InteractiveFly: GeneBrief

smt3: Biological Overview | References

BIOLOGICAL OVERVIEW

SUMO (Small Ubiquitin-like Modifier, a 90 amino acid protein) is a protein modifier that is vital for multicellular development. This study presents the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, over 140 largely novel SUMO conjugates were found in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo mutant germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, SUMO was found to be required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. It was further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in the screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, a database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development (Nie, 2009).

Post-translational protein modification adds layers of complexity to macromolecular function. One way of modifying proteins is by joining the ubiquitin family proteins to lysine residues, generating branched proteins. One such ubiquitin-like protein, SUMO (small ubiquitin-related modifier), displays remarkable versatility in modulating target protein function. Many proteins are targeted for covalent modification by SUMO, which consequently modulates many cellular processes (Geiss-Friedlander, 2007; Zhao, 2007; Martin, 2007; Nie, 2009 and references therein).

Genetic analysis has revealed essential roles for SUMO in the survival and development of organisms ranging in complexity from yeast to mammals. In S. cerevisiae, mutations in genes encoding SUMO pathway enzymes are lethal, while mutations in the corresponding genes in S. pombe severely impair growth. Deletion of genes encoding enzymes required for SUMO conjugation in C. elegans leads to embryonic lethality, while reduction of the SUMO conjugating enzyme levels in Drosophila, zebrafish, and mouse results in developmental defects (Epps, 1998; Nacerddine, 2005; Nowak, 2006; Nie, 2009 and references therein).

The Drosophila genome encodes a single form of SUMO (referred to as Drosophila SUMO, but also known as Drosophila Smt3), which shares 52% and 73% sequence identity with human SUMO-1 and SUMO-2, respectively (Huang, 1998). Drosophila and human SUMO family proteins are at least partially interchangeable, demonstrating a high level of SUMO pathway conservation between evolutionarily distant organisms (Lehembre, 2000). To date, only a few Drosophila proteins are known to be sumoylated -- the transcription factors Dorsal (Bhaskar, 2000; Bhaskar, 2002), Tramtrack (Lehembre, 2000), Vestigial {Takanaka, 2005}, SoxNeuro (Savare, 2005), and Medea (Miles, 2008); the gypsy insulator interacting proteins Mod(mdg4) and CP190 (Capelson, 2006) and the bi-functional tRNA charging enzyme glutamylprolyl-tRNA synthetase [EPRS, (Smith, 2004)]. SUMO appears to have diverse roles in the Drosophila life cycle, including the regulation of transcription and the modulation of the immune response (Nie, 2009 and references therein).

While SUMO is present throughout development, early Drosophila embryos contain particularly high concentrations of maternally contributed SUMO and the enzymes required for SUMO conjugation (Lehembre, 2000; Long, 2000; Hashiyama, 2009), suggesting that sumoylation may play particularly critical roles at this stage of fly development. Previous global analyses of SUMO substrates in S. cerevisiae and mammalian cultured cells have produced extensive lists of novel sumoylation targets. To date, however, there are no published studies that document the spectrum of sumoylated proteins in a specific developmental setting in a multicellular organism (Nie, 2009 and references therein).

To broaden understanding of the function of sumoylation in early Drosophila development, a mass spectrometry-based global identification of sumoylation targets in early embryos was performed; over 140 direct sumoylation targets were found. Among the identified SUMO target proteins are players in many processes essential to embryonic development, including proteins involved in Ras signaling, cell cycle control, and embryonic patterning. To determine the functional significance of the identified sumoylated proteins, genetic, cell culture and immunolocalization studies were carried out, obtaining evidence for roles of SUMO in these same three processes. Thus, the proteomic, genetic, and cellular studies presented in this study all converge to suggest that SUMO coordinates key aspects of early metazoan development (Nie, 2009).

The Ras signaling cascade is activated by a variety of RTKs including EGFR, and controls cell proliferation and differentiation as well as a large number of developmental patterning processes, such as patterning of the eggshell. Activation of EGFR in the dorsal follicle cells during oogenesis leads to the sequential activation of Ras, Raf, MEK, and MAPK, and results in the upregulation of RTK target genes. Complex positive and inhibitory feedback loops ultimately result in the specification of the dorsal follicle cells, which later secrete the dorsal eggshell, including the dorsal appendages (Nie, 2009 and references therein).

Previous genetic screens for mutations that enhance the eggshell ventralization phenotype of a weak hypomorphic Ras1 allele suggested a role for SUMO in the Ras pathway downstream of EGFR activation. In the current analysis of the recessive sumo mutant phenotype, fused or single dorsal appendages were observed, indicative of eggshell ventralization and consistent with the attenuation of EGFR signaling. Since the eggs under study resulted from sumo germ-line clones, the observed eggshell defect could reflect a function for SUMO upstream of EGFR in the production or secretion by the germ line of EGFR ligands. However, since sumo mutant clones are also present in the follicle cells of the germ-line clone egg chambers, the eggshell ventralization phenotype that was observed is also consistent with a role for SUMO downstream of EGFR activation in the follicle cells. Interestingly, sumoylation pathway proteins in C. elegans were also shown to interact with the Ras signaling pathway (Poulin, 2005). Cell culture experiments support a role for protein sumoylation in Ras signaling that is downstream of EGFR and upstream of, or parallel to, Ras activation. SUMO may directly modulate Ras1 function since Ras1 was found in the proteomic analysis and confirmed as a sumoylation substrate in a bacterial sumoylation assay (Nie, 2009).

Sumoylation is implicated in cell cycle regulation in many organisms. In this study, diverse nuclear cleavage defects were observed in sumo germ-line clone embryos suggestive of multiple roles for SUMO in coordinating the chromosome cycle. The phenotypes, including chromosome hypercondensation, aberrant segregation, and polyploidy, are reminiscent of the defects observed in Ubc9-deficient mouse embryos and Drosophila embryos mutant for pias, a possible SUMO ligase (Hari, 2001), indicating conservation of SUMO cell cycle functions in metazoan evolution. This study also demonstrated a requirement for SUMO in cell cycle progression in cultured cells and in larval imaginal discs by RNAi-mediated SUMO knockdown. While the cell proliferation defect in SUMO mutant wing discs could result from a requirement for SUMO for the function of many of the same cell cycle proteins found in the proteomic screen of early embryos, it could also reflect a role for SUMO in the function of Vg, a previously identified wing disc sumoylation target known to be required for wing growth (Nie, 2009).

In agreement with the diverse cell cycle defects in sumo mutant embryos and other tissues, a spectrum of cell cycle regulators involved in multiple stages of the cell cycle were identified in these SUMO proteomic screens. For example, the failure of cultured cells to progress to G2/M could reflect a role for SUMO in DNA replication, which is consistent with the finding that PCNA, RFC2, Topoisomerase I, and Topoisomerase II are all targets of sumoylation. A role for SUMO in the function of Polo kinase could further explain some of the observed cell cycle defects since Polo has multiple roles in the cell cycle. Other sumoylation targets identified in the screen, including PP2A, Arp3, Cofilin (Twinstar), Mago Nashi, and Profilin, are also consistent with multiple roles of SUMO in mitosis (Nie, 2009).

The requirement for SUMO throughout mitosis is further supported by its dynamic, mitotic stage-dependent, localization. At prometaphase and metaphase, sumoylated proteins are concentrated at the kinetochores and ICR, partially co-localizing with Polo. Ubc9 co-localizes with SUMO at the kinetochore-centromeric regions during mitosis, suggesting that active sumoylation is taking place at those locations. It is likely that many kinetochore and centromere localized proteins are targeted by SUMO, and cycles of sumoylation and de-sumoylation may help to propel unidirectional mitotic progression (Nie, 2009).

While a number of studies have connected sumoylation to centromere and kinetochore functions, spindle midbody localization of SUMO has not been widely reported. The midbody is a structure derived from the spindle midzone that contains proteins indispensable for cytokinesis. SUMO association with the midbody, which was have observed in both syncytial embryos and cultured cells beginning with anaphase and extending through cytokinesis, therefore argues for a role of sumoylation in the completion of cell division. The midbody proteome has been dissected recently in mammalian cells, revealing a large collection of proteins, including membrane associated proteins, microtubule associated proteins, and kinases. Homologs of a number of these proteins, such as Arp3, Cofilin (Twinstar), Mago Nashi, Polo, PP2A, and Profilin, were all identified in the Drosophila SUMO proteomic screens, reinforcing the notion that SUMO is involved in midbody function (Nie, 2009).

Cytokinesis does not occur in nuclear cleavage stage embryos. However, the midbody has an important role in maintaining the separation of telophase sister nuclei, a process that could be related to the formation of pseudocleavage furrows at the end of each nuclear cleavage cycle. Disruption of midbody function in SUMO deficient embryos may therefore account for some of the mitotic defects that were observed in the syncytial embryo, including polyploidy (Nie, 2009).

Diverse patterning defects were observed among the sumo germ-line clone embryos that developed a cuticle. In accordance with this observation, three absolutely critical patterning proteins, Dorsal, Bicoid, and Hunchback, are among the sumoylated proteins detected in early embryo extracts. Previous studies have shown that sumoylation of Dorsal potentiates its activity during the immune response perhaps by making it a more potent transcriptional activator. While an earlier study showed that the loss of Ubc9 results in a hunchback-like anterior patterning phenotype and defective nuclear transport of Bicoid, this study is the first to show that Hunchback, and its activator Bicoid, are direct SUMO conjugation targets. Thus, it is possible that sumoylation of these transcription factors plays a direct role in anterior patterning (Nie, 2009).

Posterior patterning and germ line specification depend upon the posterior localization of the oskar transcript. Several oskar mRNP components, including Mago Nashi, Tsunagi, Cup, Hrb27C, and Smaug, were identified as sumoylation targets, which have essential roles in the regulation of oskar mRNA localization and translation. This interesting and novel finding suggests a role of SUMO in regulating the functions of maternal mRNA by modifying components of oskar mRNP, and therefore could explain some of the pleiotropic defects observed in the embryonic patterning of embryos resulting from sumo mutant GLCs (Nie, 2009).

The oskar mRNP is one of several instances in which multiple members of the same complex appear to be direct targets of sumoylation. For example, the screen turned up several members of the multi-aminoacyl-tRNA synthetase complex, as well as multiple ribosomal proteins. Screens for sumoylation targets in S. cerevisiae have similarly detected multiple sumoylation targets in the same complex. This suggests that oligomeric protein complexes can be targeted as a whole for sumoylation and/or that sumoylation may have a general role in stabilizing protein complexes (Nie, 2009).

In contrast to previous studies in yeast and mammalian cell culture, relatively few transcription factors were identified in this study. This difference in fact accurately reflects the unique metabolic state of the pre-cellularization embryo. During the first two hours of Drosophila embryonic development, rapid nuclear divisions depend upon a complex dowry of maternally supplied proteins, as transcription of the zygotic genome has not yet begun. Instead, the proper localization and accurately regulated translation of maternally supplied mRNAs is essential for establishing the system of positional information that will later direct the spatially regulated transcription of the zygotic genome. Thus, the relatively small and selective group of sumoylated transcription factors, along with the large number of factors that control mRNA translation and localization found in the screen, is consistent with regulatory roles for SUMO in this critically important stage of fly development (Nie, 2009).

In conclusion, these genetic, cellular, and proteomic studies of sumoylation suggest mechanisms for known biological roles of the SUMO pathway and also uncover novel connections between sumoylation, signal transduction, the cell cycle, and development. Furthermore, the SUMO conjugated proteome should serve as a rich resource for those studying the roles of sumoylation in metazoan development (Nie, 2009).

Degringolade, a SUMO-targeted ubiquitin ligase, inhibits Hairy/Groucho-mediated repression

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs; see Model for SUMO-directed ubiquitination by the conserved STUbL family). STUbLs bind to SUMOylated proteins via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. This study shows that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho [Gro/transducin-like enhancer (TLE)] during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. These findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes (Abed, 2011).

Transcriptional cofactors are essential for the function of sequence-specific transcription factors and are part of the machinery required to execute temporally coordinated gene expression programs. Regulation of cofactor recruitment and activity is emerging as a major level of gene expression regulation. For example, Hairy/Enhancer of split/Deadpan (HES) family repressors are the primary transducers of the Notch signalling pathway that has a central role in patterning, stem cell development, and is misregulated in cancers. A well-studied case is the Drosophila repressor Hairy, a typical HES family member, which encodes a basic helix-loop-helix (bHLH) Orange repressor required for embryonic segmentation and adult peripheral nervous system (PNS) specification. Hairy-mediated repression is dependent on its ability to recruit cofactors. For example, Hairy recruits the corepressor Groucho (Gro) through it C-terminal WRPW domain, an interaction that is essential for periodic repression of fushi tarazu (ftz). In addition, Hairy recruits dCtBP and dSir2 through its PLSLV and basic domains, respectively. While these cofactors are required for Hairy-mediated repression, they exhibit context-dependent recruitment and function. Interestingly, some cofactors enhance Hairy-mediated repression (e.g., Gro and dSir2), whereas others are required to refine Hairy's function (e.g., dCtBP and dTopors). Consistent with this, it was found that most of the genomic loci bound by Hairy in the context of Kc cells exhibit corecruitment of dSir2 and dCtBP, but are not co-bound by Gro. However, the mechanisms that regulate context-selective cofactor association with Hairy or that may regulate cofactor activities are largely unknown (Abed, 2011).

