repression of transposons by Piwi. This depends on Panoramix (Panx); however, precisely how an RNA binding event silences transcription remains to be determined. This study shows that Nuclear Export Factor 2 (Nxf2) and its co-factor, Nxt1, form a complex with Panx and are required for co-transcriptional silencing of transposons in somatic and germline cells of the ovary. Tethering of Nxf2 or Nxt1 to RNA results in silencing of target loci and the concomitant accumulation of repressive chromatin marks. Nxf2 and Panx proteins are mutually required for proper localization and stability. This study mapped the protein domains crucial for the Nxf2/Panx complex formation and showed that the amino-terminal portion of Panx is sufficient to induce transcriptional silencing (Fabry, 2019).

The data identify Nxf2 and Nxt1 as critical mediators of co-transcriptional gene silencing, acting in concert with Panx to repress loci in response to Piwi-piRNA target engagement. The emerging model for piRNA-dependent silencing implies that target recognition by Piwi is necessary to recruit the PICTS complex onto the appropriate nascent RNA targets. Difficulties in detecting stable interactions between Piwi and PICTS components in vivo may arise from a requirement for Piwi target engagement to licence it for recruitment of silencing complexes, as has been suggested previously (Sienski, 2015; Yu., 2015). The same mechanism may underlie the difficulties experienced in observing Piwi on its target loci by ChIP (Fabry, 2019).

Panx and Nxf2 are interdependent for their protein stability and proper subcellular localization, underscoring the fact that correct assembly of the PICTS complex is essential for TGS, while the silencing capacity, per se, resides in Panx. Of note, previous work reported a partial destabilization of Nxf2 in cells depleted of Nxt1 (Herold, 2001), potentially extending the interdependency to all three proteins. RIP-seq experiments from unperturbed cells found transposon RNAs enriched only with Panx, as reported (Sienski, 2015), but not with Nxf2, possibly due to low substrate availability combined with an insensitive assay. These results are consistent with another recent report that did not detect transposon enrichment in Nxf2 CLIP-seq from wild-type cells (Batki, 2019). However, two other studies identified transposon mRNA association with Nxf2 in CLIP-seq experiments upon depletion of the previously described TGS factor, Mael (Zhao, 2019), or by using a stable cell line and depletion of endogenous Nxf2 (Murano, 2019). Considered together, these data suggest that Nxf2 might be important for stabilizing the binding of Panx to nascent RNAs. However, precisely how Nxf2 executes this function remains to be fully elucidated. Of note, Murano found that Panx interacts with Nxf2, Piwi, Mael and Arx (Murano, 2019), which could imply that other TGS factors come into contact with the PICTS complex, although the relationship between these factors and PICTS requires further investigation (Fabry, 2019).

Mutational analyses suggest that Panx and Nxf2 must normally bind Nxt1 to carry out transposon repression. Direct recruitment of any of the PICTS complex components to RNA reporters results in robust chromatin silencing. Upon tethering to DNA, however, Panx induces potent TGS, whereas Nxf2 leads to less prominent effects and Nxt1 shows no silencing capability in these assays. Interestingly, recruitment of the amino-terminal part of Panx alone is necessary and sufficient to induce reporter repression, pinpointing this domain of Panx as harboring the silencing effector function. Future investigations will be crucial to uncover the molecular mechanism by which the Panx amino terminus instructs the downstream chromatin silencing machinery (Fabry, 2019).

This work, and that of others (Batki, 2019; Murano, 2019; Zhao, 2019) indicates that piRNA-guided co-transcriptional silencing of transposons has coopted several components of the RNA export machinery, namely Nxf2 and Nxt1. Of the four NXF proteins present in flies, only two have thus far been characterized. Interestingly, while Nxf1, acting along with Nxt1, is crucial for canonical mRNA export, Nxf2 has been coopted by the piRNA pathway and functions in co-transcriptional gene silencing. Nxf3, which also is required for transposon repression in germ cells, is emerging as being critical for the export of piRNA precursors generated from dual-strand clusters in the germline. The role of Nxf4, whose expression is testis-specific, is yet to be established. This remarkable functional diversity of NXF family members correlates with tissue-specific expression patterns, and seems conserved in mammals. However, deciphering how each achieves substrate specificity will be critical to understanding how these homologs can be exclusively dedicated to different targets and confer different fates upon the RNAs that they bind (Fabry, 2019).

