InteractiveFly: GeneBrief

Suppressor of variegation 2-10: Biological Overview | References

BIOLOGICAL OVERVIEW

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation is mediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. This study shows that in Drosophila the small ubiquitin-like protein SUMO and the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. It is proposed that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites (Ninova, 2020a).

The majority of transcriptional control is achieved by transcription factors that bind short sequence motifs on DNA. In many eukaryotic organisms, transcriptional repression can also be guided by small RNAs, which (in complex with Argonaute proteins) recognize their genomic targets using complementary interactions with nascent RNA. Small RNA-based regulation provides flexibility in target selection without the need for new transcription factors and as such is well suited for genome surveillance systems to identify and repress the activity of harmful genetic elements such as transposons (Ninova, 2020a).

Transcriptional repression guided by small RNAs correlates with the deposition of repressive chromatin marks, particularly histone 3 lysine 9 methylation (H3K9me) in S. pombe, plants, and animals. In addition, plants and mammals also employ CpG DNA methylation for target silencing. Small RNA/Ago-induced transcriptional gene silencing is best understood in S. pombe, where the RNA-induced transcriptional silencing complex (RITS) was studied biochemically and genetically. In contrast to yeast, the molecular mechanism of RITS in Metazoans remains poorly understood. Small RNA-induced transcriptional repression mechanisms might have independently evolved several times during evolution and thus might mechanistically differ from that of S. pombe (Ninova, 2020a).

In Metazoans, small RNA-guided transcriptional repression is mediated by Piwi proteins, a distinct clade of the Argonaute family, and their associated Piwi-interacting RNAs (piRNAs). Both in Drosophila and in mouse, the two best-studied Metazoan systems, nuclear Piwis are responsible for transcriptional silencing of transposons. Based on the current model, targets are recognized through binding of the Piwi/piRNA complex to nascent transcripts of target genes. In both Drosophila and mouse, piRNA-dependent silencing of transposons correlates with accumulation of repressive chromatin marks (H3K9me3 and, in mouse, CpG methylation of DNA) on target sequences. These marks can recruit repressor proteins, such as HP1, providing a mechanism for transcriptional silencing. However, how recognition of nascent RNA by the Piwi/piRNA complex leads to deposition of repressive marks at the target locus is not well understood. Several proteins, Asterix (Arx)/Gtsf1, Panoramix (Panx)/Silencio, and Nxf2, were shown to associate with Piwi and are required for transcriptional silencing. Accumulation of H3K9me3 on Piwi/Panx targets requires the activity of the histone methyltransferase SetDB1 (also known as Egg). However, a mechanistic link between the Piwi/Arx/Panx/Nxf2 complex, which recognizes targets, and the effector chromatin modifier has not been established (Ninova, 2020a and references therein).

This study identified Su(var)2-10/dPIAS to provide the link between the Piwi/piRNA and the SetDB1 complex in piRNA-induced transcriptional silencing. In Drosophila, Su(var)2-10 mutation causes suppression of position effect variegation, a phenotype indicative of its involvement in chromatin repression. Su(var)2-10 associates with chromatin and regulates chromosome structure. It also emerged in screens as a putative interactor of the central heterochromatin component HP1, a repressor of enhancer function, and a small ubiquitin-like modifier (SUMO) pathway component. However, its molecular functions in chromatin silencing were not investigated. Su(var)2-10 belongs to the conserved PIAS/Siz protein family, of which the yeast, plant, and mammalian homologs act as E3 ligases for SUMOylation of several substrates. This paper reports the role of Su(var)2-10 in germ cells of the ovary, where chromatin maintenance and transposon repression are essential to grant genomic stability across generations. Germ cell depletion of Su(var)2-10 phenocopies loss of Piwi; both lead to strong transcriptional activation of transposons and loss of repressive chromatin marks over transposon sequences. Su(var)2-10 genetically and physically interacts with Piwi and its auxiliary factors, Arx and Panx. It was demonstrated that the repressive function of Su(var)2-10 is dependent on its SUMO E3 ligase activity and the SUMO pathway. These data point to a model in which Su(var)2-10 acts downstream of the piRNA/Piwi complex to induce local SUMOylation, which in turn leads to the recruitment of the SetDB1/Wde complex. SUMO modification was shown to play a role in the formation of silencing chromatin in various systems from yeast to mammals, including the recruitment of the silencing effector SETDB1 and its co-factor MCAF1 by repressive transcription factors. Together, these findings indicate that the piRNA pathway utilizes a conserved mechanism of silencing complex recruitment through SUMOylation to confer transcriptional repression (Ninova, 2020a).