A possible mechanism is that post-translational modification of Hairy regulates its association with a given cofactor and determines its overall function. One such modification is ubiquitylation that in many cases regulates the stability of transcription factors. However, ubiquitylation can also serve as a regulatory modification that does not lead to degradation, but affects protein-protein interaction or intracellular localization (Ikeda, 2008). Similarly, SUMOylation is a post-transcriptional modification that is involved in the regulation of gene expression and is mediated by the SUMO-specific E1-, E2-, and E3-SUMO ligase enzymes. Both ubiquitin and SUMO modifications are highly regulated. These two modifications can also be connected through proteins collectively termed SUMO-targeted ubiquitin ligases (STUbLs; Sun, 2007; Geoffroy, 2009). STUbLs are RING proteins that bind non-covalently to the SUMO moiety of SUMOylated proteins via their N-terminal SUMO interaction motif (SIM) domains, and subsequently target the SUMOylated protein for ubiquitylation via their RING domain. Thus, STUbLs are able to 'sense' SUMOylated targets and modify them by ubiquitylation. The observation that STUbLs are associated with transcription complexes suggests that their function is directly linked to regulation of gene expression. For example, the STUbL protein RNF4 was found to be a positive regulator of steroid hormone transcription. Importantly, STUbLs are structurally and functionally conserved, as the mouse and human RNF4 proteins can substitute for their yeast orthologs in functional assays. STUbLs are required for the correct assembly of kinetochores, for the cell's ability to cope with genotoxic stress, and for genome stability. RNF4 is highly expressed in the stem cell compartment of the developing gonads and brain, and its expression is enriched in progenitor cells, likely representing its role in 'stemness'. Recently, RNF4 was shown to regulate the SUMO- and ubiquitin-mediated degradation of PML and PML-RAR. However, the role of STUbL proteins in transcription during development of higher eukaryotes is largely unknown (Abed, 2011).

This study shows that Degringolade (Dgrn), the only Drosophila STUbL protein, physically and genetically interacts with Hairy and its cofactor Gro and antagonizes Hairy/Gro-mediated repression during segmentation and neurogenesis. Ubiquitylation of Hairy by Dgrn affects choice of cofactor by preventing Gro, but not dCtBP, from binding to Hairy. It was also found that Dgrn specifically targets SUMOylated Gro, alleviates Gro-dependent transcriptional repression, and suppresses Gro functions in vivo throughout development. DamID chromatin profiling experiments revealed that the antagonism between Dgrn and Gro is aimed at a broad array of genomic loci, suggesting that Gro-Dgrn antagonism is of general importance beyond Dgrn's interaction with Hairy (Abed, 2011).

Dgrn binds directly to Hairy and is capable of ubiquitylating Hairy in a reconstituted system and in cells. The recognition motif for Dgrn within Hairy maps to Hairy's basic region and requires a specific positive charge (Arg³³). This motif is transferable and functionally conserved, not only in Hey and other HES proteins (e.g., E(spl)m8 and Dpn), but also in dMyc and other bHLH proteins including the activator Sc. Therefore, it may reflect a general property of bHLH recognition by STUbL proteins. No evidence was found for direct SUMOylation of the HES and bHLH proteins: bacterially purified Hairy and Dgrn proteins interact, anti-SUMO antibodies fail to detect SUMOylated Hairy, Hairy's mobility in SDS-PAGE is not altered upon incubation with the dUlp1 SUMO peptidase, and mutating putative SUMOylation sites within Hairy does not alter its recognition or ubiquitylation by Dgrn. Accordingly, this study found that Dgrn's interaction with Hairy is mediated through Dgrn's RING motif independent of the SIM domains. Similarly, the yeast STUbL Slx5-Slx8 recognizes the MATα2 repressor independent of SUMOylation (Xie, 2010). Hairy recognition by Dgrn/RNF4 is also different from its recognition of substrates, such as GST-SUMO or PML, that involves direct SUMOylation of the targeted protein and requires the Dgrn/RNF4 SIM domains (Sun, 2007; Wang, 2009; Abed, 2011 and references therein).

Importantly, SUMOylation and the SIM motifs are necessary for Dgrn to target SUMOylated Gro and for Dgrn's suppression of HES/Gro repression in vivo, it is likely that the SIM domains interact with the poly-SUMO chain itself (Geoffroy, 2010). Dgrn possessing two separate recognition modules is reminiscent of the dual recognition properties described for the RING protein UBR1 (E3alpha). As the current dogma is that STUBLs recognize (via their SIM domains) poly SUMO chain(s) rather than the substrate, the dual recognition mechanism observed with Dgrn may further substantiate substrate recognition and specificity (Abed, 2011).

The contribution of each SIM domain is additive, and a Dgrn mutant harbouring a single SIM domain is capable of binding to GST-SUMO, as well as conjugating Hairy, although to a lesser extent than wild-type Dgrn. Correspondingly, it was found that elevated levels of SUMOylated proteins are detected in dgrn null embryos (Barry, 2011; Abed, 2011).

As an ubiquitin ligase, Dgrn catalyses the formation of mixed poly-ubiquitin chains on Hairy. This ubiquitylation does not map to Hairy's basic region, its putative SUMOylation sites, or to a single Lys residue. Importantly, this poly-site ubiquitylation does not affect Hairy protein stability or integrity, but rather selectively inhibits Gro binding to Hairy. Furthermore, in cells in which Dgrn protein levels are reduced via RNAi, Hairy protein levels are also decreased compared with control cells, suggesting that Dgrn is likely required for Hairy expression. This is different from dTopors, a Hairy-associated PHD-RING finger protein, which catalyses Lys⁴⁸-linked chains and regulates Hairy turnover. Further work will be required to determine the exact molecular events and the role that specific ubiquitin chain linkage has in Dgrn's ability to inhibit Gro from binding to Hairy in vivo (Abed, 2011).

Despite extensive efforts, ubiquitylated Gro forms were not identifed in this study. Nonetheless, the data suggest that Dgrn specifically targets the SUMO chains on Gro, which likely serve as a signal for Gro sequestration by as yet to be identified machinery (Abed, 2011).

In transcription assays, Dgrn is a potent activator of ac and Sxl transcription, a function that requires its catalytic activity. Dgrn antagonizes Hairy-, Dpn-, and Gro-mediated repression in vivo. Dgrn specifically targets SUMOylated Gro, Dgrn function inversely correlates with SUMOylation, and a reduction in SUMO levels impairs Dgrn's ability to fully alleviate repression. Thus, Dgrn's activity suppresses the local repressive chromatin structure generated by repressors, their associated cofactors, and the SUMO pathway. It was also found that expression of Dgrn^HC/AA can inhibit the activation mediated by Da/Sc, suggesting that Dgrn is required to alleviate repression by endogenous repressors and/or corepressors. This fits well with the observation that reduction in Dgrn protein levels via RNAi impairs Da/Sc-mediated activation. While this study focused on Dgrn's effects on the repressive machinery, it is also possible that part of Dgrn ligase activity enhances the function of activators and/or coactivators. For example, Dgrn efficiently ubiquitylates the pro-neural activator Sc, and significant activation of the ac or Sxl promoters requires only Dgrn along with either Da or Sc (Abed, 2011).

These data suggest that part of Dgrn's activity is aimed specifically at the Gro corepressor that is shared by all HES proteins. First, Dgrn-mediated ubiquitylation of Hairy prevents Gro recruitment to Hairy. Second, Dgrn specifically targets SUMOylated Gro and its associated Gro oligomers for sequestration. Specifically, it was found that the detected level of Gro protein is dependent on Dgrn and the method of protein extraction. For example, in embryos that lack Dgrn (dgrn^DK) and when protein extracts are made in RIPA buffer, the detected levels of Dgrn in dgrn^DK embryos is higher compared with that of wild type. However, if the extraction is performed in 4% SDS buffer, the detected levels of Gro protein in wild-type and dgrn^DK embryo extracts is equal. Likewise, the signal detected for Gro using immunostaining in embryos is highly complementary to the milder RIPA extraction. dgrn^DK embryos show an increased signal compared with wild-type embryos (as in the absence of Dgrn, less Gro is sequestered and more Gro molecules are available for detection by the antibody). The majority of Gro appears to be sequestered. Since only 90% of Gro can be recovered after co-transfection of Dgrn using SDS extraction, the possibility cannot be ruled out that a fraction of the SUMOylated Gro is degraded. All together, these data suggest that Dgrn is required for Gro sequestration and that loss of Dgrn 'liberates' sequestered Gro (Abed, 2011).

While the data support a model in which Dgrn targets SUMOylated Gro for sequestration, Dgrn may also regulate the molecular machinery that is required for Gro SUMOylation and subsequently sequestration. Furthermore, while it is established that STUbL targets SUMOylated proteins for ubiquitylation and degradation, it is also possible that Dgrn has an impact on the SUMO pathway and SUMO isopeptidases (Abed, 2011).

Gro and its mammalian orthologs, the transducin-like enhancers of split (TLE1-4) proteins, repress transcription via several mechanisms, including oligomerization to generate local repressive chromatin structures, and are negatively regulated by phosphorylation. This study found that site-specific phosphorylation used by RTK signalling to inactivate Gro is not a prerequisite for Dgrn activity. However, the details surrounding other phosphorylations, the role of site-specific SUMOylation of Gro, and the molecular machinery mediating sequestration, as well as Dgrn's effects on specific Gro-dependent repressive mechanisms await further studies (Abed, 2011).

In vivo, it was found that Dgrn antagonism of Gro is highly relevant for embryonic segmentation, PNS development, and sex determination, processes that are regulated by Gro (Barry, 2011). Indeed, Dgrn can suppress the gain-of-function phenotypes of Gro, as well as rescue the phenotypes associated with tissue-specific inactivation of Gro using RNAi transgenes. The genomic targets of Gro and Dgrn are distinct from that of dCtBP or dSir2, and that 38% of Gro direct targets are shared with Dgrn. Thus, it is predicted that Dgrn will be involved in other HES-independent, but Gro-regulated, processes as well. It is likely that both proteins have unique regulatory roles during early development. This notion stems from observations that each of the factors has exclusive, non-overlapping, genomic binding sites, and that neither of the two genes can functionally rescue the embryonic lethality associated with mutants of the other protein (i.e., Gro cannot rescue the female sterility associated with dgrn null females, and reducing the dose of Dgrn does not rescue the lethality associated with the gro^E48 mutant) (Abed, 2011).

Finally, an open question is how can the activity of a general corepressor be temporally and spatially regulated during development. The data to date suggest a model in which Dgrn has a regulatory role. Since it is suggested that SUMOylation enhances Gro-mediated repression (Ahn, 2009), one can imagine that ATP-dependent SUMOylation of Gro within the repressor complex will result in local augmented repression. However, concomitantly, SUMOylation will promote Dgrn recruitment, and subsequent inactivation of the repression complex on chromatin or in its vicinity, ensuring that local SUMO-augmented repression is limited in time and space. It is speculated that this type of transcriptional regulation will be instrumental to define and sharpen patterning borders throughout development (Abed, 2011).

Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg

The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. Recent findings have identified PcG proteins both as targets for modification by the small ubiquitin-like modifier (SUMO) protein and as catalytic components of the SUMO conjugation pathway. This study found that the SUMO-conjugating enzyme Ubc9 binds to Scm and that this interaction, which requires the Scm C-terminal sterile α motif (SAM) domain, is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx), and efficient PRE recruitment requires an intact Scm SAM domain. Global reduction of sumoylation augments binding of Scm to the PRE. This is likely to be a direct effect of Scm sumoylation because mutations in the SUMO acceptor sites in Scm enhance its recruitment to the PRE, whereas translational fusion of SUMO to the Scm N terminus interferes with this recruitment. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced, decreased expression of Ubx and partial haltere-to-wing transformation phenotypes were observed. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE (Smith, 2011).

Alternative sumoylation sites in the Drosophila nuclear receptor Usp

The Ultraspiracle protein (Usp), together with an ecdysone receptor (EcR) forms a heterodimeric ecdysteroid receptor complex, which controls metamorphosis in Drosophila. Although the ecdysteroid receptor is considered to be a source of elements for ecdysteroid inducible gene switches in mammals, nothing is known about posttranslational modifications of the receptor constituents in mammalian cells. Up until now there has been no study about Usp sumoylation. Using Ubc9 fusion-directed sumoylation system, Usp was identified as a new target of SUMO1 and SUMO3 modification. Mutagenesis studies on the fragments of Usp indicated that sumoylation can occur alternatively on several defined Lys residues, i.e., three (Lys16, Lys20, Lys37) in A/B region, one (Lys424) in E region and one (Lys506) in F region. However, sumoylation of one Lys residue within A/B region prevents modification of other residues in this region. This was also observed for Lys residues in carboxyl-terminal fragment of Usp, i.e. comprising E and F regions. Mass spectrometry analysis of the full-length Usp indicated that the main SUMO attachment site is at Lys20. EcR, the heterodimerization partner of Usp, and muristerone A, the EcR ligand, do not influence sumoylation patterns of Usp. Another heterodimerization partner of Usp - HR38 fused with Ubc9 interacts with Usp in HEK293 cells and allows sumoylation of Usp independent of the direct fusion to Ubc9. Taken together, it is proposed that sumoylation of DmUsp can be an important factor in modulating its activity by changing molecular interactions (Bielska, 2012).

Sumoylation of Drosophila transcription factor STAT92E

STAT92E is an essential transcription factor in Drosophila for the development of several organs and the immune system. The JAK/STAT pathway employs different evolutionary conserved regulatory mechanisms to control biological processes. Numerous transcription factors in both mammals and invertebrates have been shown to be either activated or inhibited by a covalent modification with a small ubiquitin-like modifier (Sumo). This study show that Drosophila STAT92E is modified by Sumo at a single lysine residue 187 in S2 cells. Mutation of Lys187 increases the transcriptional activity of STAT92E, thus suggesting that sumoylation of STAT92E has a repressive role in the regulation of the JAK/STAT pathway in Drosophila (Grönholm, 2010).