Importantly, the fate of the nascent transcript that is detected by Piwi and instructed by PICTS for silencing remains unclear. One hypothesis is that instead of being exported, these targets undergo degradation by the nuclear exosome. Such mechanism would be contrary to yeast, where the TREX complex subunit Mlo3 was shown to oppose siRNA-mediated heterochromatin formation at gene loci, and suggests that different lineages have evolved different silencing mechanisms. In any case, it is possible that a single transcript from a locus that is marked for silencing might pose a lesser threat than an unsilenced locus and, therefore, not be capable of exerting evolutionary pressure for the detemination of its fate (Fabry, 2019).

piRNA-mediated regulation of transposon alternative splicing in the soma and germ line

Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. This study shows that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, it was shown that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a [HP1a; encoded by Su(var)205]. Furthermore, this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. These findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development (Teixeira, 2017).

Hybrid dysgenesis is a syndrome that affects progeny in a non-reciprocal fashion, being normally restricted to the offspring of crosses in which males carry transposable elements but which females lack. In Drosophila, the dysgenic traits triggered by the P-element DNA transposon are restricted to the germ line and include chromosomal rearrangements, high rates of mutation, and sterility. The impairment is most prominent when hybrids are grown at higher temperatures, with adult dysgenic females being completely sterile at 29°C. Despite the severe phenotypes, little is known about the development of germ cells during P-M dysgenesis. To address this, germline development was characterized in the progeny obtained from reciprocal crosses between w1118 (P-element-devoid strain) and Harwich (P-element-containing strain) flies at 29°C. In non-dysgenic progeny, germline development progressed normally throughout embryonic and larval stages, leading to fertile adults. Although the development of dysgenic germline cells was not disturbed during embryogenesis, germ cells decreased in number during early larval stages, leading to animals with no germ cells by late larval stages. These results indicate that the detrimental effects elicited by P-element activity are triggered early on during primordial germ cell (PGC) development in dysgenic progeny, leading to premature germ cell death (Teixeira, 2017).

Maternally deposited small RNAs cognate to the P-element are thought to provide the 'P-cytotype' by conferring the transgenerationally inherited ability to protect developing germ cells against P-elements. Small RNA-based transposon regulation is typically mediated by either transcriptional silencing or post-transcriptional clearance of mRNAs, both of which result in a decrease in the accumulation of transposon mRNA. To understand how maternally provided small RNAs control P-elements in germ cells, this study focused on embryonic PGCs sorted from 4- to 20-h-old embryos generated from reciprocal crosses between w1118 and Harwich strains. Surprisingly, the accumulation of P-element RNA as measured by quantitative reverse transcription PCR (RT-qPCR) showed no change in dysgenic PGCs when compared to non-dysgenic PGCs. This indicates that P-cytotype small RNAs exert their function by means other than regulating P-element mRNA levels (Teixeira, 2017).

P-element activity relies on production of a functional P-element transposase protein, the expression of which requires precursor mRNA (pre-mRNA) splicing of three introns. To analyse P-element RNA splicing in germ cells during hybrid dysgenesis, primers were designed that specifically anneal to spliced mRNA transcripts. The accumulation of spliced forms for the first two introns (IVS1 and IVS2) did not show changes in dysgenic PGCs when compared to non-dysgenic PGCs. By contrast, the accumulation of spliced transcripts for the third intron (IVS3) was substantially increased in dysgenic germ cells. Given that the overall accumulation of P-element mRNA showed no changes, the results indicate that the maternally provided P-cytotype can negatively regulate P-element IVS3 splicing and therefore inhibits the production of functional P-transposase in germ cells (Teixeira, 2017).