In both insect and mammals, piRNA-guided transcriptional silencing is associated with the deposition of repressive chromatin marks on genomic targets. In Drosophila, the conserved histone methyltransferase SetDB1 (Egg) is responsible for deposition of the silencing H3K9me3 mark at Piwi targets (Sienski, 2015, Yu, 2015). However, the molecular mechanism leading to the recruitment of SetDB1 by the Piwi/piRNA complex remained unknown. Thus study showed that in Drosophila SUMO and the SUMO E3 ligase Su(var)2-10 act together downstream of the piRNA-guided complex to recruit the histone methyltransferase complex SetDB1/Wde and cause transcriptional silencing. The results suggest a model for the molecular mechanism of piRNA-guided transcriptional silencing in which Su(var)2-10 provides the connection between the target recognition complex composed of piRNA/Piwi/Panx/Arx and the chromatin effector complex composed of SetDB1 and Wde (Ninova, 2020a).

This study has identified a new role for the SUMO pathway in piRNA-guided transcriptional silencing. The SUMO pathway plays important roles in heterochromatin formation and maintenance, and genome stability in different organisms from yeast to humans. Among different functions, SUMO is required for recruitment and activity of the histone methyltransferase complex composed of SetDB1 and MCAF1 (Wde in Drosophila), which confers transposon silencing in mammals. Remarkably, SUMO-dependent recruitment of SetDB1 to TEs in mammalian somatic cells does not require piRNAs but is instead mediated by the large vertebrate-specific family of Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs) that bind specific DNA motifs. Although distinct members of the KRAB-ZFP family recognize different sequence motifs in target transposons, repression of all targets by various KRAB-ZFPs requires the universal co-repressor KAP1/TIF1b (KRAB-associated protein 1). KAP1 is a SUMO E3 ligase, and its auto-SUMOylation leads to SetDB1 recruitment. The current results suggest that Drosophila Su(var)2-10 can be SUMOylated, and SetDB1 and Wde have functional SIMs, suggesting that Su(var)2-10 auto-SUMOylation might induce SetDB1/Wde recruitment. These results suggest that two distinct transposon repression pathways, by DNA-binding proteins and by piRNAs, both rely on SUMO-dependent recruitment of the conserved silencing effector to the target (Ninova, 2020a).

The results in Drosophila and studies in mammals (Ivanov, 2007) suggest that in both clades self-SUMOylation of SUMO E3 ligases might be involved in recruitment of SetDB1 to chromatin. However, these results do not exclude the possibility that the recruitment of SetDB1 is facilitated by SUMOylation of additional chromatin proteins by Su(var)2-10. Studies in yeast led to the 'SUMO spray' hypothesis that postulates that SUMOylation of multiple different proteins localized in physical proximity promotes the assembly of multi-unit effector complexes. Local concentration of multiple SUMO moieties leads to efficient recruitment of SUMO-interacting proteins. According to this hypothesis, multiple SUMO-SIM interactions within a protein complex act synergistically, and thus SUMOylation of any single protein is neither necessary nor sufficient to trigger downstream processes. Assembly of such 'SUMO spray' on chromatin might be governed by the same principles of multiple weak interactions as was recently recognized for the formation of various phase-separated liquid-droplet compartments in the cell. The presence of Su(var)2-10 on a chromatin locus might lead to SUMOylation of multiple chromatin-associated proteins that are collectively required for the recruitment of effector chromatin modifiers. The SUMOylation consensus (ψKxE/D) is very simple and therefore quite common in the fly proteome. Consistent with this, several hundred SUMOylated proteins were identified in proteomic studies in Drosophila. Thus, it is possible that collective SUMOylation of multiple chromatin-associated proteins contributes to recruitment and stabilization of the SetDB1 complex on chromatin (Ninova, 2020a).

The cascade of events leading to repression initiated by target recognition by piRNA/Piwi, followed by interaction with Su(var)2-10 and subsequent SUMO-dependent recruitment of SetDB1/Wde, suggests that the three complexes tightly cooperate. But do these three complexes (Piwi, Su(var)2-10, and SetDB1) always work together, or does each complex have additional functions independent of the other two? Genome-wide analysis suggests that the vast majority of Piwi targets are repressed through SUMO/Su(var)2-10 and, likely, SetDB1/Wde, suggesting that Piwi always requires these other complexes for its function in transcriptional silencing. On the other hand, multiple instances were found of host genes that are repressed by Su(var)2-10 and SetDB1 but do not require piRNAs (Ninova, 2019). Su(var)2-10 and SetDB1 are also expressed outside of the gonads and were implicated in chromatin silencing in somatic tissues that lack an active piRNA pathway. It is speculated that Su(var)2-10 might bind to specific targets directly through its SAP domain or might get recruited by specific DNA-binding proteins, similar to the way SetDB1 is recruited to ERVs by KRAB-ZFP in mammals, though specific factors are yet to be uncovered (Ninova, 2020a).