Removal of endogenous Sumo E3 ligase dPIAS by dsRNA has been shown to increase the STAT92E activity on TotM promoter approximately to the same level as K187R mutation in STAT92E, suggesting that sumoylation of STAT92E is involved in dPIAS-mediates inhibition of STAT92E. The mechanisms of how sumoylation is affecting STAT92E are presently unknown, but several possible mechanisms can be envisioned. The sumoylation site Lys187 is localized in the coiled coil domain, which in the mammalian system is involved in nuclear transport of STATs. The coiled coil domain of STATs is composed of 4 α-helixes that are pointing out from the DNA-bound STAT dimer, forming a hydrophilic surface able to interact with other molecules. Thus, sumoylation of Lys187 may interrupt the interaction between STAT92E and its transcriptional coregulators or the proteins involved in its nuclear translocation. Alternatively, sumoylation may lead to the recruitment of histone deacetylases to the promoter or allow the interaction with a transcription repression complex similarly to Drosophila Sp3. The effect of sumoylation on DNA-binding properties of STAT92E was not analyzed, but the coiled coil domain is not contacting DNA, suggesting that direct effects upon the promoter-binding activity are less likely (Grönholm, 2010).

SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation

Chromatin insulators are special regulatory elements involved in modulation of enhancer-promoter interactions. The best studied insulators in Drosophila require Suppressor of Hairy Wing [Su(Hw)], Modifier of mdg4 [Mod(mdg4)] and centrosomal 190 kDa (CP190) proteins to be functional. These insulator proteins are colocalized in nuclear speckles named insulator bodies. This study demonstrates that post-translational modification of insulator proteins by small ubiquitin-like modifier (SUMO) and intact CP190 protein is crucial for insulator body formation. Inactivation of SUMO binding sites in Mod(mdg4)-67.2 leads to the inability of the mutant protein and Su(Hw) to be assembled into insulator bodies. In vivo functional tests show that a smaller amount of intact Mod(mdg4)-67.2, compared with the mutant protein, is required to restore the normal activity of the Su(Hw) insulator. However, high expression of mutant Mod(mdg4)-67.2 completely rescues the insulator activity, indicating that sumoylation is not necessary for enhancer blocking. These results suggest that insulator bodies function as a depot of sumoylated proteins that are involved in insulation and can facilitate insulator complex formation, but are nonessential for insulator action (Golovnin, 2012).

Posttranslational modification by SUMO has been shown to regulate subcellular localization of many targets, including RanGAP, PML, SATB2 and others. This study presents data that SUMO is necessary for co-localizing the Su(Hw), Mod(mdg4)-67.2, and CP190 proteins in nuclear speckles, named insulator bodies. Previously an opposite model has been proposed according to which sumoylation of Mod(mdg4)-67.2 and CP190 leads to disruption of insulator bodies. This model was mainly based on the observation that, in diploid cells from the larval brain, mutations in the gene encoding Ubc9 restored aggregation of the CP190 protein in the mod(mdg4)^u1 background. This study found that inactivation of Mod(mdg4)-67.2 did not affect the ability of CP190 to form insulator bodies in S2 cells (Golovnin, 2012).

mod(mdg4)^u1 mutation also did not affect CP190 incorporation into the insulator bodies in diploid cells of wing and eye imaginal discs. Thus, the significance of Mod(mdg4)- 67.2 for CP190 recruitment to the insulator bodies is confined to diploid cells of the larval brain. To test the role of sumoylation in the formation of insulator bodies, the lwr5 mutation, generated by a single amino acid substitution in the Ubc9 region (R104H) located on the loop between strand 7 and helix B, has been used. This region of Ubc9 is required for the interaction of its active site with the substrate. Although untested, it appears that R104H makes the surface of the mutant enzyme (Ubc95) more hydrophobic, thereby strengthening binding interactions for certain enzyme-substrate pairs. Thus, lwr5 is not a null-mutation in the gene, and Ubc95 can either increase or decrease sumoylation, depending on the protein substrate. Therefore, additional studies are required to demonstrate role of Ubc95 in the formation of insulator bodies in the imaginal disks of larvae (Golovnin, 2012).

The data provide evidence for a critical role of CP190 and a passive role of Su(Hw), a DNA-binding protein, in the formation of insulator bodies. In addition to Su(Hw), CP190 forms complexes with dCTCF that is also co-localized in the insulator bodies. Thus, it is likely that Mod(mdg4)-67.2 and CP190 proteins recruit DNA-binding dCTCF and Su(Hw) proteins to the insulator bodies (Golovnin, 2012).

As shown previously, SUMO is necessary for the formation of PML nuclear bodies (PML-NBs). These bodies are formed due primarily to the self-assembly ability of the PML N-terminal domain. Moreover, SUMO-1 modification of PML was shown to target the protein from the nucleoplasm to the NBs. The occurrence of both sumoylation sites and SUMO-interacting motifs (SIMs) in the PML protein provides a basis for the network of interactions that constitute the nucleation event for subsequent recruitment of sumoylated proteins and SIM-containing proteins (Golovnin, 2012).

Cells that lack PML are unable to form NBs, with other NB components remaining diffusely distributed in the nucleus. While analysis of the CP190 sequence suggests the presence of two SIMs, no direct interaction was observed between CP190 and SUMO in vitro. At the same time, CP190 and Mod(mdg4)-67.2 contain several protein-protein interaction domains, including BTB/POZ that might be involved in direct interaction with many DNA-binding transcription factors, such as Su(Hw) and dCTCF, to facilitate their assembly into the insulator bodies. It is noteworthy that heat shock has proved to induce redistribution of CP190 to the nuclear periphery, in complex with SUMO. This is evidence that the formation of insulator bodies requires interactions with additional proteins, which are disrupted as a result of heat shock treatment (Golovnin, 2012).

Sumoylation is essential for the functional activity of proteins in transcriptional repression, activation, and recruitment of modifying complexes. This study has demonstrated that inactivation of sumoylation sites in the Mod(mdg4)-67.2 protein does not affect its functional activity in the insulator complex. This finding is in accordance with the previous observation that only 10% of Su(Hw) binding sites coincide with SUMO on polytene chromosomes (Golovnin, 2012).

This study confirms the role of Mod(mdg4)-67.2 in recruiting the Su(Hw) protein to the insulator bodies and insulators. When the mutant Mod(mdg4)-67.2 protein was expressed at a low level, Su(Hw) binding was reduced, whereas low expression of the wild-type Mod(mdg4)-67.2 protein was sufficient for completely restoring Su(Hw) binding to insulators. Therefore, the assembly of the Su(Hw) and Mod(mdg4)-67.2 proteins in insulator bodies is essential for subsequent recruitment of insulator complexes to DNA. A higher level of the mutant Mod(mdg4)-67.2 protein increases the probability of formation of the Su(Hw)/Mod(mdg4)-67.2 complex out of insulator bodies, thereby providing for more effective binding of the Su(Hw) and mutant Mod(mdg4)-67.2 proteins to the insulators (Golovnin, 2012).

Taken together, these results support the model of insulator bodies as a depot of proteins involved in transcription regulation and insulation. According to these results, the insulator proteins can interact and form complexes without SUMO. However, partial sumoylation of the Mod(mdg4)-67.2 and CP190 proteins lead to further aggregation of the protein complexes in insulator bodies. The sumoylated Mod(mdg4)-67.2 and CP190 proteins interact with Su(Hw) and recruit it to the insulator bodies. The insulator bodies possibly protect the insulator complex from degradation and facilitate the formation of complexes between Su(Hw)/Mod(mdg4)-67.2/CP190 and other transcription factors. 'Mature' insulator complexes may then transiently interact with the chromatin fibril and detach from the insulator bodies by means of desumoylation. As was suggested for PML bodies, proteins deposited in the insulator bodies may be used during cell stress. For example, it was found that heat shock treatment induced relocation of CP190 from the insulator bodies to the nuclear periphery but did not affect the insulator complexes bound to DNA. Such an unusual relocation of the CP190 protein resulted in a diffuse distribution of the Su(Hw) and Mod(mdg4)-67.2 proteins. Thus, it appears that insulator proteins may have an as yet unknown yet role in cell response to heat shock stress. During DNA replication, a large amount of insulator proteins is required for newly synthesized chromosomes. It is possible that desumoylation of insulator bodies during DNA replication results in the release of protein complexes that form functional insulators on the newly synthesized DNA.Further studies are required to verify this model (Golovnin, 2012).

The SUMO pathway promotes bHLH proneural factor activity via a direct effect on the Zn finger protein

During development, proneural transcription factors of the bHLH family are required to commit cells to a neural fate. In Drosophila neurogenesis, a key mechanism promoting sense organ precursor (SOP) fate is the synergy between proneural factors and their coactivator Senseless in transcriptional activation of target genes. This study presents evidence that post-translational modification by SUMO enhances this synergy via an effect on Senseless protein. Senseless is a direct target for SUMO modification, and mutagenesis of a predicted SUMOylation motif in Senseless reduces Senseless/proneural synergy both in vivo and in cell culture. It is proposed that SUMOylation of Senseless via lysine 509 promotes its synergy with proneural proteins during transcriptional activation, and hence regulates an important step in neurogenesis leading to the formation and maturation of the SOPs (Powell, 2012).

SUMO enhances SENS's ability to promote proneural activity in reporter gene assays and to promote neurogenesis in vivo. The data suggest that SUMO modification promotes proneural gene autoregulation and is also likely to be important in the regulation of downstream proneural target genes. SUMOylation has a positive effect and deSUMOylation a negative effect on transcriptional activation by proneural/DA/SENS ternary complexes in S2 cells. In contrast, no effect was observed on proneural protein activity in the absence of SENS, suggesting that SENS is the target for SUMO. This is supported by the interactions between SUMO and SENS in the HeLa cell relocalisation and yeast two-hybrid assays, the direct covalent interaction between SENS and SUMO detected in S2 cells and the in vitro SUMOylation assay, and the effect of mutating a putative SUMOylation motif in SENS Zn finger 4 (Powell, 2012).

The latter identified a lysine (K509) in the fourth Zn finger as a candidate for a major SUMOylation site in the SENS sequence. Mutation of this lysine to arginine (K509R) resulted in disruption of SUMO-dependent SENS interaction in the HeLa cell assay, a SENS protein refractory to SUMO stimulation in the S2 cell transcriptional assay, and reduced genetic interaction between SENS and SUMO in vivo. Furthermore, evidence from yeast two-hybrid assays and from analysis of S2 lysates for SENSK509R suggested that additional unidentified lysines may also be SUMOylated. Interestingly, the basal transcriptional synergy between SENS and proneural/DA heterodimers observed in S2 cells appears to be largely dependent on endogenous SUMOylation, as the synergy is strongly reduced by ULP1 cotransfection. Consistent with this, proteomic analysis has shown that S2 cells express SUMO, UBC9 and UBA2 (SAE1) proteins (Powell, 2012).

SUMO affects the activity of the proneural/DA/SENS ternary complex. While the evidence suggests that SENS is the target of SUMOylation, the possibility that the other proteins of the complex may also be SUMOylated is not ruled, but at present there is no evidence for this. Notably, the ATO sequence has no ΨKxD/E motifs, while SC has been shown to be unaffected by SUMOylation in a separate study. DA has three potential SUMOylation motifs, but mutation of each of these does not affect proneural/DA/SENS synergy (Powell, 2012).

The evidence suggests that SUMOylation of SENS enhances transcriptional synergy via an effect on the proneural/SENS ternary complex itself. How might SENS SUMOylation mediate this increase in transcriptional synergy? SUMOylation can exert a positive effect on transcriptional activation by various mechanisms including alteration of subcellular localisation and mediation of interaction with transcriptional coactivators or DNA. In the present case, it is suggested either (1) SUMOylation increases the affinity of SENS for the proneural protein heterodimer hence favouring formation of the more transcriptionally active ternary complex, (2) SUMOylation increases the transcriptional activation or DNA-binding ability of the ternary complex perhaps by inducing a conformational change or (3) SUMO simply stabilises SENS. SUMO is known to modulate protein-protein interactions in other systems: for example SUMOylation of RanGAP1 promotes binding of RanB2 either by creating or exposing a binding site, while NMR studies have provided direct evidence of a SUMOylation-induced conformational change in Thymine-DNA glycosylase. The identified SUMO site of SENS (K509) is within the fourth Zn finger. This is significant because the Zn finger has been shown to be unimportant for DNA binding by SENS but contributes to the transcriptional synergy mediated by proneural/SENS interaction. It is conceivable therefore that SUMOylation at this site increases the affinity of SENS for proneural/Da heterodimers. This would be similar to the proposed enhanced interaction between the TEA family transcription factor Scalloped and its coactivator Vestigial upon SUMOylation of the latter (Powell, 2012).

A major effect of SENS (and therefore SUMO) in promoting SOP specification appears to be via promoting proneural/DA activation of autoregulatory enhancers. This proneural/SENS autoregulatory synergy is thought to have an important role in bypassing the negative regulatory effects of the Hairy/E(SPL) (HES) bHLH repressor proteins downstream of Notch signalling. It is interesting to note therefore that another role for SUMOylation in SOP specification has recently been identified in relation to HES repressors. A model has been proposed in which the repressive activity of HES proteins during neurogenesis (as well as segmentation and sex determination) is disrupted by the SUMO-targeted Ubiquitin ligase (STUbL), Degringolade (DGRN). DGRN binds to SUMOylated Groucho (GRO), the corepressor of HES. This interaction, as well as ubiquitination of the HES proteins, is thought to disrupt the HES-GRO interaction, leading to increased neurogenesis. Hence these two SUMO-dependent mechanisms (i.e. increased SENS coactivation and decreased HES repression) may work in a complementary manner to enhance neurogenesis. It will be important to determine how SOP-specific SUMOylation is regulated in order to elucidate the developmental mechanisms involved (Powell, 2012).