Analysis of publically available small RNA sequencing data from 0-2-h-old embryos laid by Harwich females indicated that two classes of small RNAs cognate to the P-element are maternally transmitted: small interfering RNAs (siRNAs, 20-22-nucleotides long) and piRNAs (23-29 nucleotides long). To test the role of distinct small RNA populations on P-element expression, mutants were analyzed uniquely affecting each small RNA biogenesis pathway in the Harwich background. Mutations that disrupt siRNA biogenesis components Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), or mutations ablating components of the piRNA biogenesis pathway, such as the Argonautes piwi, aubergine (aub), and Argonaute 3 (AGO3), as well as the RNA helicase vasa (vas) and spindle E (spn-E), did not affect P-element mRNA accumulation in adult ovaries as measured by RT-qPCR. However, mutations that disrupted piRNA biogenesis, and not the siRNA pathway, led to a strong and specific increase in the accumulation of IVS3-spliced mRNAs. RNA sequencing (RNA-seq) analysis on poly(A)-selected RNAs from aub and piwi mutant adult ovaries confirmed the specific effect on IVS3 splicing. To examine transposon expression in tissue, RNA fluorescent in situ hybridization (FISH) was performed using probes specific for the P-element and for the Burdock retrotransposon, a classic target of the germline piRNA pathway. In mutants affecting piRNA biogenesis, increased abundance of Burdock RNA was readily observed in germline tissues, with most of the signal accumulating close to the oocyte. By contrast, no difference was detected in the P-element RNA FISH signal in piRNA biogenesis mutants compared to control. Nuclear RNA foci observed in nurse cells were of similar intensity and number regardless of the genotype, and cytoplasmic signal showed no detectable difference. Therefore, the results indicate that in germ cells, piRNAs specifically modulate IVS3 splicing. This regulation is reminiscent of the well-documented mechanism that restricts P-element activity to germline tissues, which involves the expression of a host-encoded RNA binding repressor protein that negatively regulates IVS3 splicing in somatic tissues (Teixeira, 2017).

In somatic tissues, P-element alternative splicing regulation is mediated by the assembly of a splicing repressor complex on an exonic splicing silencer element directly upstream of IVS3. To test whether the P-element IVS3 and flanking exon sequences were sufficient to trigger the piRNA-mediated splicing regulation in germ cells, a transgenic reporter system for IVS3 splicing was used in which a heterologous promoter (Hsp83) drives the expression of an IVS3-lacZ-neo fusion mRNA specifically in the germ line. Using RT-qPCR, the F1 progeny from reciprocal crosses between w1118 and Harwich flies were analyzed in the presence of the hsp83-IVS3-lacZ-neo reporter. The fraction of spliced mRNAs produced from the transgenic reporter was substantially increased in dysgenic compared to non-dysgenic adult ovaries, in agreement with previously reported results. Most importantly, genetic experiments confirmed that the repression of IVS3 splicing in germ cells relies on piRNA biogenesis, as the splicing repression observed with this reporter in non-dysgenic progeny was specifically abolished in adult ovaries of aub and vas mutants (Teixeira, 2017).

Mechanistically, piRNA-mediated splicing regulation may be achieved through direct action of piRNA complexes on target pre-mRNAs carrying the IVS3 sequence or indirectly by piRNA-mediated changes in chromatin states. Piwi-interacting proteins such as Asterix (Arx) and Panoramix (Panx) are dispensable for piRNA biogenesis but are essential for establishing Piwi-mediated chromatin changes, possibly by acting as a scaffold to recruit histone-modifying enzymes and chromatin-binding proteins to target loci. To test the role of these chromatin regulators on P-element splicing, germline-specific RNA interference (RNAi) knockdown experiments were performed in the Harwich background. Similar to what was observed for the piRNA biogenesis components, germline knockdown of Arx and Panx showed no change in the accumulation of P-element RNA, but a strong and specific effect on IVS3 splicing in adult ovaries. The same pattern on IVS3 splicing was observed in the germline knockdown of HP1a and Maelstrom (Mael), both of which act downstream of Piwi-mediated targeting to modulate chromatin structure. The same genetic requirement for Panx for IVS3 splicing control was also confirmed when using the transgenic IVS3 splicing reporter, further indicating that Piwi-mediated chromatin changes at the target locus are involved in IVS3 splicing regulation. At target loci, Piwi complexes are known to mediate the deposition of the classic heterochromatin mark histone H3 lysine 9 trimethylation (H3K9me3). To assess the effect of piRNA-targeting on P-element chromatin marks directly, H3K9me3 chromatin immunoprecipitation was performed followed by sequencing (ChIP-seq) or quantitative PCR on adult ovaries of progeny from reciprocal crosses between w1118 and Harwich strains (to avoid developmental defects, ChIP was performed on F1 progeny raised at 18°C. This analysis revealed a specific loss of global H3K9me3 levels over P-element insertions in dysgenic progeny when compared to non-dysgenic progeny (Teixeira, 2017).