Though both Drosophila and mouse have nuclear Piwi proteins involved in transcriptional silencing of transposons, these proteins, PIWI and MIWI2, are not one-to-one orthologs. Unlike Drosophila, other insects including the silkworm Bombyx mori, the flour beetle Tribolium castaneum, and the honeybee Apis mellifera encode only two Piwi proteins, and at least in B. mori, these proteins do not localize to the nucleus. These observations suggest that the nuclear Piwi pathway in Drosophila has evolved independently in this lineage. In light of this evolutionary interpretation, the interaction of the Piwi complex and the E3 SUMO ligase Su(var)2-10 indicates that in Drosophila the nuclear piRNA pathway co-opted an ancient mechanism of SUMO-dependent recruitment of the histone-modifying complex for transcriptional silencing of transposons. The molecular mechanism of piRNA-induced transcriptional repression in other clades such as mammals might have evolved independently of the corresponding pathway in flies. It will be interesting to investigate if mammals also use SUMO-dependent recruitment of silencing complexes for transcriptional repression of piRNA targets (Ninova, 2020a).

The SUMO ligase Su(var)2-10 controls hetero- and euchromatic gene expression via establishing H3K9 trimethylation and negative feedback regulation

Chromatin is critical for genome compaction and gene expression. On a coarse scale, the genome is divided into euchromatin, which harbors the majority of genes and is enriched in active chromatin marks, and heterochromatin, which is gene-poor but repeat-rich. The conserved molecular hallmark of heterochromatin is the H3K9me3 modification, which is associated with gene silencing. This study found that in Drosophila, deposition of most of the H3K9me3 mark depends on SUMO and the SUMO ligase Su(var)2-10, which recruits the histone methyltransferase complex SetDB1/Wde. In addition to repressing repeats, H3K9me3 influences expression of both hetero- and euchromatic host genes. High H3K9me3 levels in heterochromatin are required to suppress spurious transcription and ensure proper gene expression. In euchromatin, a set of conserved genes is repressed by Su(var)2-10/SetDB1-induced H3K9 trimethylation, ensuring tissue-specific gene expression. Several components of heterochromatin are themselves repressed by this pathway, providing a negative feedback mechanism to ensure chromatin homeostasis (Ninova, 2020b).

This study shows that in addition to the effects on TE silencing (Ninova, 2020a), Su(var)2-10 and H3K9me3 influence the expression of protein-coding genes. Su(var)2-10-dependent H3K9me3 deposition on TEs affects the expression of genes located in heterochromatin and of euchromatic genes adjacent to TE insertions. Su(var)2-10 is also involved in TE-independent H3K9me3 deposition on host genes, which is essential for the suppression of ectopic expression of tissue-specific genes, thereby conferring correct cell type identity (Ninova, 2020b).

Approximately half of the human genome comprises TE sequences, and the TE fraction is as high as 90% in several plant species. One new TE insertion per generation is estimated to propagate to the offspring. Somatic TE insertions, although difficult to detect, are likely even more prevalent. Thus, TE activity is a major source of genetic variation that can occur on a very short timescale. The effects of TEs on the host transcriptome have been the subject of many studies ever since Barbara McClintock identified 'control' elements that regulate gene expression before genome compositions were known. TEs can disrupt gene expression by inserting into coding regions or into or close to cis-regulatory sequences. TE insertions are not always disruptive: insertions into non-coding regions can bring new regulatory elements that change gene expression patterns, resulting in increased fitness. Instances of positive selection for TE insertions are well documented in Drosophila. TE-derived promoters also drive the expression of numerous mouse and human genes, suggesting that TE insertions can be co-opted into gene regulatory pathways (Ninova, 2020b).

In addition to changes in the DNA sequence, TE insertions may introduce local epigenetic effects. Active TEs are transcriptionally silenced by H3K9 trimethylation and/or DNA methylation. The H3K9me3 mark can spread several kilobases outside the TE region, affecting adjacent cis-regulatory elements of host genes, and thereby interfering with their normal expression. TE insertions with high levels of H3K9me3 are strongly selected against, supporting a model that TEs can alter the expression of host genes through epigenetic changes (Ninova, 2020b).