As well as acting as a proneural coactivator, SENS directly represses some target genes via binding to S box motifs. It is therefore conceivable that SUMO can relieve SENS repression of its targets by its promotion of ternary proneural-SENS formation, effectively sequestering SENS from binding to its target S boxes. For example, SENS directly represses the SOP-specific gene, rhomboid (rho), activation of which is crucial for the EGFR-dependent recruitment of secondary SOPs during neurogenesis. ATO indirectly activates rho expression in larval abdominal SOPs by binding SENS and preventing it from binding and repressing the rho enhancer. If for example SUMOylation enhances SENS binding to ATO, then it may play a role in activation of rho and other direct targets of SENS repression (Powell, 2012).

SENS belongs to the GPS (Gfi1/Pag-3/SENS) family of proteins and its mammalian orthologues are the oncogenes, Gfi1 and Gfi1b. The Gfi proteins differ from SENS in containing transcriptional repression SNAG domains near their N-termini, and Gfi1 and Gfi1b have been 03 reported to act mainly as transcriptional repressors. Despite these differences, in the mammalian peripheral nervous system Gfi1 functions in close connection to proneural factors. For example it works in concert with Atoh1 (the mammalian homologue of ATO) in the specification of inner ear hair cells. Gfi1 also has a crucial role in formation of retinal ganglion cells in the mammalian eye, working downstream of a different ATO homologue, Atoh7. Gfi1 also has key developmental roles in the lung and intestine, working together with the mammalian AC/SC homologue Ascl1 in pulmonary neuroendocrine cell production and with Atoh1 in the production of secretory cells of the intestine (Powell, 2012).

It has been suggested that Gfi1 and the mammalian proneural proteins may act as transcriptional coactivators in a similar way to the Drosophila proteins although direct evidence for this is lacking. If corroborated, such interactions could conceivably be modulated by SUMO in a similar mechanism to that which has been found in Drosophila. Interestingly, this is supported by the observation that, like SENS, Gfi1 associates with SUMO pathway proteins including the SUMO-conjugating enzyme UBC9 in a yeast two-hybrid assay although no other evidence has so far been reported for SUMOylation of Gfi1. While SENS has four C-terminal Zn fingers, 19 Gfi has six. The sixth Zn finger of Gfi1 is not needed for DNA binding, and is equivalent to the fourth Zn finger of SENS. This is the location of the putative SUMOylated lysine (K509) in SENS which is completely conserved in the context of the SUMOylation motif in Zn finger 6 of Gfi1. In conclusion, it is possible that Gfi1 activity is modulated by SUMOylation, and this could have an effect via a molecular mechanism similar to that which have been identified for SENS (Powell, 2012).

The SUMO protease Verloren regulates dendrite and axon targeting in olfactory projection neurons

Sumoylation is a post-translational modification regulating numerous biological processes. Small ubiquitin-like modifier (SUMO) proteases are required for the maturation and deconjugation of SUMO proteins, thereby either promoting or reverting sumoylation to modify protein function. This study shows a novel role for a predicted SUMO protease, Verloren (Velo), during projection neuron (PN) target selection in the Drosophila olfactory system. PNs target their dendrites to specific glomeruli within the antennal lobe (AL) and their axons stereotypically into higher brain centers. This study uncovered mutations in velo that disrupt PN targeting specificity. PN dendrites that normally target to a particular dorsolateral glomerulus instead mistarget to incorrect glomeruli within the AL or to brain regions outside the AL. velo mutant axons also display defects in arborization. These phenotypes are rescued by postmitotic expression of Velo in PNs but not by a catalytic domain mutant of Velo. Two other SUMO proteases, DmUlp1 and CG12717, can partially compensate for the function of Velo in PN dendrite targeting. Additionally, mutations in SUMO (smt3) and lesswright (which encodes a SUMO conjugating enzyme) similarly disrupt PN targeting, confirming that sumoylation is required for neuronal target selection. Finally, genetic interaction studies suggest that Velo acts in SUMO deconjugation rather than in maturation. This study provides the first in vivo evidence for a specific role of a SUMO protease during neuronal target selection that can be dissociated from its functions in neuronal proliferation and survival (Berdnik, 2012).

Protein sumoylation plays an important role in a wide range of cellular processes, including transcription, chromosome organization and function, DNA repair, nuclear transport, signal transduction, and cell cycle progression. Since its discovery, several hundred sumoylation substrates have been identified, including proteins localized to the nucleus, cytoplasm, or at the plasma membrane. Recent studies have shown that many of these sumoylated substrates are crucial for neuronal development and function. However, because the major components of the sumoylation pathway are essential for cell viability, it is challenging to examine the specialized functions of these enzymes and hence the effects of sumoylation in vivo (Berdnik, 2012).

In this study, from a forward genetic screen using a powerful mosaic analysis technique, a predicted SUMO protease, Velo, was identified that regulates dendrite and axon targeting in postmitotic neurons in vivo. Several lines of evidence indicate that Velo controls neuronal morphogenesis by regulating protein sumoylation. First, the catalytic domain of the protease is required for its function in neurons. Second, the dendrite targeting phenotypes can partially be rescued by two other predicted SUMO proteases from two evolutionarily separable branches. SUMO proteases from the Ulp1 family predominantly function in SUMO maturation and deconjugation of SUMO from mono-sumoylated substrates, while Ulp2-like proteases deconjugate SUMO protein^S from poly-SUMO chains. Interestingly, overexpression of both Ulp1 and Ulp2 family proteases was able to rescue the velo dendrite phenotypes. Third, two other components of the sumoylation pathway, SUMO itself and the unique E2 conjugating enzyme Lesswright (Lwr), are also required cell-autonomously for PN dendrite targeting. Fourth, SUMO and Lwr exhibit dosage-sensitive interactions with Velo; velo mutant dendrite phenotypes were suppressed by reducing SUMO or lwr gene dosage by half. Indeed, the nature of these genetic interactions suggests that Velo acts primarily to reverse sumoylation via SUMO deconjugation rather than to promote sumoylation via SUMO maturation (Berdnik, 2012).

It has previously been shown that the knockdown of the Drosophila SUMO protease Ulp1 and overexpression of human SENP7 result in a change of total SUMO conjugates in cultured cells (Smith, 2004; Shen, 2009). Similar experiments were performed to test the biochemical activity of Velo as a SUMO protease by overexpressing Velo in cultured cells. No significant changes were detected in the overall spectrum of SUMO conjugates upon Velo overexpression. It is possible that Velo activity requires a cofactor that is absent in cultured cells, or that Velo's substrates are absent in cultured cells. For these reasons and other technical hurdles, such as the lack of a Velo-specific antibody and difficulty to express the large Velo protein in bacteria, biochemical evidence for Velo acting as a SUMO protease is still missing. The possibility cannot be ruled out that some of the effects of Velo on PN dendrite and axon targeting are caused by its action on substrates unrelated to the SUMO pathway (Berdnik, 2012).

Although velo, SUMO and lwr mutant PNs exhibit aberrant dendrite targeting, their phenotypes are not identical. One possibility for the phenotypic differences could be due to the redundant action of SUMO proteases either between members of the same branch or even the two distinct branches. For example, CG12717, the closest homolog of Velo, or the Ulp1-related DmUlp1 could act redundantly with Velo during PN target selection. This is consistent with the fact that the overexpression of transgenes for both proteases can partially revert velo mutant dendrite phenotypes. However, the Drosophila genome contains only one gene encoding for SUMO and one for an E2 conjugating enzyme. Therefore, their loss-of-function phenotypes are more severe. Another possibility for the phenotypic differences observed in velo, SUMO and lwr mutant PNs could be attributed to the differential perdurance of these proteins in single neurons generated by MARCM. Finally, the two members of the sumoylation pathway examined in this study act in opposite ways with Velo: SUMO and Lwr promote, whereas Velo reverts, sumoylation. This feature implies that the dynamics of sumoylation are essential for dendrite and axon targeting: too much or not enough sumoylation are both harmful to PNs and cause neuronal mistargeting. Although all three possibilities can contribute, the last one might contribute most to the observed phenotypic differences (Berdnik, 2012).

The closest human homolog to Velo is SENP7. SENP7 localizes to the nucleoplasm, consistent with the findings regarding Velo protein distribution. The catalytic domain of SENP7 is essential for its protease activity. Biochemical assays revealed that this protease functions preferably during deconjugation of poly-SUMO chains (Lima, 2008; Shen, 2009). The biological role of poly-SUMO chains is still largely unknown in eukaryotes and few substrates have been identified. SUMO chain formation requires internal lysines within sumoylation consensus sites and is not required for viability in budding yeast during vegetative growt. However, SUMO polymers play a structural role during meiosis in yeast and mitosis in mammalian cells. Moreover, the attachment of poly-SUMO chains to a substrate can promote its subsequent ubiquitylation and degradation, thereby acting as ubiquitylation signals in the turnover of SUMO targets. It is speculated that Velo acts likely in the deconjugation of poly-SUMO chains because of the sequence similarities to SENP7. However, roles for poly-SUMO chains in neurons and crosstalks between sumoylation and ubiquitination pathways during neuronal target selection remain to be determined (Berdnik, 2012).

Further elucidation of the mechanism by which Velo regulates PN dendrite and axon targeting requires identification of its target substrate(s). Because Velo-HA localizes to the nucleus, the potential substrate is likely a nuclear protein. Numerous studies have demonstrated a role for sumoylation regulating transcription. For example, the E3 SUMO ligase and transcriptional coregulator Protein Inhibitor of Activated Stat3 (Pias3) controls rod photoreceptor development and differentiation in the mouse retina by regulating transcription factors via sumoylation. Furthermore, several transcription factors have been shown to regulate PN dendrite target selection when misexpressed or mutated. Another likely set of substrates for Velo includes factors involved in chromosome organization and function. Indeed, it has recently been shown that SMC1, a cohesin subunit required for sister chromatid cohesion during mitosis and meiosis, and the chromatin remodeling factor Rpd3, a class 1 histone deacetylase (HDAC1) involved in chromatin integrity, play roles during PN targeting. Future studies on candidate genes that exhibit similar neuronal targeting errors, together with biochemical and proteomic approaches, might uncover potential Velo substrates, and provide further insight into how sumoylation participates in the precise wiring of the olfactory circuit (Berdnik, 2012).

Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen

Polycomb group (PcG) proteins dynamically define cellular identities through epigenetic repression of key developmental genes. PcG target gene repression can be stabilized through the interaction in the nucleus at PcG foci. This study report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. In the absence of SUMO, Pc foci coagulate into larger aggregates. Conversely, loss of function of the SUMO peptidase Velo disperses Pc foci. Moreover, SUMO and Velo colocalize with PcG proteins at PREs, and Pc SUMOylation affects its chromatin targeting, suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci (Gonzalez, 2014).

Heterochromatic breaks move to the nuclear periphery to continue recombinational repair

Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. This study shows that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/Smc6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote (Eyu, 2015).

Nuclear architecture contributes to HR repair of certain types of DSBs in budding yeast. Specifically, most DSBs exhibit Brownian motion and remain in the nucleoplasm during HR, but persistent DSBs are shunted to the nuclear periphery after resection. This relocalization has been observed in conditions where HR repair is effectively stalled, such as in the absence of a donor sequence for repair or after fork collapse. Whether relocalization is a physiological response to DSBs is still controversial, and the existence of similar roles for the nuclear periphery in multicellular eukaryotes has not been addressed (Eyu, 2015).

Pericentromeric heterochromatin occupies about 30% of fly and human genomes and is characterized by large contiguous stretches of repeated sequences (transposons and 'satellite' repeats) and the 'silent' epigenetic marks H3K9me2/3 and Heterochromatin Protein 1 (HP1a in Drosophila). While pericentromeric heterochromatin is absent in budding yeast, it represents a major threat to genome stability in multicellular eukaryotes. Thousands to millions of identical repeated sequences on different chromosomes can engage in ectopic recombination and generate chromosome rearrangements (e.g., acentric and dicentric chromosomes) during DSB repair. Previous work has identified a mechanism that promotes HR repair while preventing aberrant recombination in Drosophila. Early HR steps (resection and ATRIP/TopBP1 recruitment) occur quickly within the heterochromatin domain, but later steps (Rad51 recruitment) occur only after repair sites have relocalized to outside the domain. Relocalization of heterochromatic DSBs also occurs in mouse cells, suggesting that this mechanism is conserved. It is proposed that relocalization prevents aberrant recombination by separating damaged DNA from similar repeats on non-homologous chromosomes, while promoting 'safe' exchanges with the sister chromatid or homolog. Removing heterochromatic proteins (e.g., Smc5/6) results in relocalization defects, abnormal recruitment of Rad51 inside the heterochromatin domain, and massive aberrant recombination between heterochromatic sequences, revealing the importance of this pathway to genome stability. Whether heterochromatic DSBs relocalize to a specific subnuclear compartment was unclear, and the mechanisms responsible for relocalization and the regulation of HR progression were unknown (Eyu, 2015).