To analyse the chromatin structure at individual P-element insertions, DNA sequencing (DNA-seq) data was used to identify all euchromatic insertions in the Harwich strain, and RNA-seq analysis was used to define transcriptionally active insertions. At transcriptionally active P-element euchromatic insertions, the spreading of H3K9me3 into the flanking genomic regions was readily observed in non-dysgenic progeny, but was completely absent in dysgenic offspring. Similarly, a reduction in H3K9me3 modification levels was also observed over the IVS3 transgenic reporter in dysgenic progeny when compared to non-dysgenic progeny. Interestingly, euchromatic insertions with no evidence of transcriptional activity were devoid of an H3K9me3 signal in both non-dysgenic and dysgenic crosses, providing further evidence for a model initially suggested in yeast and more recently proposed for Drosophila and mammals, in which H3K9me3 deposition by piRNA complexes would require transcription of the target loci. Mechanistically different from the well-described somatic repression, the results uncovered the existence of an unexpected piRNA-mediated, chromatin-based mechanism regulating IVS3 alternative splicing in germ cells (Teixeira, 2017).

To expand the analysis, the literature was searched for other cases of transposon splicing regulation. Drosophila Gypsy elements are retrotransposons that have retrovirus-like, infective capacity owing to their envelope (Env) protein. These elements are expressed in somatic ovarian cells, in which they are regulated by the flamenco locus, a well-known piRNA cluster that is a soma-specific source of antisense piRNAs cognate to Gypsy. Interestingly, it has been shown that mutations in flamenco not only elicited the accumulation of Gypsy RNA, but also modulated pre-mRNA splicing, favouring the production of the env mRNA and therefore germline infection. To test whether the piRNA pathway, in addition to its role in regulating the accumulation of Gypsy RNA, is also responsible for modulating the splicing of Gypsy elements in somatic tissues, publically available RNA-seq data from poly(A)-selected RNAs extracted from in vivo cultures of ovarian somatic cells (OSCs) was analyzed. The analysis indicates that piwi knockdown was sufficient to modulate Gypsy splicing, favouring the accumulation of env-encoding mRNA. In agreement with a chromatin-mediated regulation of alternative splicing, RNAi depletion of Arx, Panx, HP1a and Mael, as well as knockdown of the histone linker H1, was sufficient to favour Gypsy splicing, recapitulating the effect caused by Piwi depletion. Notably, this was also the case for the H3K9 methyltransferase Setdb1, but not for the H3K9 methyltransferases Su(var)3-9 and G9a, indicating specific genetic requirements. Taken together, the results indicate that the piRNA pathway, through its role in mediating changes in chromatin states, regulates the splicing of transposon pre-mRNAs in both somatic and germline tissues (Teixeira, 2017).

Using P-M hybrid dysgenesis as a model, this study hasa uncovered splicing regulation elicited by chromatin changes as a previously unknown mechanism by which the piRNA pathway protects the genome from the detrimental effects of transposon activity. Splicing control at piRNA-target loci is likely to be mechanistically different from what has been observed for germline piRNA clusters given the low enrichment of the HP1 homologue Rhino (also known as HP1D) protein, which is required for piRNA cluster RNA processing, over the endogenous P-element insertions in the Harwich genome or over the transgenic IVS3 splicing reporter in non-dysgenic and dysgenic progeny (as measured by ChIP-qPCR). Because small RNA-based systems leading to chromatin mark changes at target loci are pervasive in eukaryotes, it is expected that this new type of targeted regulation is of importance in settings far beyond the scope of the piRNA pathway and Drosophila. Indeed, small RNA-guided DNA methylation over the LINE retrotransposon Karma was recently shown to modulate alternative splicing in oil palm, disrupting nearby gene expression and ultimately affecting crop yield. In this context, small RNA-based control of chromatin structure may be crucially important in genomes with a high content of intronic transposon insertions, such as the human genome, by providing a mechanism to suppress exonization of repeat elements. Although the means by which piRNA-mediated changes in chromatin states could regulate alternative splicing remain to be determined, it is tempting to speculate that piRNA pathway components do so by co-transcriptionally modulating interactions between RNA polymerase II and the spliceosome (Teixeira, 2017).

Panoramix enforces piRNA-dependent cotranscriptional silencing

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based innate immune system that defends germ cell genomes against transposons. In Drosophila ovaries, the nuclear Piwi protein is required for transcriptional silencing of transposons, though the precise mechanisms by which this occurs are unknown. This study shows that the CG9754 protein is a component of Piwi complexes that functions downstream of Piwi and its binding partner, Asterix, in transcriptional silencing. Enforced tethering of CG9754 to nascent messenger RNA transcripts causes cotranscriptional silencing of the source locus and the deposition of repressive chromatin marks. CG9754 has been named 'Panoramix,' and it is proposed that this protein could act as an adaptor, scaffolding interactions between the piRNA pathway and the general silencing machinery that it recruits to enforce transcriptional repression (Yu, 2015).