The finding that Su(var)2-10 is responsible for the deposition of H3K9me3 on TE bodies and flanking sequences allows separation if the effect of direct damage to cis-regulatory elements from the effect on chromatin. Evidence was found that TE insertions can lead to H3K9me3-dependent changes in gene expression, as shown for the jheh3 and frl loci. Notably, the BARI insertion at the jheh3 locus was shown to be positively selected in the D. melanogaster population, indicating that Su(var)2-10-dependent epigenetic silencing caused by a TE insertion can be used for beneficial rewiring of host gene regulatory networks (Ninova, 2020b).

The current results suggest that TEs can rewire gene regulatory networks on a short timescale, at least in part via their effects on chromatin. Euchromatic H3K9me3 peaks due to TE insertions are widespread in Drosophila, indicating that TE insertions may be a common cause of gene regulatory variation. New TE insertions during development generate genomic diversity between different cell types in human and mouse with implications for tumorigenesis and brain development. Future studies are required to elicit the epigenetic effects of somatic TE insertions on gene regulatory networks (Ninova, 2020b).

Heterochromatin domains include nearly 30% of the fly genome. Although relatively gene-poor, heterochromatin hosts several hundred protein-coding genes. Studies of chromosomal rearrangements suggested that heterochromatic localization is required for the proper expression of heterochromatic genes. However, the molecular mechanism of the positive effect of the heterochromatin environment on expression is not fully understood (Ninova, 2020b).

Consistent with previous studies, this study observed many active genes in H3K9me3-rich heterochromatic regions and found that for many active heterochromatic genes, Su(var)2-10-induced H3K9 methylation is not only permissive but also required for proper expression (Ninova, 2020b).

How can the same chromatin mark lead to the repression of genes in euchromatin and activation in heterochromatin? H3K9me3 is present over the gene bodies and regions flanking heterochromatic genes, but is depleted at promoters, which instead carry typical active marks such as H3K4me3 and Pol II occupancy. Thus, H3K9me3 over gene bodies appears to be compatible with transcription. H3K9me3 loss upon Su(var)2-10 GLKD correlated with increased levels of intronic RNAs and the appearance of H3K4me2/3 and Pol II signals in introns, indicating the upregulation of spurious transcripts originating from within host-gene introns. One possible source of such transcripts is the activation of TE promoters that are highly abundant within introns and flanking sequences of heterochromatic genes. It is proposed that transcription from TE promoters located in introns and flanking sequences interferes with proper gene expression through transcriptional interference (Ninova, 2020b).

H3K9me3 loss also disrupted the normal isoform regulation of heterochromatic genes, as was observed both truncated and extended mRNA isoforms with coding potential distinct from the canonical gene mRNA upon the depletion of Su(var)2-10. The activation of cryptic promoters may disrupt proper gene expression through multiple mechanisms, such as reduction in canonical mRNA output or dominant negative effects of the extended or truncated protein isoforms. Not all heterochromatic genes that lose H3K9me3 upon Su(var)2-10 germline knockdown (GLKD) show signs of interfering transcripts or cryptic promoters, indicating that H3K9me3 may have other functions in heterochromatic gene activation. For example, the compaction of heterochromatin by HP1 may bring distant enhancers of heterochromatic genes into physical proximity of promoters to activate expression. The results, combined with previous studies, indicate that genes positioned in heterochromatin require high H3K9me3 levels for proper expression and isoform selection (Ninova, 2020b).

Discrete Su(var)2-10-dependent H3K9me3 peaks are present in a number of euchromatic genes. Some of these peaks have no TEs in their vicinity, and their H3K9me3-based repression is conserved between D. melanogaster and D. virilis, two species that separated >45 million years ago and have no common TE insertions. The expression of many of these TE-independently repressed genes is restricted to specific tissues such as testis, the digestive system, or the CNS, and the loss of H3K9me3 leads to ectopic expression in the female germline. The finding is in line with a recent report that SetDB1 depletion in the female germline was associated with the loss of H3K9me3 and the mis-expression of male-specific genes (Smolko, 2018). H3K9me3, SetDB1, and the SUMO pathway were also implicated in lineage-specific gene expression and cell fate commitment in mammals. These data suggest that a TE-independent H3K9me3 deposition via the SUMO-SetDB1 pathway plays an evolutionarily conserved role in restricting gene expression to proper cell lineages (Ninova, 2020b).