These studies reveal the nuclear periphery as a specialized site for repairing heterochromatic DSBs in Drosophila. DSBs leave the heterochromatin domain and relocalize to nuclear pores or INMPs to continue HR repair, and this process is mediated by STUbL/RENi proteins associated with these nuclear periphery components. This study identified the Nup107-160 sub-complex and Koi and Spag4 INMPs as specific anchoring sites for the STUbL/RENi complex Dgrn/dRad60 and for repair sites. Further, recruitment of dRad60 to the nuclear periphery relies on Dgrn, and both physically associate with Smc5/6 in response to damage. This suggests that interactions between Smc5/6 and Dgrn/dRad60 stabilize the association of heterochromatic DSBs with the nuclear periphery. Finally, Nse2 and dPIAS SUMO ligases and SUMO are required for both relocalizing DSBs and preventing Rad51 recruitment inside the heterochromatin domain (Eyu, 2015).

It is proposed that SUMOylation of one or more HR components after resection, generates a temporary block to Rad51 recruitment inside the heterochromatin domain to prevent ectopic recombination. Relocalization to the nuclear periphery isolates the broken DNA, presumably together with its homologous template (sister chromatid and/or homolog) to complete 'safe' repair. STUbL might mediate the removal of this block by ubiquitylating poly-SUMOylated components, and inducing their proteasome-mediated degradation or recognition by other repair proteins. Potential SUMOylated targets include histones, RPA, Mdc1/Mu2, Smc5/6 subunits, Blm, and other repair and heterochromatin components. Inactivation of this pathway causes instability of repeated sequences and chromosome aberrations, revealing its critical role in heterochromatin repair and genome integrity. Importantly, inactivation of this pathway also leads to disrupted micronuclei, potentially contributing to DNA damage and genome instability in cancer cells (Eyu, 2015).

Aspects of this pathway are surprisingly similar to the mechanism that targets persistent DSBs to the nuclear periphery in S. cerevisiae, including the role of Smc5/6 and SUMO. This likely results from common signaling mechanisms, such as SUMOylation of repair components following extensive resection. However, this similarity is unexpected because budding yeast lacks the long stretches of pericentromeric repeats that present a major challenge for DSB repair in Drosophila and human cells, as well as H3K9 methylation and HP1 proteins that are required for spatial and temporal regulation of heterochromatic HR repair. Remarkably, a pathway utilized by yeast to deal with a rare class of 'persistent' DSBs, collapsed forks, or eroded telomeres, is now emerging as one of the most important mechanisms to safeguard genome stability in multicellular eukaryotes (Eyu, 2015).

Su(var)2-10 and the SUMO pathway link piRNA-guided target recognition to chromatin silencing

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 (Eggless)/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. 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 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. 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 (Eggless)/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. 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 and promotes its methyltransferase activity. 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. 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. 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. 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).

Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae

To counter systemic risk of infection by parasitic wasps, Drosophila larvae activate humoral immunity in the fat body and mount a robust cellular response resulting in encapsulation of the wasp egg. Innate immune reactions are tightly regulated and are resolved within hours. To understand the mechanisms underlying activation and resolution of the egg encapsulation response and examine if failure of the latter develops into systemic inflammatory disease, parasitic wasp-induced changes in the Drosophila larva were correlated with systemic chronic conditions in sumoylation-deficient mutants. It has been reported that loss of either Cactus, the Drosophila (IkappaB) protein or Ubc9, the SUMO-conjugating enzyme, leads to constitutive activation of the humoral and cellular pathways, hematopoietic overproliferation and tumorogenesis. This study reports that parasite infection simultaneously activates NF-kappaB-dependent transcription of Spätzle processing enzyme (SPE) and cactus. Endogenous Spätzle protein (the Toll ligand) is expressed in immune cells and excessive SPE or Spätzle is pro-inflammatory. Consistent with this function, loss of Spz suppresses Ubc9- defects. In contrast to the pro-inflammatory roles of SPE and Spätzle, Cactus and Ubc9 exert an anti-inflammatory effect. Ubc9 maintains steady state levels of Cactus protein. In a series of immuno-genetic experiments, the existence of a robust bidirectional interaction between blood cells and the fat body was demonstrated, and it is proposed that wasp infection activates Toll signaling in both compartments via extracellular activation of Spätzle. Within each organ, the IkappaB/Ubc9-dependent inhibitory feedback resolves immune signaling and restores homeostasis. The loss of this feedback leads to chronic inflammation. These studies not only provide an integrated framework for understanding the molecular basis of the evolutionary arms race between insect hosts and their parasites, but also offer insights into developing novel strategies for medical and agricultural pest control (Paddibhatla, 2010).

Parasitic wasps are a large group of insects that typically attack other insects. Because of the absolute dependence on their insect hosts, parasitic wasps are of enormous commercial interest and can replace insecticides to control insect pests. The motivation of this study was to gain a clearer understanding of how insect larvae respond to attacks of these natural enemies. Using an immuno-genetic approach in Drosophila, this study found that the same Toll-dependent NF-kappaB mechanism that rids Drosophila of microbial infections also defends the host against metazoan parasites. However, because of critical differences in their size and mode of entry, the combination of immune responses summoned in the two cases is different. While phagocytosis and systemic humoral responses (the latter originating from the fat body and in the gut) are the principal mechanisms of host defense against bacteria and fungi, the development of parasitic wasp eggs is blocked primarily by encapsulation response (Paddibhatla, 2010).

Data is presented that demonstrate the critical requirement of the humoral arm in both the activation and resolution of egg encapsulation. The bi-directional interaction between the blood cells and the fat body occurs via cell non-autonomous effects of SPE/Spz, where these secreted proteins synthesized in one compartment can activate immune signaling in the other. Recent reports corroborate a signaling role for Spz derived from blood cells in the expression of antimicrobial peptides from the larval fat body in response to microbes. Because activation/deactivation of both immune arms is accomplished via the IkappaB/Ubc9-dependent feedback loop that has both, cell autonomous and cell non-autonomous effects, it is proposed that this shared mechanism allows efficient coordination between the immune organs and helps restore normal immune homeostasis within the infected host (Paddibhatla, 2010).

The mechanism that coordinates the activation and resolution of both immune arms after parasite infection involves a balance between the positive (SPE) and negative (Cactus) components. Infection induces nuclear localization of Dorsal and Dif, and the transcription of both SPE (which resolves over time) and cactus (transcription levels off). This Cactus-dependent regulation is essential for the downregulation of SPE transcription and the termination of the encapsulation response. The negative feedback loop of Cactus in flies is similar to the one identified for IkappaBα in mammalian cells (Paddibhatla, 2010).

In Ubc9 mutants, the stability of Cactus protein is compromised, and Toll signaling persists during the extended larval life. Accordingly, knockdown of Cactus in blood cells (Hml>cactus^RNAi) promotes inflammation, aggregation and melanization. It is proposed that loss of immune homeostasis leads to constitutive SPE expression and activation of Spätzle, which promotes the development of chronic inflammation. Thus, sumoylation serves an anti-inflammatory function in the fly larva (Paddibhatla, 2010).

This study has identified at least two distinct biological roles of sumoylation: first, an essential role in blood cells, where the post-translational modification curbs proliferation in the lymph gland in the absence of infection. This conclusion is also strongly supported by restoration of normal hematopoietic complement in mutants expressing wild type Ubc9 only within a limited lymph gland population. Second, sumoylation is essential to sustain significant, steady state levels of Cactus. In mammalian cells, sumoylation of IkappaBα protects it from antagonistic, ubiquitination-mediated degradation. The results are consistent with the mammalian model where Cactus sumoylation would be expected to modulate its half-life (Paddibhatla, 2010).

Cytokine activation and function are hallmarks of the normal inflammatory response in mammals. A key finding of this study is that active Spz serves a pro-inflammatory function in fly larvae. This first report of any pro-inflammatory molecule in the fly confirms that cytokines activate inflammation across phyla. As with mammalian cytokines that act as immuno-stimulants, Spz is expressed, and is therefore likely to activate the blood cells surrounding the parasite capsule. Active Spz promotes blood cell division, migration and infiltration much like high levels of Dorsal and Dif, suggesting that the cell biological changes triggered by SPE/Spz are mediated by target genes of Dorsal and Dif. It is intriguing that the integrity of the basement membrane (as visualized by Collagen IV expression pattern) appears to be important for orchestrating blood cells to the site of 'diseased self' (the mutant fat body in this study) in a manner that may be similar to recognition of the non-self parasitic egg, underscoring the parallel roles of basement membrane proteins in the origin and development of inflammation in both flies and mammals (Paddibhatla, 2010).

Although excessive (active) Spz is proinflammatory, its loss leads to reduction in the hematopoietic complement. For example mutants lacking spz (spz^rm7/spz^rm7) exhibit a 40% reduction in circulating blood cell concentration and these animals do not encapsulate wasp eggs as efficiently as their heterozygous siblings. These observations suggest that active Spz's normal proliferative/pro-survival functions, required for maintaining the normal hematopoietic complement, are fundamentally linked to its immune function for the activation and recruitment of blood cells to target sites. Thus, the autocrine and paracrine hematopoietic and inflammatory effects of Spz are amplified in the presence of hyperactive Toll receptor, excessive Dorsal/Dif, or the loss of Cactus/Ubc9 inhibition, resulting in production of hematopoietic tumors. It is possible that mutations in other, unrelated, genes that yield similar inflammatory tumors arise due to the loss of Toll-NF-kappaB dependent immune homeostasis (Paddibhatla, 2010).

These results highlight the central role of the Dorsal/Dif proteins not only in immune activation, but also in the resolution of these responses. Proteomic studies have confirmed that Dorsal is a bona fide SUMO target and its transcriptional activity is affected by sumoylation. Dorsal and Dif exhibit genetic redundancy in both the humoral and cellular responses. It is possible that this redundancy ensures that immune reactions against microbes and parasites are efficiently resolved to allow proper host development (Paddibhatla, 2010).

In nature, parasitic wasps are continually evolving to evade or suppress the immune responses of their hosts. To this end, they secrete factors or produce protein complexes with specific molecular activities to block encapsulation. These studies provide the biological context in which the effects of virulence factors produced by pathogens and parasites on primordial immune pathways can be more clearly interpreted. The molecular identity of wasp factors which actively suppress humoral and cellular responses (e.g., those in L. heterotoma remains largely unknown. Such virulence factors are likely to be 'anti-inflammatory' as they clearly interfere with host physiology that ultimately disrupts the central regulatory immune circuit defined in these studies (Paddibhatla, 2010).

Encapsulation reactions of non-self (wasp egg) or diseased self tissues (fat body) of the kind in the Drosophila larva are not only reported in other insects, but the reaction is likely to be similar to mammalian granulomas, which are characterized by different forms of localized nodular inflammation. Furthermore, the phenotypes arising from persistent signaling in mutants recapitulate the key features of mammalian inflammation: i.e., reliance on conserved signaling mechanism, the requirement for cytokines, and sensitivity to aspirin. These studies also reveal a clear link between innate immunity and the development and progression of hematopoietic cancer in flies, as has been hypothesized from work in mammalian systems. In the past, genetic approaches in Drosophila have served well to dissect signaling mechanisms governing developmental processes in animals. The fly model with hallmarks of acute and chronic mammalian inflammatory responses will provide deep insights into signaling networks and feedback regulatory mechanisms in human infections and disease. It can also be used to test the potency and mechanism of action of pesticides, anti-inflammatory and anti-cancer agents in vivo (Paddibhatla, 2010).

Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo

Morphogens are secreted signaling molecules that form concentration gradients and control cell fate in developing tissues. During development, it is essential that morphogen range is strictly regulated in order for correct cell type specification to occur. One of the best characterized morphogens is Drosophila Decapentaplegic (Dpp), a BMP signaling molecule that patterns the dorsal ectoderm of the embryo by activating the Mad and Medea (Med) transcription factors. This study demonstrates that there is a spatial and temporal expansion of the expression patterns of Dpp target genes in SUMO pathway mutant embryos. Med is identified as the primary SUMOylation target in the Dpp pathway; failure to SUMOylate Med leads to the increased Dpp signaling range observed in the SUMO pathway mutant embryos. Med is SUMO modified in the nucleus, and evidence is provided that SUMOylation triggers Med nuclear export. Hence, Med SUMOylation provides a mechanism by which nuclei can continue to monitor the presence of extracellular Dpp signal to activate target gene expression for an appropriate duration. Overall, these results identify an unusual strategy for regulating morphogen range that, rather than impacting on the morphogen itself, targets an intracellular transducer (Miles, 2008).

Together, these data suggest a model whereby Med enters the nucleus either by shuttling in a signal-independent manner or through pathway activation, leading to its SUMOylation. Since less SUMOylated Med is detected in the presence of signal, it is proposed that pMad slows the rate of Med SUMOylation, possibly via an effect on Ubc9 recruitment [Ubc9 is encoded by the lesswright (lwr, also called semushi) gene in flies]. FRAP data and imaging of Med in lwr mutant embryos suggest that SUMO modification of Med acts as a trigger to promote its mobility and nuclear export. This finding could explain the necessity for pMad to delay SUMO modification of Med, in order that active Smad complexes have sufficient time to activate transcription. It has been reported previously that TGF-β signaling decreases the nuclear mobility of vertebrate Smad4. It is proposed that this decrease may reflect a slower rate of Smad4 SUMOylation in the presence of phosphorylated R-Smad, which in turn retains Smad4 in an unmodified immobile form (Miles, 2008).

Like Med, the pMad domains are also expanded in lwr mutant embryos and those with non-SUMOylatable Med. More pMad was associated with the non-SUMOylatable MedABC mutant than with wild-type Med. Therefore, the loss of nuclear Med upon SUMOylation appears to promote loss of pMad, even though pMad can accumulate in the nucleus without Med interaction. Recently, pyruvate dehydrogenase phosphatase (PDP) has been shown to terminate Dpp signaling through dephosphorylation of pMad. Although it is presently unclear if PDP dephosphorylates pMad in the nucleus or cytoplasm, the Smad2/3 phosphatase PPM1A acts in the nucleus, resulting in Smad2/3 nuclear export. Therefore, it is possible that SUMO and PDP function together in the nucleus to terminate Dpp signaling. The expanded pMad domains observed when Med SUMOylation is prevented suggest a model in which Med SUMO modification in a wild-type embryo precedes pMad dephosphorylation. This model is consistent with the evidence that dephosphorylation of the receptor-activated Smad promotes complex dissociation and export (Miles, 2008).