The Piwi interacting RNA (piRNA) pathway controls transposons through a number of distinct, but likely interlinked, mechanisms. Whereas cytoplasmic Piwi proteins silence their targets posttranscriptionally through piRNA-directed cleavage and the ping-pong cycle, nuclear Piwi-piRNA complexes function at the transcriptional level. Piwi-directed repression of transcription is thought to be dependent on piRNA-guided recognition of nascent transposon transcripts. Transcriptional gene silencing (TGS) correlates with the presence of histone H3 lysine 9 trimethylation (H3K9me3) marks, yet the mechanism through which Piwi binding promotes the deposition of these marks remains enigmatic. With the exception of the zinc finger protein Asterix (also known as DmGTSF1), the components of Piwi effector complexes at target loci are largely unexplored (Yu, 2015).

This study systematically mined candidate genes from RNA interference (RNAi) screens for potential TGS effector proteins and identified CG9754 in three independently published screens as being critical in both the germ cells and follicle cells for transposon silencing. Loss of CG9754 had essentially no effect on the abundance or content of piRNA populations or on the nuclear localization of Piwi protein, suggesting that it is probably an effector component. CG9754 encodes a ~60-kD nuclear protein with no identifiable domains. The expression of CG9754 is restricted to the female gonads, as is seen for other core piRNA pathway components such as Asterix (Yu, 2015).

To examine global effects on transposon expression, RNA sequencing (RNA-seq) was used to measure steady-state RNA levels from ovaries with germline-specific knockdowns of either CG9754 or Piwi. Piwi knockdown caused a sharp rise in transposon transcripts, with minimal effects on protein-coding gene expression. Knockdown of CG9754 caused effects very similar to those of Piwi, with most transposon targets being shared. Changes in steady-state RNA levels could have resulted from alterations in either element transcription or the stability of transposon mRNAs. Global run-on sequencing (GRO-seq) was used to measure nascent RNA synthesis following gene knockdown. Loss of either CG9754 or Piwi produced very similar profiles, suggesting that CG9754 is specifically required for transcriptional silencing of transposons targeted by Piwi (Yu, 2015).

Piwi-mediated TGS correlates with the presence of H3K9me3 marks at silenced transposons. Depletion of either CG9754 or Piwi resulted in nearly identical losses of H3K9me3 over transposons. Four independent frameshift mutations of CG9754 generated via the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein system were isolated. A consistent global up-regulation of transposable elements and the corresponding loss of H3K9me3 marks in CG9754 mutant ovaries were observed without changes in piRNA levels. Similarly to other core piRNA pathway mutants, female flies lacking CG9754 were sterile. Moreover, flies double-mutant for CG9754 and Asterix showed transposon derepression comparable to that of flies with either single mutation, suggesting that both genes act in the same pathway. Thus, CG9754 functions along with Piwi and Asterix in the repression of transposon transcription (Yu, 2015).

Next it was asked whether the presence of CG9754 at a target locus might be sufficient to induce its silencing. Because piRNAs likely direct binding to nascent RNAs rather than to the DNA of their targets, CG9754 was delivered via protein-RNA interactions. A series of luciferase reporters was constructed with BoxB sites in their 3' untranslated regions (UTRs) and they were used to create transgenic reporter flies. BoxB sites are bound by the λN protein, which can also bring other components to the RNA as part of a fusion. Flies were also generated expressing λN proteins fused to CG9754, Asterix, nuclear Piwi, and, as a negative control, a cytoplasmic Piwi missing its nuclear localization signal (dN-Piwi). When coexpressed with the reporter, the dN-Piwi fusion failed to induce any change in luciferase expression. Of the remainder, only the CG9754 fusion considerably reduced luciferase activity. Silencing appeared to be dosage-dependent, as the degree of repression correlated with the number of BoxB binding sites inserted into the reporter mRNA. Consistent with a role for CG9754 in transcriptional silencing, the abundance of the reporter mRNAs was significantly reduced upon tethering (Yu, 2015).