SUMO- and Su(var)2-10-dependent H3K9me3 repression also regulates several factors involved in heterochromatin formation and maintenance, such as SUMO (smt3), Wde, Sov, and CG30403. Wde is the homolog of the mammalian MCAF1/ATF7IP, which is required for the nuclear localization and stability of SetDB1 (Koch, 2009, Timms, 2016) and promotes its methyltransferase activity (Wang, 2003). Drosophila Wde also associates with SetDB1, and their germline depletion results in a similar phenotype, supporting the role of Wde as a conserved SetDB1 co-factor (Koch, 2009, Smolko, 2018). The current data in Drosophila and studies in mammals suggest that SUMO is involved in SetDB1/Wde recruitment to its targets. HP1 is an H3K9me3 reader that is responsible for the structural properties of heterochromatin and also serves as a hub for many other heterochromatin proteins. Both Sov and CG30403 interact with HP1, and Sov is critical for heterochromatin maintenance (Ninova, 2020b).

The genes encoding Wde, SUMO, Sov, and CG30403 reside in euchromatin and are repressed by local H3K9me3. Unlike tissue-restricted genes, which are often completely repressed by Su(var)2-10 in the female germline, these factors are not fully silenced, although they are upregulated upon Su(var)2-10 depletion. The results indicate that these four genes are part of a negative feedback mechanism that controls heterochromatin formation. Negative feedback in biological circuits maintains protein levels within a certain range, providing homeostatic regulation. It is proposed that SUMO-dependent repression of heterochromatin proteins provides such homeostatic regulation to maintain the proper ratio and boundaries of hetero- and euchromatin. According to this model, specific genes, such as wde, act as sensors of the overall H3K9me3 level. Insufficient levels of H3K9 methylation lead to elevated sensor gene expression due to decreased H3K9me3 at their promoters, which in turn enhances H3K9me3 deposition and heterochromatin formation throughout the genome. Concomitant repression of sensor genes ensures that H3K9me3 is restricted to proper genomic domains and does not spread to euchromatic regions that should remain active. Inspection of ENCODE data showed that the mammalian homolog of wde, ATF7IP, is decorated by H3K9me3 in some human cell lines, suggesting that this mode of regulation may be deeply conserved (Ninova, 2020b).

A reminiscent negative feedback loop was identified in yeast. The single H3K9 methyltransferase clr4 is suppressed by H3K9me3 to restrict ectopic spreading of silencing chromatin (Wang, 2015). In mammals, genes encoding proteins from the KRAB-ZFP family of transcriptional repressors reside in H3K9me3- and HP1-enriched loci. Thus, autoregulation of heterochromatin effectors is a conserved mode of chromatin regulation, although the genes involved in the feedback mechanism differ between different organisms. In the future, it will be important to dissect the network architecture of heterochromatin regulation. As heterochromatin formation and maintenance was reported to be disrupted in cancer and during aging, this mechanism may be a promising target of therapeutic interventions (Ninova, 2020b).

H3K9me3 writer enzymes are targeted to genomic loci by different mechanisms. In the case of TE repression in germ cells, piRNAs bound to nuclear Piwi proteins serve as sequence-specific guides that bind complementary nascent transcripts and recruit Su(var)2-10, which induces H3K9me3 deposition by SetDB1. Su(var)2-10 identifies non-TE targets in a piRNA-independent fashion, in agreement with a broader function of Su(var)2-10 in development (Hari, 2001). The observation that H3K9me3 peaks at homologous euchromatic genes are also present in the distantly related D. virilis points to a conserved mechanism of H3K9me3 deposition in host-gene regulation (Ninova, 2020b).

The molecular mechanism of piRNA-independent recruitment of Su(var)2-10 remains to be explored. Su(var)2-10 has a putative DNA binding SAP domain that may be sufficient for its binding to DNA. However, motif enrichment analysis failed to identify a common sequence motif among TE-independent Su(var)2-10 targets (MEME-ChIP), suggesting that different partners may recruit Su(var)2-10 to distinct targets. In mammals, a large family of transcription factors, the KRAB-ZFPs, are responsible for SetDB1 recruitment and H3K9me3 deposition on many different targets, primarily endogenous retroviruses. Individual members of the KRAB-ZFP family influence distinct targets due to differences in DNA-binding specificities of their zinc-finger DNA-binding domains. Notably, SetDB1 recruitment through KRAB-ZFPs occurs through a SUMO-dependent mechanism. The KRAB-ZFP family is vertebrate specific, and there are no known proteins in D. melanogaster that can recruit H3K9me3 activity. A preliminary search for direct Su(var)2-10 interactors using a yeast two-hybrid screen identified several proteins with putative DNA-binding domains. Thus, it is proposed that analogous to the KRAB-ZFP pathway in mammals, Su(var)2-10 may link DNA-binding proteins to the SetDB1 silencing machinery. Future studies are necessary to identify the proteins that guide Su(var)2-10 to target loci and to elucidate TE-independent recruitment mechanisms of the silencing machinery (Ninova, 2020b).