SUMO-dependent export of Med from the nucleus following signal activation provides a mechanism to ensure that cells activate Dpp-dependent transcription only in response to the continual receipt of an extracellular Dpp signal. Removal of this sensing mechanism in lwr mutant embryos leads to an inappropriate signaling duration as detected by prolonged zen expression and the cuticle phenotypes (Miles, 2008).

The fate of SUMOylated Med is currently unknown. However, as Ulp1, one of the major SUMO deconjugating enzymes in Drosophila, is localized to the nuclear pore complex (Smith, 2004), it is likely that Med is deSUMOylated upon export. It is suggested that ultimately SUMOylated Med is either recycled following deSUMOylation or degraded. Despite the apparently large cytoplasmic pool of Med, overexpression of wild-type Med expands Dpp target gene expression and the number of amnioserosa cells in early and late stage embryos, respectively. These observations suggest that Med is limiting for signaling, in which case failure to recycle SUMO-modified Med would have a significant impact on the Med pool (Miles, 2008).

Med, which constitutively shuttles between the nucleus and cytoplasm in the absence of signal, is also SUMO modified in the nucleus. There is evidence that in the absence of signal, the Sno corepressor is recruited to nuclear Smad4 to prevent signal-independent transcriptional activation. By limiting Med’s time in the nucleus, SUMO-mediated nuclear export may be an additional strategy deployed to further protect against inappropriate transcriptional responses. Interestingly, the results suggest that activation of the Dpp pathway inhibits Med constitutive shuttling. This scenario is different from that described for vertebrate Smad4, which can shuttle independently of an R-Smad upon active TGF-β signaling. Recently, basal shuttling of Smad4 has been shown to require Importin7/8, whereas the mechanism of nuclear import of constitutively shuttling Med is independent of Moleskin, the Drosophila ortholog of Importin7/8. These findings provide further support to the conclusion that there are inherent differences between the constitutive shuttling properties of Med and Smad4 (Miles, 2008).

These data identify a central role for SUMO in modulating the nuclear-cytoplasmic partitioning of the Smad transcription factors. Precedents already exist for SUMO in regulating both the import and export of proteins. For example, SUMO has been implicated in promoting the nuclear retention of the Elk-1 transcription factor, adenoviral E1B-55K protein, and CtBP1 corepressor. In terms of SUMO promoting nuclear export, as the data suggest for Med, examples include the TEL repressor protein, MEK1 kinase, ribosome biogenesis factors, and p53 transcription factor (Miles, 2008).

Following genotoxic stress, SUMOylation of the IkappaB kinase regulator NEMO triggers a cascade of additional modifications including phosphorylation and ubiquitination that ultimately promote NEMO’s nuclear export. Ectodermin, a nuclear ubiquitin ligase, constrains BMP signaling by promoting nuclear clearance of Smad4. Whether the fly ortholog of Ectodermin has a similar role, and indeed if there is any interplay between Ectoderminmediated ubiquitination and SUMOylation of Med in its nuclear export, remains to be determined. An alternative mechanism by which SUMO promotes Med export is based on that described for p53. p53 is monoubiquitinated by MDM2, which exposes the NES and allows recruitment of the PIASy E3 ligase leading to p53 SUMOylation. As a result, MDM2 dissociates and p53 nuclear export occurs. SUMOylation may re-expose the Med NES that has been inactivated upon signaling (Watanabe, 2000), promoting nuclear export. The location of the Med NES in between SUMO sites A and B may lend itself to this type of regulation. Interestingly, SUMO sites A and B are the two that are conserved in vertebrate Smad4, as is the position of the NES. It is speculated that SUMOylation will also direct nuclear export of vertebrate Smad4 (Miles, 2008).

Although SUMO modification of Smad4 has been postulated to have both positive and negative effects on gene expression, Med SUMOylation leads to a reduction in its transcriptional activity in the context of Dpp signaling in the Drosophila embryo. These differences may reflect promoter-specific effects or particular characteristics of the transcription factor complex that depend on which receptor-activated Smad is associated with Med/Smad4 (Miles, 2008).

Studies of extracellular signals such as Dpp and Hedgehog support the generation of different gene activity thresholds by a 'French flag' model of positional information. Signal concentration provides positional information so that cells located nearest the source activate a peak threshold of gene activity and adopt a specific cell fate, whereas cells located further from the source express different threshold responses and assume distinct fates. Morphogen concentration at the source and sink is therefore crucial, and mechanisms that have been characterized for regulating patterning by morphogens have intuitively focused on the morphogen itself. However, the current results identify a twist on the French flag model whereby the positional information provided by a specific concentration of morphogen can be refined by modulating the activity of an intracellular transducer. In this way the French flag floats in relation to Dpp activity, since the absolute amount of Dpp required for each fate is influenced by the activity of the SUMOylation pathway. Although this study has concentrated on the SUMO post-translational modification, any mechanism that hones the activity or distribution of an intracellular transducer will affect the interpretation of positional information and pattern formation in a similar way. Moreover, it is predicted that SUMO itself will be used to modulate the signaling outputs by other morphogens in different developmental contexts. A good candidate appears to be the Wnt morphogen, as links between SUMO and the Wnt pathway during Xenopus development been suggested (Miles, 2008).

The spatial and temporal range of the Dpp/BMP signal is controlled not only by Med SUMOylation but also by PDP dephosphorylation of pMad and dSmurf-dependent ubiquitination of cytoplasmic Mad. Therefore, multiple mechanisms exist for constraining the activity of the Smad transcription factors, all of which are wasteful in terms of signal. Although wasteful, having a dedicated dampener in the form of SUMO modification may be tolerated so that the Dpp signaling pathway can be controlled somewhat in the event of inappropriate activation. This may be essential given the potency of Dpp signaling in inducing cell fates. Another possibility is that the disadvantage of losing signal through this built-in dampener is far outweighed by its use as a mechanism through which the presence of an extracellular signal can constantly be sensed (Miles, 2008).

In addition to the central role of Med/Smad4 in mediating the appropriate transcriptional outputs in response to signaling by all TGF-β ligands, the function of Smad4 as an essential tumor suppressor protein in humans has been well documented. As well as SUMOylation, ubiquitination of the Med/Smad4 transcription factor has been described. Therefore, it appears that multiple mechanisms are deployed during development to harness the activity of this pivotal signal-responsive transcription factor (Miles, 2008).

SUMO conjugation attenuates the activity of the gypsy chromatin insulator

Chromatin insulators have been implicated in the establishment of independent gene expression domains and in the nuclear organization of chromatin. Post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) has been reported to regulate their activity and subnuclear localization. Evidence is presented suggesting that two protein components of the gypsy chromatin insulator of Drosophila melanogaster, Mod(mdg4)2.2 and CP190, are sumoylated, and that SUMO is associated with a subset of genomic insulator sites. Disruption of the SUMO conjugation pathway improves the enhancer-blocking function of a partially active insulator, indicating that SUMO modification acts to regulate negatively the activity of the gypsy insulator. Sumoylation does not affect the ability of CP190 and Mod(mdg4)2.2 to bind chromatin, but instead appears to regulate the nuclear organization of gypsy insulator complexes. The results suggest that long-range interactions of insulator proteins are inhibited by sumoylation and that the establishment of chromatin domains can be regulated by SUMO conjugation (Capelson, 2006).

Two protein components of the gypsy chromatin insulator, Mod(mdg4)2.2 and CP190, were found to be modified by SUMO in vitro and in vivo. dTopors was observed to interfere with their sumoylation by possibly disrupting the contacts between the SUMO E2 enzyme Ubc9 and substrate insulator proteins. The inhibitory effect of dTopors, although relatively subtle, is consistent across the various assays utilized such that any time dTopors was introduced at higher levels, either by direct addition in vitro or by increasing expression in vivo, it was found to result in reduced sumoylation of Mod(mdg4)2.2 and CP190. Disruption of SUMO conjugation by mutations in genes coding for Ubc9 and SUMO exerts a positive effect on gypsy insulator activity, suggesting that the normal role of SUMO modification is to antagonize insulator function. A fraction of chromatin-bound insulator proteins appears to be associated with SUMO, yet mutations in the SUMO pathway are not seen to affect the chromatin-binding properties of CP190 or Mod(mdg4)2.2. Instead, sumoylation interferes with the formation of nuclear insulator bodies, such that overexpression of Ubc9 leads to breakdown of nuclear insulator structures, whereas lower levels of Ubc9 and sumoylation result in a partial recovery of coalescence lost in the absence of Mod(mdg4)2.2 (Capelson, 2006).

These findings suggest that modification of CP190 and Mod(mdg4)2.2 by SUMO may prevent self-association and thus interfere with long-range interactions between distant insulator complexes required to form insulator bodies. Thereby, sumoylation may preclude formation of closed chromatin loops and the consequent establishment of autonomous gene expression domains (Capelson, 2006).

Multiple lines of evidence point to a role for SUMO modification in transcriptional repression. Sumoylation of histones has been characterized as a mark of repressed chromatin, whereas SUMO conjugation to certain transcriptional regulators leads to their association with histone deacetylases, which remove the active acetylation marks from histones. SUMO modification of the Polycomb group (PcG) protein SOP-2 is required for its function in stable repression of Hox genes, and another PcG repressor, Pc2, acts as a SUMO E3 ligase. Modification of gypsy insulator proteins by SUMO does not seem to associate them exclusively with transcriptional repression, as reduction of sumoylation in lwr/smt3 mutants results in the upregulation of expression from the omb^P1-D1 locus, but in the downregulation of transcription at y² and ct⁶. In these cases, transcriptional output appears to correlate only with the enhancer-blocking activity of the insulator. Nevertheless, it is possible that one of the roles of sumoylation involves association of selected insulator sites in the genome with transcriptional repression. Sumoylated insulator complexes may not participate in the formation of expression domains, but instead, could target silencing factors to the surrounding chromatin (Capelson, 2006).

In mammalian nuclei, the homolog of dTopors localizes to PML bodies, which are enriched in the SUMO conjugation machinery. If inhibition of sumoylation is also a property of mammalian Topors, it may play a role in preventing further sumoylation of factors that are targeted to these nuclear compartments. In this manner, ICP0 also localizes to the PML bodies, where it causes desumoylation of two primary components, PML and SP100. It has been reported that Topors may function as a SUMO E3 ligase for the tumor suppressor p53 protein. This apparent contradiction with the current results may be due to several reasons. Topors and dTopors may have diverged their functions regarding the SUMO pathway, such that Topors functions as a SUMO E3 while dTopors interferes with SUMO addition due to its conserved interaction with Ubc9. Alternatively, the involvement of dTopors in the SUMO pathway may be substrate-specific, since it may bind to Ubc9 in ways that allow for interaction with a given target protein or prevent it. In the context of the gypsy insulator, the interference of dTopors with sumoylation is consistent with previous observations that dTopors promotes insulator activity, whereas sumoylation appears to disrupt it (Capelson, 2006).

It has been suggested that SUMO conjugation may affect the function of the modified protein even after the SUMO tag itself has been removed, creating a cellular memory for protein regulation. This idea has arisen partly to explain the commonly observed contradiction between the small percentage of a given protein that is modified by SUMO and the dramatic consequences of the modification on the protein's cellular function. Sumoylation may be needed for proteins to enter stable complexes or functional states, but the persistence of the SUMO modification may not be required after the initial establishment. Thus, the actual effect of sumoylation may far exceed that of the detectable sumoylated population since the function of a much larger proportion of molecules has been altered by SUMO conjugation and subsequent deconjugation. Similarly to other reported cases, the sumoylated forms of Mod(mdg4)2.2 and of CP190 represent a small fraction of the total pool of the insulator proteins, yet the phenotypic effects of the loss of these forms are quite striking. It is possible that SUMO attachment regulates the initial organization of chromatin domains, perhaps in earlier development or following mitosis, yet once established, the domains may be stably maintained without SUMO. Additionally, the rapid conjugation and deconjugation cycle of the SUMO tag implies that sumoylation may be used by processes that require reassembly upon signal. In that sense, SUMO modification seems particularly suitable for the regulation of gene expression domains as it can result in 'remembered' yet flexible states (Capelson, 2006).

SUMO enhances Vestigial function during wing morphogenesis

The conjugation of the ubiquitin-like protein SUMO to lysine side chains plays widespread roles in the regulation of nuclear protein function. Since little information is available about the roles of SUMO in development, a screen was performed of a collection of chromosomal deficiencies to identify developmental processes regulated by SUMO. Flies heterozygous for a deficiency uncovering vestigial (vg) and mutations in any of several genes encoding components of the SUMO conjugation machinery exhibit severe wing notching. This phenotype is due to an interaction between sumo and vg since it is suppressed by expression of Vg from a transgene, and is also observed in flies doubly heterozygous for vg hypomorphic alleles and sumo. In addition, the ability of Vg to direct the formation of ectopic wings when misexpressed in the eye field is enhanced by simultaneous misexpression of SUMO. In S2 cell transient transfection assays, overexpression of SUMO and the SUMO conjugating enzyme Ubc9, but not a catalytically inactive form of Ubc9, results in sumoylation of Vg and augments the activation of a Vg-responsive reporter. These findings are consistent with the idea that sumoylation stimulates Vg function during wing morphogenesis (Takanaka, 2005).