Although CG9754-triggered repression appeared to be independent of chromatin context, integration of the reporter into genomic DNA appeared to be critical for repression. Transient cotransfection of reporter constructs into the OSS cell line, which contains an active piRNA pathway, resulted in little to no detectable silencing. In contrast, tethering Drosophila Ago1 (λN-dAgo1) to the luciferase reporter mRNA in OSS cells caused substantial repression of the same reporter. These results indicate that CG9754 can function properly only in the context of chromatin, likely acting at the transcriptional level, by interacting with nascent transcripts (Yu, 2015).

To test the hypothesis that λN-CG9754 acts on nascent transcripts, a reporter was generated for which the BoxB binding sites were located within the intron of the primary transcript. λN-CG9754 maintained the ability to repress this reporter but not a similar transcript carrying BoxB sites in the antisense orientation integrated into the same genomic locus. Because the spliced, mature reporter transcripts lack the BoxB sites, it was reason that λN-CG9754 must be able to exert its effects by binding to unspliced precursor mRNAs. Given that splicing occurs cotranscriptionally, this implies that CG9754 confers its effects by interaction with the nascent transcript (Yu, 2015).

If CG9754 mediates Piwi-dependent transcriptional silencing, delivery of CG9754 alone might recapitulate hallmarks of piRNA-directed repression. Tethering of CG9754 had a highly specific effect, changing levels of only the reporter mRNA. Repression occurred at the transcriptional level, as GRO-seq indicated a loss of nascent RNA from the integrated reporter. Repression by CG9754 also correlated with specific deposition of H3K9me3 marks over the reporter locus. Tethering of CG9754 failed to trigger piRNA production from the reporter, as has been seen previously for some loci that become targets of the piRNA pathway. Of note, spreading of H3K9me3 marks to other regions of the reporter gene was observed, as described previously for regions flanking piRNA-targeted transposon insertions. Thus, delivering CG9754 to the nascent RNA causes repression of a locus in a manner that mimics targeting by the piRNA pathway (Yu, 2015).

Whether CG9754 might be a component of Piwi complexes, as predicted by epistasis experiments, was tested. Functional GFP-Piwi fusion proteins copurified with hemagglutinin (HA)-tagged CG9754 from OSS cells, but not with a negative-control fusion (HA-mKate2). Conversely, Flag-tagged CG9754 was able to specifically precipitate endogenous Piwi proteins from OSS cell lysates, confirming the interaction between these two proteins. Given its properties, CG9754 was named 'Panoramix,' after the mentor who empowers the French comic book character Asterix to perform his feats of strength (Yu, 2015).

The identification of Panoramix as a key mediator of piRNA-directed TGS presented an opportunity to use the tethering assay to dissect the mechanism of transcriptional silencing. RNAi was used to deplete selected piRNA pathway genes in flies in which λN-Panoramix was tethered to the luciferase-BoxB reporter. Knockdown of Panoramix itself weakened the repression significantly, as compared with a control knockdown (mCherry). Silencing of factors required for piRNA biogenesis (Zuc and Armi) or those that are expected to act upstream of Panoramix (Piwi and Asterix) did not significantly affect repression. Depletion of dLSD1/Su(var)3-3 and its cofactor, CoREST, which normally form a complex that removes H3K4me2 marks from promoters, had significant effects on the ability of Panoramix to repress the reporter. Because H3K4me2 marks actively transcribed genes, it is possible that dLSD1-mediated removal of these marks is a key step in Panoramix-mediated transcriptional silencing. This raises a potential parallel with piRNA-directed silencing in mice, wherein engagement by DNMT3L, which is necessary for piRNA-induced DNA methylation, requires removal of such marks. Similarly, knockdown of HP1a caused derepression, in agreement with its role as a constitutive heterochromatin component required for transposon silencing and with the observation that the presence of Panoramix is correlated with the deposition of H3K9me3 marks at target loci. The H3K9 methyltransferase Eggless/dSETDB1 and its cofactor Windei appeared to be required specifically for Panoramix-mediated silencing, as knockdown of G9a, another H3K9 methyltransferase, showed no effect on the reporter. In eggless mutants, essentially complete derepression of the reporter was observed, despite Panoramix tethering. In contrast, the piRNA biogenesis mutant zuc showed little to no effect on the repression of the reporter, as also observed in zuc RNAi experiments. These data raise the possibility that Eggless could be one of the enzymes responsible for the deposition of H3K9me3 marks over silenced transposons in a Piwi-targeted fashion (Yu, 2015).