Transcripts immunoprecipitated with Sxl protein in primordial germ cells of Drosophila embryos

In Drosophila, Sex lethal (Sxl), an RNA binding protein, is required for induction of female sexual identity in both somatic and germline cells. Although the Sxl-dependent feminizing pathway in the soma was previously elucidated, the downstream targets for Sxl in the germline remained elusive. To identify these target genes, transcripts associated with Sxl in primordial germ cells (PGCs) of embryos were selected using RNA immunoprecipitation coupled to sequencing (RIP-seq) analysis. A total of 308 transcripts encoded by 282 genes were obtained. Seven of these genes, expressed at higher levels in PGCs as determined by microarray and in situ hybridization analyses, were subjected to RNAi-mediated functional analyses. Knockdown of Neos, Kap-alpha3, and CG32075 throughout germline development caused gonadal dysgenesis in a sex-dependent manner, and Su(var)2-10 knockdown caused gonadal dysgenesis in both sexes. Moreover, as with knockdown of Sxl, knockdown of Su(var)2-10 in PGCs gave rise to a tumorous phenotype of germline cells in ovaries. Because this phenotype indicates loss of female identity of germline cells, Su(var)2-10 is considered to be a strong candidate target of Sxl in PGCs. These results represent a first step toward elucidating the Sxl-dependent feminizing pathway in the germline (Ota, 2017).

A Drosophila PIAS homologue negatively regulates stat92E

Transcriptional activation by (and therefore the physiologic impact of) activated tyrosine-phosphorylated STATs (signal transducers and activators of transcription) may be negatively regulated by proteins termed PIAS (protein inhibitors of activated stats), as shown by experiments with mammalian cells in culture. By using the genetic modifications in Drosophila, in vivo functional interaction of the Drosophila homologs stat92E and a Drosophila PIAS gene (dpias) have been demonstrated. A loss-of-function allele was used and dpias was conditionally overexpressed in JAK-STAT pathway mutant backgrounds. It is concluded that the correct dpias/stat92E ratio is crucial for blood cell and eye development (Betz, 2001).

By matching the available flanking sequence of the P element insertion of the stock l(2)03697 with the 5' untranslated region (UTR) of a cDNA highly homologous to the mammalian PIAS genes, a putative mutant allele of the Drosophila PIAS gene was identified. The P element insertion at the dpias locus (the dpias⁰³⁶⁹⁷ allele) blocks all mRNA formation. Therefore, the dpias⁰³⁶⁹⁷ allele constitutes a strong LOF or a null allele of the dpias gene (Betz, 2001).

Coimmunoprecipitation of mammalian PIAS and tyrosine-phosphorylated STATs has been established, and the interacting region of PIAS3 with STAT3 lies in the center of the molecule embracing a portion of a putative zinc finger domain. By using an in vitro protein association assay, it was found that a similar region of a dPIAS-GST fusion molecule binds to a FLAG-tagged STAT92E protein. The interaction depends on prior activation of the STAT92E protein brought about by the inhibition of tyrosine dephosphorylation with vanadate/peroxide, which was used because natural activation of STAT92E has not been accomplished in cell culture (Betz, 2001).

Because the dpias⁰³⁶⁹⁷ allele is a homozygous lethal, genetic interaction crosses were designed in which flies heterozygous for the recessive dpias⁰³⁶⁹⁷ allele were scored for the possible enhancement or suppression of known phenotypes in JAK-STAT pathway mutants. hop^Tum-l is a dominant hyperactive allele (increased HOP activity at elevated temperature) that causes tumor formation. This tumor formation, which is suppressed by stat92E LOF mutants, results from excessive proliferation of blood cells (plasmatocytes) that form melanotic abdominal tumors in larvae and pupae that can be scored in adults. At 25°C, 37% of heterozygous hop^Tum-l adult females had at least one abdominal tumor. Reduction of a negative activating regulator of this pathway should cause an increase in tumors. The percentage of flies with at least one tumor more than doubled in the hop^Tum-l/+;dpias⁰³⁶⁹⁷/+ genotype compared with the progeny with two WT dpias alleles. Experiments on tumor frequency support the conclusion that dPIAS interacts negatively with the JAK-STAT pathway made overactive by hop^Tum-l: this leads to tumor formation. It is concluded that dPIAS decreases the transcriptional impact of the overactive STAT92E (Betz, 2001).