Thus, sumo loss-of-function mutations act as genetic enhancers of vg loss-of-function mutations. For example, flies doubly heterozygous for recessive hypomorphic vg alleles and recessive sumo or ubc9 alleles exhibit wing notching that is as severe as that exhibited by flies homozygous for the vg mutant alleles. In addition, co-overexpression of SUMO and Vg in the wing or eye significantly exacerbates the phenotype due to overexpression of Vg alone. These findings are consistent with the idea that the SUMO machinery acts to augment Vg function. However, attempts to further confirm this idea by generating homozygous SUMO loss-of-function clones in discs have failed, probably because SUMO is required for cell cycle progression or cell survival (Takanaka, 2005).

Transient transfection assays further support the idea that the sumoylation machinery can potentiate Vg/Sd transactivation. Specifically, cotransfection of Ubc9 and SUMO augments the Vg/Sd dependent activation of the VgQ-luciferase reporter. This effect requires a catalytically active form of Ubc9 strongly suggesting that it is dependent upon sumoylation (Takanaka, 2005).

Attempts were made to map the SUMO acceptor lysine in Vg. There is only a single lysine (Lys 180) that falls in a sequence context with any resemblance to the consensus sumoylation site. Lys 180 falls in the sequence TKEE, while the sumoylation consensus is ψKxE (with ψ signifying a hydrophobic residue). Surprisingly, however, mutagenesis of this lysine to arginine does not significantly reduce the ability of Vg to serve as a target for sumoylation in S2 cells. Apparently, sumoylation occurs at non-consensus sites in Vg. There are multiple precedents for such non-consensus sites in other sumoylation targets (Takanaka, 2005).

The mechanism by which sumoylation renders Vg a more potent activator appears to be distinct from the mechanism by which sumoylation regulates a number of transcription factors. There are numerous examples in which sumoylation of a transcription factor alters the subcellular localization of a factor by directing it to the PODs, resulting in either the activation or inhibition of the factor. However, immunofluorescence studies reveal no evidence for an effect of sumoylation on Vg subcellular localization. There are also numerous examples in which sumoylation upregulates a transcription factor by disrupting an interaction with a negative regulatory factor. Although the existence of a similar negatively acting factor in the case of Vg cannot be ruled out, there is no direct evidence for such a factor. An alternative intriguing possibility, which remains to be explored, is that the sumoylation of Vg enhances transcription by enhancing the interaction between Vg and Sd (Takanaka, 2005).

This study represents one of only a few efforts using genetic approaches to illuminate the biological role of SUMO conjugation in a multicellular organism. Previous genetic analyses have demonstrated a role for the sumoylation machinery in embryonic patterning. For example, in C. elegans embryos, loss of SUMO, Ubc9, or the SUMO activating enzyme results in homeotic transformations apparently due to a role for sumoylation in the function of the Polycomb group protein SOP-2. In Drosophila embryos, loss of Ubc9 results in the deletion of variable numbers of thoracic and anterior abdominal segments, but in this case the relevant sumoylation target is not known. Previous genetic analysis also suggests a role for sumoylation in immune system function as mutations in sumo or ubc9 compromise the Drosophila innate immune response by attenuating the LPS-induced expression of genes encoding anti-microbial peptides such as Cecropin A1. This is consistent with the finding that sumoylation significantly stimulates the function of the Drosophila rel family protein Dorsal since rel family proteins play critical roles in both vertebrate and invertebrate innate immunity. Finally, a recent yeast two-hybrid screen indicates that Dof, a cytoplasmic components of the FGF signaling pathway, interacts with multiple components of the SUMO conjugation pathway. This suggests possible roles for SUMO conjugation in the morphogenetic processes controlled by FGF receptors such as mesodermal and tracheal morphogenesis. Thus, the finding of a likely role for sumoylation in wing development adds to a growing body of evidence suggesting pleiotropic roles for sumoylation in the development and function of multicellular organisms (Takanaka, 2005).

SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors

Sry high mobility group (HMG) box (Sox) transcription factors are involved in the development of central nervous system (CNS) in all metazoans. Little is known on the molecular mechanisms that regulate their transcriptional activity. Covalent posttranslational modification by small ubiquitin-like modifier (SUMO) regulates several nuclear events, including the transcriptional activity of transcription factors. This study demonstrates that SoxNeuro, an HMG box-containing transcription factor involved in neuroblast formation in Drosophila, is a substrate for SUMO modification. SUMOylation assays in HeLa cells and Drosophila S2 cells reveal that lysine 439 is the major SUMO acceptor site. The sequence in SoxNeuro targeted for SUMOylation, IKSE, is part of a small inhibitory domain, able to repress in cis the activity of two adjacent transcriptional activation domains. These data show that SUMO modification represses SoxNeuro transcriptional activity in transfected cells. Overexpression in Drosophila embryos of a SoxN form that cannot be targeted for SUMOylation strongly impairs the development of the CNS, suggesting that SUMO modification of SoxN is crucial for regulating its activity in vivo. Finally, evidence is presented that SUMO modification of group B1 Sox factors was conserved during evolution, because Sox3, the human counterpart of SoxN, is also negatively regulated through SUMO modification (Savare, 2005).

This report shows that the SoxN and its human counterpart Sox3, both involved in CNS development, are SUMO modified in vivo. Ootential SUMOylation sites (ψKXE motif) were sought in all mammalian and Drosophila Sox proteins. One or several ψKXE motifs are present in some but not all Sox genes, these motifs being usually conserved within a given subgroup between Drosophila and humans. These include group B1 (H.s Sox1/2/3 and D.m SoxN), group C (H.s Sox11 and D.m SoxC), group D (H.s Sox5/6/13), group E (H.s Sox8/9/10 and D.m Sox100B), group F (H.s Sox17), and group H (H.s Sox30). Recently, Sox9 was shown to be SUMO modified, and SUMO modification was associated with transcriptional repression. In all the other groups (B2, F, and G), no ψKXE motif is present (except Drosophila SoxB2-2, human group C Sox11 and group F Sox17), suggesting that these proteins are not SUMO modified. To confirm this, the same SUMOylation assay was used as described in this report for SoxN and Sox3, and no SUMO modified human Sox7, mouse Sox15 and Drosophila Dichaete (respectively, group F, G, and B) was detected. Thus, based on the data and the presence of ψKXE motif in various Sox, one can postulate that SUMO modification might be used to regulate several Sox group genes (Savare, 2005).

The results show that SUMO modification of the CNS-specific group B1 SoxN and Sox3 proteins was conserved during evolution to regulate their transcriptional capacity. Based on the presence of ψKXE motif in group B1 proteins (SoxN in Drosophila and Sox1/2/3 in humans), and its absence in group B2 (Dichaete in Drosophila and Sox14/21 in humans), it is tempting to speculate that these two subgroups differ in their ability to be regulated by SUMOylation. This is particularly interesting because in Drosophila, SoxN and Dichaete were shown to partially overlap in their expression and function within the neuroectoderm, suggesting that these genes are to some extent functionally redundant in the developing CNS but that there must exist molecular mechanisms responsible for their specificity of action in restricted areas of the CNS (interactions with specific partners? posttranslational modifications?). Furthermore, it has been shown in chick that group B2 Sox14/21 could bind and differentially regulate δ1-crystallin gene regulatory sequences, known to be regulated by group B1 Sox1/2/3 factors in vivo. These observations suggested that target of group B genes might be regulated by the counterbalance of activating and repressing Sox proteins in restricted sites of the developing CNS. In light of these results, SUMOylation might be one of the mechanisms used for this purpose (Savare, 2005).

As shown in this study, substitution of lysine 439 to arginine within SoxN IKSE motif impaired SoxN SUMO modification in both transfected HeLa and S2 cells. SoxN transcriptional activity was dramatically enhanced in three conditions: in the substitution mutant K439R, in the deletion mutants where the IKSE motif was deleted, and when the dominant negative form of Ubc9 was used to interfere with the endogenous SUMO machinery. This correlation between transcriptional repression and the ability of SoxN to be SUMOylated strongly suggests that SUMO conjugation to SoxN results in transcriptional repression. Similar results were obtained for its human counterpart Sox3. Many of the SUMO-modified proteins identified to date are transcription factors, and in most cases, SUMO modification has been associated with transcriptional repression. Nevertheless, the molecular mechanisms underlying this repression are still a matter of debate. In some cases, SUMO modification was associated with the relocalization of the targeted factor to specialized repressive subnuclear structures such as PML bodies. In SoxN and Sox3, data in HeLa and S2 cells suggest that SUMOylation is apparently not associated with major changes in the nuclear localization of these proteins. This was also evident in vivo, because the wild-type and K439R SoxN forms both localized similarly in the nuclei (Savare, 2005).

In both SoxN and Sox3, it was found that the ψKXE motif is targeted for SUMOylation, and constitutes an inhibitory domain able to affect the activity of adjacent TADs. Interestingly, this motif is surrounded by conserved proline residues, reminiscent of the SC synergy domain (consensus P-X0-4-ψKXE-X0-3-P) found in several transcription factors, including SP3, c-myb, C/EBP, and Sox9. Potential SC motifs also are found in other Sox: H.s Sox6, H.s Sox8, and H.s Sox30. SC motif is both necessary and sufficient to limit transcriptional synergy, because its disruption selectively enhances synergistic activation at compound response elements without altering the activity driven from a single site. Thus, SUMOylation of the SC domain is believed to modulate higher order interactions among transcriptional regulators. This motif in Sox proteins might behave as SC domain, because these factors are known to pair off with specific partners to exert full and synergistic activity in a context dependent manner. Because SUMO modification is believed to modulate protein-protein interactions, it will be of interest to examine whether Sox SUMOylation is able to interfere with their ability to interact with their partners (Savare, 2005).

Using transgenic Drosophila lines, strong evidence was obtained that SUMOylation regulates the activity of SoxN in vivo. Indeed, overexpressing the SUMO-deficient K439R SoxN form resulted in strong defects in embryonic CNS. Because the GAL4 driver used for embryonic overexpression is ubiquitous, these results are interpreted as the capacity of the nonSUMOylable form to interfere with endogenous SoxN in the cells were SoxN is expressed (neuroblasts and neurons). In addition, the experiments where the wild-type and K439R SoxN proteins were overexpressed in larvae clearly showed that the two forms display different activity in vivo, further demonstrating the functional relevance of SoxN SUMOylation in vivo. Because the K439R form is a strong transcriptional activator as observed in luciferase assays in transfected cells, it can be postulated that the repressing activity of SoxN is important for the proper development of embryonic CNS. Further work will be required to demonstrate whether SUMOylation regulates SoxN activity in all the different cell types where the protein is expressed (embryonic, larval and adult CNS, larval and adult eyes, and larval leg imaginal discs) (Savare, 2005).

Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO conjugates

SUMO is a small ubiquitin-like protein that becomes covalently conjugated to a variety of target proteins, the large majority of which are found in the nucleus. Ulp1 is a member of a family of proteases that control SUMO function positively, by catalyzing the proteolytic processing of SUMO to its mature form, and negatively, by catalyzing SUMO deconjugation. In Drosophila S2 cells, depletion of Ulp1 by RNA interference results in a dramatic change in the overall spectrum of SUMO conjugates, indicating that SUMO deconjugation is substrate-specific and plays a critical role in determining the steady state targets of SUMO conjugation. Ulp1 normally serves to prevent the accumulation of SUMO-conjugated forms of a number of proteins, including the aminoacyl-tRNA synthetase EPRS. In the presence of Ulp1, most SUMO conjugates reside in the nucleus. However, in its absence, SUMO-conjugated EPRS accumulates in the cytoplasm, contributing to an overall shift of SUMO from the nucleus to the cytoplasm. The ability of Ulp1 to restrict SUMO conjugates to the nucleus is independent of its role as a SUMO-processing enzyme because Ulp1-dependent nuclear localization of SUMO is even observed when SUMO is expressed in a preprocessed form. Studies of a Ulp1-GFP fusion protein suggest that Ulp1 localizes to the nucleoplasmic face of the nuclear pore complex. It is hypothesize that, as a component of the nuclear pore complex, Ulp1 may prevent proteins from leaving the nucleus with SUMO still attached (Smith, 2004).

Depletion of Ulp1 from S2 cells by RNAi results in increased levels of SUMO conjugation indicating that deconjugation rather than SUMO maturation is the dominant role of Ulp1. Furthermore, Ulp1 depletion results in a change in the spectrum of SUMO-conjugated proteins indicating that the specificity with which proteins are selected for deconjugation may play an important role in the specificity of SUMO targeting. Substrate-specific deconjugating enzymes may prevent limiting amounts of SUMO from becoming irreversibly conjugated to inappropriate targets that encounter the conjugation machinery. In this way, specific SUMO conjugation could be achieved even if the specificity of the enzymes responsible for conjugation was relatively low. This contrasts with ubiquitin conjugation in which the specificity of conjugation is ensured by a myriad of ubiquitin ligases, which select specific ubiquitin conjugation targets in response to a huge variety of extrinsic and intrinsic cues. While analogous SUMO ligases do exist, they appear to be fewer in number than ubiquitin ligases, and may not always be required for SUMO conjugation (Smith, 2004).

There are multiple ways in which SUMO deconjugation could be rendered substrate specific. For example, the deconjugating enzymes could recognize specific sequence or structural motifs in the deconjugation targets. However, it is difficult to see how a limited number of deconjugating enzymes could specifically recognize all the proteins (probably the majority of cellular proteins) that should not be conjugated to SUMO. An alternative strategy might be to control deconjugation specificity by targeting the deconjugating enzymes to specific subcellular locales (Smith, 2004).