Panoramix functions downstream of Piwi and Asterix and is both necessary and sufficient to elicit transcriptional repression when bound to nascent transcripts. Panoramix represents an example in metazoans of a protein inducing cotranscriptional silencing when recruited to the nascent transcript from a locus. In fact, only cotranscriptional silencing can resolve the conundrum of a target being transcriptionally repressed while transcripts from that target locus are responsible for recruiting their own repressors. Orthologs of some of the general silencing factors that act with Panoramix to deposit and interpret repressive chromatin marks have also been implicated in mammalian transposon silencing, in which the pathway functions by causing heritable DNA methylation. Though one cannot identify a mammalian ortholog of Panoramix based on primary sequence alone, the overall conservation of the piRNA-mediated transcriptional machinery suggests that a protein with an equivalent function likely exists in mammals (Yu, 2015).

Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. This study characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, it is suggested that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery (Sienski, 2015).

The formation of heterochromatin (with the resulting transcriptional silencing of the locus) downstream from nuclear Argonaute proteins is an intriguing process. On the one hand, this is because the target RNA, which is essential for Argonaute targeting, will be suppressed in its own production. On the other hand, the process must deal with the fact that transcripts are only transiently attached to their originating chromatin locus; transcript maturation or transcription termination should in principle lead to the release of the target RNA, including bound Argonaute complexes. A wealth of pioneering work in S. pombe on centromeric heterochromatin formation downstream from Ago1 has provided significant insight into these questions. However, how these findings relate to the molecular steps that are involved in transcriptional target silencing downstream from animal PIWI clade proteins is only poorly understood. In fact, the involvement of several non-conserved proteins between yeast and animals suggests significant differences (Sienski, 2015).

This study has characterized CG9754/Silencio as an essential piRNA pathway factor required for nuclear Piwi-piRNA complexes to mediate transcriptional silencing and heterochromatin formation. The central conclusions from this work are as follows: (1) Silencio is the first protein in the piRNA pathway that triggers heterochromatin formation and silencing if tethered ectopically to RNA or DNA. This work provides a powerful entry point into the mechanistic dissection of the silencing process in a controlled setting. (2) The two central H3K9 methyltransferases, Su(var)3-9 and Eggless/SetDB1, are acting downstream from Piwi yet in different and nonredundant manners: While SetDB1 is required for heterochromatin formation downstream from Piwi, Su(var)3-9 appears to be involved in spreading the H3K9me3 to flanking genomic areas (Sienski, 2015).

The primary sequence of Silencio does not reveal insights into its molecular roles in the piRNA-guided silencing process. Silencio does not harbor any domain of known function. Also, orthologs of Silencio are not identifiable outside Drosophilids. However, considering that heterochromatin formation downstream from nuclear PIWI proteins also occurs in mammals, the existence of a factor with equivalent function seems very plausible outside Drosophilids (Sienski, 2015).

How Silencio triggers heterochromatin formation is unclear. The data suggest that it directly or indirectly recruits SetDB1 to target loci or licenses SetDB1’s methylation activity. The specificity for this process almost certainly relies on Piwi-piRNA complexes. In fact, Silencio interacts with target-engaged Piwi. In this sense, Silencio could be considered as an adaptor protein that bridges Piwi to basic cellular activities involved in the establishment and maintenance of heterochromatin. Such a model is highly reminiscent of the biology of TE silencing in mammals, where, besides the gonadal piRNA pathway, a large family of KRAB-type zinc finger transcription factors recruits the adaptor protein KAP1/Trim28 to target TE sequences. KAP1 acts as a binding platform for various chromatin effector proteins, including SetDB1 and HP1a. The concept of an adaptor protein is also evident for small RNA-guided post-transcriptional silencing, where GW182 bridges the sequence-specific AGO-miRNA complex to the cellular pathways that mediate mRNA turnover and translational repression (Sienski, 2015).

The most remarkable aspect about Silencio is its ability to trigger heterochromatin formation if tethered to the nascent RNA. In fact, overcoming the transient interaction of the target RNA with chromatin does not seem to be a trivial task, as tethering of HP1a, which is a potent silencer if recruited directly to promoter regions, is unable to induce heterochromatin formation and silencing under these conditions. Whether Silencio links to activities that tether the target RNA to chromatin or whether this indicates that Silencio and HP1a signal to different downstream effectors with different silencing capacities or kinetics is unclear. In this respect, it is interesting to note that tethering of Ago1 as well as other RITS (RNA-induced transcriptional silencing) complex components to nascent transcripts in S. pombe is very inefficient in initiating heterochromatin as well. A recent report indicates that impairing transcription elongation by loss of the Paf1 complex enables trans-silencing by RITS, presumably as the residence time of the nascent transcript at the target locus is increased (Sienski, 2015).