The role of dpias in eye development was examined because hypomorphic mutants of hop and os have small eyes. Two different lines, GMR-Gal4 and ey-Gal4, in which dpias overexpression depends on Gal4 activation at different times during eye development, were used. When the GMR-Gal4 line was used to drive UAS-dpias(537), no obvious effect on eye size or texture was observed. When UAS-dpias(537) was activated with the ey-Gal4 driver, eye size was severely reduced and the remaining small eye had a rough texture. A doubling of the transgene dosage further aggravated this phenotype and resulted in complete loss of the eyes in most of the surviving progeny. Because ey-Gal4 is active very early in eye development (before cellular differentiation) and GMR-Gal4 at later stages (during cellular differentiation), it is concluded that overexpression of dpias(537) has an effect primarily on cells in the early proliferating eye disc (Betz, 2001).

Whether this occurs because of a decreased activity of the JAK-STAT pathway was investigated. To this end Small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies were crossed to a stock carrying a heat shock-inducible stat92E gene (hs-stat92E) and the progeny were raised under mild heat-shock conditions. A significant rescue of eye size and texture was observed only in progeny that carried the hs-stat92E transgene but not in genotypes without the hs-stat92E transgene segregating from the same cross. Moreover, a similar eye-size rescue effect was achieved by crossing the hop^Tum-l stock with small-eyed UAS-dpias(537)/CyO;ey-Gal4 flies, further bolstering the notion that activated STAT92E is required for eye development and that dPIAS counteracts the activated STAT92E (Betz, 2001).

The effect of replacing both WT copies of the dpias gene with the mutant dpias⁰³⁶⁹⁷ alleles in eyes was examined. The yeast recombinase (flipase) system was used to generate clonal patches of mutant homozygous dpias⁰³⁶⁹⁷ cells (from now on called dpias^-/-) within heterozygous phenotypically WT flies. The lens structure completely fails to develop and is replaced by a heterogeneous bulged-out surface lacking bristles. Partially differentiated lenses surround the border of the clone (Betz, 2001).

The lack of, or abnormal differentiation of, lens structure observed on the surface of dpias^-/- clones, in particular in the clonal border areas, appears to be phenotypically similar to Notch GOF phenotypes. Overexpression of activated Notch delays the differentiation of cone cells, the cells that secrete the lens material. Therefore, it is inferred that cone cell differentiation in surviving dpias^-/- clones might be similarly affected (Betz, 2001).

Sections through dpias^-/- clones reveal that cellular differentiation into photoreceptors and other cell types have failed, especially in the center of these clones. Along the clonal borders, partially differentiated ommatidia could be seen with incomplete sets of photoreceptors. In sections through dpias^-/- clones, retinal cellular differentiation fails and is replaced by a heterogeneous cell mass. Other clones have apparently undergone either apoptotic or necrotic cell death, as indicated by frequent scars. Which of these diverse phenotypes might be caused by unopposed overactive STAT92E remains to be seen. It will be important to learn whether members of the mammalian PIAS genes are playing related roles in STAT-dependent tumor suppression, cell death, and differentiation (Betz, 2001).

Thus, with regard to eye development, a dramatic developmental role of dpias-stat92E interaction is found. Overexpression of dPIAS early (driven by ey-Gal4) aborts eyes, but loss of stat92E function later (by overexpression of dPIAS under the control of GMR-Gal4) has no apparent detrimental effect on cell growth or survival. Therefore, factors controlling stat92E function must be normally balanced in a critical time window in early eye development. Further increased expression of dpias or coupling with heterozygosity for the stat92E LOF allele stat⁰⁶³⁴⁶ leads to transformation events with antennae frequently replacing eyes. LOF alleles of the Drosophila JAK-kinase hop of increasing severity cause the same sequence of increasing phenotypic abnormalities. Moreover, os¹, a hypomorphic LOF allele of a JAK-STAT pathway ligand, results in small eyes. This phenotype can be partially suppressed and the eye size increased by reducing the dpias gene dosage, implying that with no transgenic intervention, dPIAS and STAT92E naturally interact in eye formation and eye determination (Betz, 2001).

The overgrowth of plasmatocytes and melanotic abdominal tumor formation caused by the hop^Tum-l allele presumably depends on too much activated STAT92E, because stat92E LOF mutants such as stat^HJ suppress tumor formation. By the same logic, it is inferred that dPIAS regulates the number of active STAT92E molecules, because increased dPIAS decreases tumor formation and decreased dPIAS increases tumor formation, indicating that HOP, STAT92E, and dPIAS act together in this pathway. This type of behavior -- genetic removal increasing tumorigenesis and overexpression reducing tumorigenesis -- is characteristic of genes in mammals that are labeled tumor-suppressor genes. By this definition, dpias would be a tumor suppressor. Recent widespread reports of persistently active STAT3 in a variety of human tumors and the demonstration of an engineered constitutively active STAT3 as an oncogene coupled with the present results predict that mutations in human PIAS3 might very well allow for persistent activation of STAT3, resulting in tumor formation. This interpretation is further supported by recent findings (Hari, 2001). Certain transheteroallelic dpias [Su(var)2-10] LOF alleles in otherwise genetically WT backgrounds caused melanotic tumors in third instar larvae (Betz, 2001).