These data point to a major role of Eggless/SetDB1 in the establishment of H3K9me3-marked heterochromatin downstream from nuclear Piwi and Silencio. Consistent with this, loss of SetDB1, but not of Su(var)3-9, leads to the derepression of piRNA targeted TEs as well as a genome-wide loss of H3K9me3 at targeted TE insertions. In contrast to Su(var)3-9, which harbors an H3K9me3binding chromodomain, SetDB1 seems to require other specificity determinants as a guide (e.g., piRNAs and KRAB zinc fingers). Whether SetDB1 is required for only the initiation or also the maintenance of the H3K9me3 domain is currently unclear and will require sophisticated experimental systems similar to those established in yeast. In terms of TE repression, Su(var)3-9 plays a less important role downstream from Piwi. Instead, it seems to primarily mediate the spreading of the H3K9me3 mark to genomic regions that are flanking piRNA targets. This is very consistent with a central role of Su(var)3-9 in position effect variegation. Also, phenotypically, the described division of labor between these two methyltransferases is consistent: eggless/SetDB1 mutant flies are subviable, and escapers exhibit severe oogenesis defects and are sterile. Instead, Su(var)3-9 mutant flies are viable and fertile (Sienski, 2015).

The precise role of H3K9 methylation in piRNA-guided heterochromatin formation is not clear. It might serve as a binding platform for downstream effector complexes such as chromatin remodelers or histone deacetylase complexes, as has been shown previously. In mammals, it is probably also involved in guiding the de novo DNA methylation at CpG dinucleotides (Matsui et al. 2010). Alternative/additional roles of the H3K9me3 mark could be to aid higher order chromatin structures or control the subnuclear localization of silenced loci as well as prevent the ectopic recombination of TE loci that are identical in sequence at many genomic locations (Sienski, 2015).

Clearly, the molecular events that occur downstream from a Piwi-piRNA complex upon interacting with a complementary target transcript at chromatin is only beginning to be understood. Understanding the mechanistic logic of piRNA-guided heterochromatin formation promises to provide a more general insight into the connections and interplay between heterochromatin formation and transcription (Sienski, 2015).


Search PubMed for articles about Drosophila Panoramix

Batki, J., Schnabl, J., Wang, J., Handler, D., Andreev, V. I., Stieger, C. E., Novatchkova, M., Lampersberger, L., Kauneckaite, K., Xie, W., Mechtler, K., Patel, D. J. and Brennecke, J. (2019). The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation. Nat Struct Mol Biol 26(8): 720-731. PubMed ID: 31384064

ElMaghraby, M. F., Andersen, P. R., Puhringer, F., Hohmann, U., Meixner, K., Lendl, T., Tirian, L. and Brennecke, J. (2019). A heterochromatin-specific RNA export pathway facilitates piRNA production. Cell 178(4): 964-979 e920. PubMed ID: 31398345

Fabry, M. H., Ciabrelli, F., Munafo, M., Eastwood, E. L., Kneuss, E., Falciatori, I., Falconio, F. A., Hannon, G. J. and Czech, B. (2019). piRNA-guided co-transcriptional silencing coopts nuclear export factors. Elife 8. PubMed ID: 31219034

Murano, K., Iwasaki, Y. W., Ishizu, H., Mashiko, A., Shibuya, A., Kondo, S., Adachi, S., Suzuki, S., Saito, K., Natsume, T., Siomi, M. C. and Siomi, H. (2019). Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing. EMBO J: e102870. PubMed ID: 31368590

Ninova, M., Chen, Y. A., Godneeva, B., Rogers, A. K., Luo, Y., Fejes Toth, K. and Aravin, A. A. (2020a). Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing. Mol Cell 77(3): 556-570. PubMed ID: 31901446

Sienski, G., Batki, J., Senti, K. A., Donertas, D., Tirian, L., Meixner, K. and Brennecke, J. (2015). Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery. Genes Dev 29(21): 2258-2271. PubMed ID: 26494711

Teixeira, F. K., Okuniewska, M., Malone, C. D., Coux, R. X., Rio, D. C. and Lehmann, R. (2017). piRNA-mediated regulation of transposon alternative splicing in the soma and germ line. Nature 552(7684): 268-272. PubMed ID: 29211718

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Biological Overview

date revised: 11 June 2022

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