The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family

The conserved heterochromatic location of centromeres in higher eukaryotes suggests that intrinsic properties of heterochromatin are important for chromosome inheritance. Based on this hypothesis, mutations in Drosophila melanogaster that alter heterochromatin-induced gene silencing were tested for effects on chromosome inheritance. This study describes the characterization of the Su(var)2-10 locus, initially identified as a Suppressor of Position-Effect Variegation. Su(var)2-10 is required for viability, and mutations cause both minichromosome and endogenous chromosome inheritance defects. Mitotic chromosomes are improperly condensed in mutants, and polytene chromosomes are structurally abnormal and disorganized in the nucleus. Su(var)2-10 encodes a member of the PIAS protein family, a group of highly conserved proteins that control diverse functions. SU(VAR)2-10 proteins colocalize with nuclear lamin in interphase, and little to no SU(VAR)2-10 is found on condensed mitotic chromosomes. SU(VAR)2-10 is present at some polytene chromosome telomeres, and FISH analyses in mutant polytene nuclei revealed defects in telomere clustering and telomere-nuclear-lamina associations. It is proposed that Su(var2-10 controls multiple aspects of chromosome structure and function by establishing/maintaining chromosome organization in interphase nuclei (Hari, 2001).

Search PubMed for articles about Drosophila Su(var)2-10

Betz, A., et al. (2001). A Drosophila PIAS homologue negatively regulates stat92E. Proc. Natl. Acad. Sci. 98: 9563-9568. PubMed ID: 11504941

Hari, K. L., Cook, K. R. and Karpen, G. H. (2001). The Drosophila Su(var)2-10 locus regulates chromosome structure and function and encodes a member of the PIAS protein family. Genes Dev 15(11): 1334-1348. PubMed ID: 11390354

Koch, C. M., Honemann-Capito, M., Egger-Adam, D. and Wodarz, A. (2009). Windei, the Drosophila homolog of mAM/MCAF1, is an essential cofactor of the H3K9 methyl transferase dSETDB1/Eggless in germ line development. PLoS Genet 5(9): e1000644. PubMed ID: 19750210

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

Ninova, M., Godneeva, B., Chen, Y. A., Luo, Y., Prakash, S. J., Jankovics, F., Erdelyi, M., Aravin, A. A. and Fejes Toth, K. (2020b). The SUMO ligase Su(var)2-10 controls hetero- and euchromatic gene expression via establishing H3K9 trimethylation and negative feedback regulation. Mol Cell 77(3): 571-585 e574. PubMed ID: 31901448

Ota, R., Morita, S., Sato, M., Shigenobu, S., Hayashi, M. and Kobayashi, S. (2017). Transcripts immunoprecipitated with Sxl protein in primordial germ cells of Drosophila embryos. Dev Growth Differ 59(9):713-723. PubMed ID: 29124738

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

Smolko, A. E., Shapiro-Kulnane, L. and Salz, H. K. (2018). The H3K9 methyltransferase SETDB1 maintains female identity in Drosophila germ cells. Nat Commun 9(1): 4155. PubMed ID: 30297796

Timms, R. T., Tchasovnikarova, I. A., Antrobus, R., Dougan, G. and Lehner, P. J. (2016). ATF7IP-mediated stabilization of the histone methyltransferase SETDB1 is essential for heterochromatin formation by the HUSH complex. Cell Rep 17(3): 653-659. PubMed ID: 27732843

Wang, H., An, W., Cao, R., Xia, L., Erdjument-Bromage, H., Chatton, B., Tempst, P., Roeder, R. G. and Zhang, Y. (2003). mAM facilitates conversion by ESET of dimethyl to trimethyl lysine 9 of histone H3 to cause transcriptional repression. Mol Cell 12(2): 475-487. PubMed ID: 14536086

Wang, J., Reddy, B. D. and Jia, S. (2015). Rapid epigenetic adaptation to uncontrolled heterochromatin spreading. Elife 4. PubMed ID: 25774602

Yu, Y., Gu, J., Jin, Y., Luo, Y., Preall, J. B., Ma, J., Czech, B. and Hannon, G. J. (2015). Panoramix enforces piRNA-dependent cotranscriptional silencing. Science 350(6258): 339-342. PubMed ID: 26472911

date revised: 2 January 2023

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