In support of this latter possibility, it has been found that Drosophila Ulp1 localizes to the nucleoplasmic face of the NPC where it is apparently required to prevent the accumulation of SUMO-conjugated cytoplasmic proteins. Upon depletion of Ulp1 from S2 cells, there is a dramatic shift in the localization of SUMO from the nucleus to the cytoplasm, which at least partly reflects the attachment of SUMO to high molecular mass cytoplasmic proteins such as EPRS and MRS. While these aminoacyl-tRNA synthetases are predominantly located in the cytoplasm, a growing body of evidence suggests that they also spend time in the nucleus, where they may catalyze an initial round of tRNA aminoacylation. These enzymes may help to channel aminoacyl-tRNA directly to the cytoplasmic translational elongation factor EF1, and thus it is possible that they shuttle in and out of the nucleus. Since the SUMO conjugation machinery is concentrated inside the nucleus, enzymes like EPRS that may transiently enter the nucleus could become inappropriately conjugated to SUMO during the time spent inside the nucleus. The localization of Ulp1 to the nucleoplasmic face of the NPC might then assure that SUMO deconjugation occurs during re-export. If this was the case, then it inhibition of EPRS export might be expected to result in the accumulation of SUMO-conjugated forms of EPRS. However, the mechanism of EPRS re-export is not understood and attempts to block EPRS export by the specific inhibition or RNAi-mediated depletion of Crm1, the best characterized of the exportins, were not successful (Smith, 2004).

With one possible exception, previous studies of other SUMO proteases have also demonstrated localization to specific nuclear subcompartments. Yeast Ulp1 and human SENP2 are found at the nuclear pores, and furthermore, the targeting of yeast Ulp1 to the nuclear membrane is required to maintain the normal spectrum of SUMO-conjugated proteins. Yeast Ulp2 is found in the nucleoplasm. Murine SUMO protease-1 localizes to the nuclear bodies. Finally, human SENP3 (SMT3IP1) localizes to the nucleolus. Localization may be a means to limit the access of these proteases to proteins in particular subcellular compartments thereby targeting them to a particular subset of SUMO-conjugated proteins. In addition to the evidence presented in this study, further evidence for this hypothesis comes from experiments in which removal of the NPC-targeting sequence from the human SUMO protease SENP2 was found to result in a significant change in the intracellular spectrum of sumoylated proteins (Smith, 2004).

The findings suggest that one purpose of Ulp1 is to prevent the conjugation of SUMO to cytoplasmic proteins such as EPRS, which spend most of their life in the cytoplasm, but which may encounter the SUMO conjugation machinery during transient passage through the nucleus. Thus, it is possible that EPRS sumoylation plays no beneficial cellular role. However, this study found that EPRS also becomes sumoylated upon cellular stress such as heat shock, raising the possibility that aminoacyl-tRNA synthetase sumoylation plays a role in the stress response. Given the variety of functions that have been associated with aminoacyl-tRNA synthetases, sumoylation of these enzymes could help mediate the stress response in any number of ways. For example, both EPRS and MRS are components of the MSC, and the rep domain, which is the region of EPRS targeted for sumoylation, is required for the formation of this complex. The functional significance of the MSC is unclear, although it may play a role in the trafficking of tRNA from the nucleus to the ribosome. Cellular stress and the resulting protein damage may lead to a need for increased protein synthesis, which in turn, would require increased levels of aminoacyl-tRNA at the ribosome. By regulating the function of the MSC, sumoylation could therefore help cells up-regulate protein synthesis to replace proteins damaged during cellular stress (Smith, 2004).

Whereas a few SUMO target proteins may be exclusively extranuclear, the vast majority of such proteins including NPC-associated factors and numerous transcription factors spend some or most of their life in the nucleoplasm or at the nuclear periphery. For example, in vertebrates, SUMO seems to play a role in the structure and/or function of discrete intranuclear foci called PML oncogenic domains (PODs). Anti-SUMO staining of Drosophila cells reveals punctate nuclear dots above a diffuse background of SUMO throughout the nucleus. These dots may represent the Drosophila counterpart of the PODs (Lehembre, 2000). Interestingly, it was found that when SUMO is excluded from the nucleus by interference with Ulp1 expression, the dots relocalize to the cytoplasm. While it is uncertain whether these dots are truly equivalent to the nuclear dots seen in normal cells, this finding suggests that dot formation may be an intrinsic property of SUMO itself that is independent of SUMO nuclear localization (Smith, 2004).

Restriction of SUMO conjugation to the nucleus is largely achieved by the localization of the SUMO conjugation machinery to the nucleus. However, the findings presented in this study indicate that Ulp1 plays an important role in enforcing this nuclear restriction through substrate-specific deconjugation (Smith, 2004).

Smrt3 conjugation machinery and transport into the nucleus

To identify proteins that regulate the function of Dorsal, a yeast two-hybrid screen was used to search for genes encoding Dorsal-interacting proteins. Six genes have been identified, including two that encode previously known Dorsal-interacting proteins (Twist and Cactus); three that encode novel proteins, and one that encodes Drosophila Ubc9 (DmUbc9: lesswright). The name 'Ubc9' reflects the homology of this protein to ubiquitin-conjugating enzymes. However, recent studies on yeast and human Ubc9 have shown that this enzyme primarily conjugates the yeast protein Smt3p or its human homologs SMT3A, SMT3B, and SMT3C rather than ubiquitin to proteins. DmUbc9 binds and conjugates Drosophila Smt3 (DmSmt3) to Dorsal. In cultured cells, DmUbc9 relieves inhibition of Dorsal nuclear uptake by Cactus, allowing Dorsal to enter the nucleus and activate transcription. The effect of DmUbc9 on Dorsal activity is potentiated by the overexpression of DmSmt3. A DmSmt3-activating enzyme, DmSAE1/DmSAE2, has been identified, and found to further potentiate Dorsal-mediated activation (Bhaskar, 2000).

Smt3 homologs have been cloned from eukaryotes as diverse as yeast, Arabidopsis, and humans. In general, these proteins display greater than 50% identity with one another but also roughly 20% identity with ubiquitin. The identification of the components of the Smt3 conjugation pathway in yeast, humans, and now Drosophila has revealed that Smt3 conjugation and ubiquitin conjugation proceed by similar pathways. Both pathways require an activating enzyme, or E1 protein, which becomes covalently attached to ubiquitin or Smt3 via a high energy thioester bond, and a conjugating enzyme, or E2 protein, which accepts ubiquitin or Smt3 from the E1 protein forming a second thioester-linked covalent complex. Ubiquitin or Smt3 is then transferred to an epsilon-amino group on a final protein substrate. The transfer of ubiquitin from the E2 protein to the final substrate often requires a ubiquitin ligase, or E3 protein. In contrast, an E3-type protein is apparently not required for Smt3 conjugation (Bhaskar, 2000 and references therein).

Although ubiquitin conjugation targets proteins for proteasomal degradation, Smt3 conjugation appears to serve other purposes. Originally identified in yeast as an enzyme required for proper cell cycle progression, Ubc9 has been found to physically interact with a diverse array of proteins, including RanGAP1, PML (promyelocytic leukemia protein), bleomycin hydrolase, E2A, androgen receptor, and c-Rel. Association of human Ubc9 with RanGAP1 results in the conjugation of RanGAP1 to the Smt3 homolog SMT3C/SUMO-1 (small ubiquitin-related modifier), allowing it to bind RanBP2 at the nuclear periphery. This allows RanGAP1 to stimulate GTP hydrolysis by Ran. Only SUMO-1-conjugated RanGAP1 binds to RanBP2, implying that SMT3C and Ubc9 are required for nuclear import. In the case of PML, interaction with Ubc9 and subsequent SUMO-1 conjugation is essential for targeting PML to discreet subnuclear structures known as PML-bodies or nuclear dots. In acute promyelocytic leukemia cells, the subnuclear localization of PML is altered, suggesting that improper SUMO-1 conjugation may trigger oncogenesis. These studies argue that one function of Smt3 conjugation is to regulate the subcellular localization of proteins (Bhaskar, 2000 and references therein).

Although Smt3 conjugation may play a role in regulating Dorsal activity, a number of reports have implicated Ubc9 in the modulation of transcriptional activation by other Rel family proteins. For example, SUMO-1-conjugated IkappaB is resistant to degradation and, accordingly, SUMO-1 and Ubc9 work together to inhibit activation of an NFkappaB-dependent reporter. This contrasts with the current findings, which show that the Smt3 conjugation pathway activates Dorsal-dependent reporters. This difference could relate to inherent differences between the NFkappaB/IkappaB and Dorsal/Cactus pathways. However, an earlier report suggests that mammalian Ubc9 can enhance Rel protein function via an interaction with NFkappaB and/or IkappaB. Thus, an alternative explanation for the different effects of Smt3 conjugation on Rel protein activity could be that different Smt3 family proteins have different functions. An alignment of DmSmt3 with the three members of the human SMT3 family reveals that DmSmt3 displays significantly higher homology to SMT3A and SMT3B (77% and 75%, respectively) than to SMT3C/SUMO-1 (55%). Thus, DmSmt3, SMT3A, and SMT3B appear to define an Smt3 subfamily that is distinct from SMT3C/SUMO-1. Perhaps SMT3C/SUMO-1 antagonizes transcriptional activation by Rel proteins, whereas SMT3A/B-like proteins (such as DmSmt3) enhance Rel protein function (Bhaskar, 2000 and references therein).

The Smt3 conjugation system may also function at other levels in the regulation of Rel family protein activity. For example, Ubc9 has been shown to associate with the type I TNFalpha receptor and MEKK1 and to synergize with MEKK1 to activate an NFkappaB-dependent reporter. Although no DmSmt3-Dorsal conjugate could be detected in cells that were simultaneously co-transfected with Dorsal, DmUbc9, and DmSmt3, the level of conjugation is low: no more than about 10% of the Dorsal protein is found in the DmSmt3-conjugated form. Perhaps the conjugation of DmSmt3 to Dorsal is transient. Perhaps Dorsal and DmSmt3 are deconjugated as soon as Dorsal enters the nucleus. In accord with this idea, recent observations suggest that a dynamic equilibrium may exist between Smt3-conjugated and unconjugated protein species. In yeast, the vast majority of cellular Smt3p is conjugated to other proteins, although the population of proteins that is covalently modified changes during the cell cycle. Furthermore, a yeast enzyme capable of catalyzing the deconjugation reaction has been identified, and homologs of this enzyme appear to exist in many other eukaryotic species (Bhaskar, 2000 and references therein).

A genetically defined locus, termed semushi (Epps, 1998) is identical with DmUbc9. Experiments employing the semushi allele suggest that DmUbc9 may be necessary for the nuclear import of the anteroposterior patterning morphogen Bicoid. Embryos lacking maternally supplied DmUbc9 have multiple patterning defects of varying penetrance. Because of the complex nature of these defects, their characterization will require extensive phenotypic analysis and the generation of additional DmUbc9 alleles. The possibility that DmUbc9 has pleiotropic developmental roles is not surprising given increasing evidence for wide spread roles of Smt3 conjugation in transcription factor function and in the targeting of proteins to their proper subcellular locales (Bhaskar, 2000 and references therein).

A variety of transcription factors are targets for conjugation to the ubiquitin-like protein Smt3 (also called SUMO). While many such factors exhibit enhanced activity under conditions that favor conjugation, the mechanisms behind this enhancement are largely unknown. The Drosophila rel family factor Dorsal is a substrate for Smt3 conjugation. The conjugation machinery enhances Dorsal activity at least in part by counteracting the Cactus-mediated inhibition of Dorsal nuclear localization. Smt3 conjugation occurs at a single site in Dorsal (lysine 382), requires just the Smt3-activating and -conjugating enzymes, and is reversed by the deconjugating enzyme Ulp1. Mutagenesis of the acceptor lysine eliminates the response of Dorsal to the conjugation machinery and results in enhanced levels of synergistic transcriptional activation. Thus, in addition to controlling Dorsal localization, Smt3 also appears to regulate Dorsal-mediated activation, perhaps by modulating an interaction with a negatively acting nuclear factor. Finally, since Dorsal contributes to innate immunity, the role of Smt3 conjugation in the immune response was investigated. The conjugation machinery is required for lipopolysaccharide-induced expression of antimicrobial peptides in cultured cells and larvae, suggesting that Smt3 regulates Dorsal function in vivo (Bhaskar, 2002).

Covalent modification of the transcriptional repressor Tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies

The ubiquitin-related SUMO-1 modifier can be covalently attached to a variety of proteins. To date, four substrates have been characterized in mammalian cells: RanGAP1, IkappaBalpha, and the two nuclear body-associated PML and Sp100 proteins. SUMO-1 modification has been shown to be involved in protein localization and/or stabilization and to require the activity of specialized E1-activating and E2 Ubc9-conjugating enzymes. SUMO-1 homologs have been identified in various species and belong to the so-called Smt3 family of proteins. The Drosophila homologs of mammalian SUMO-1 and Ubc9 (termed dSmt3 and dUbc9/lesswright, respectively) have been characterized. dUbc9 is the conjugating enzyme for dSmt3 and dSmt3 can covalently modify a number of proteins in Drosophila cells in addition to the human PML substrate. The dSmt3 transcript and protein are maternally deposited in embryos, where the protein accumulates predominantly in nuclei. Similar to its human counterpart, dSmt3 protein is observed in a punctate nuclear pattern. Tramtrack 69 (Ttk69), a repressor of neuronal differentiation, is a bona fide in vivo substrate for dSmt3 conjugation. Both the modified and unmodified forms of Ttk69 can bind to a Ttk69 binding site in vitro. Moreover, dSmt3 and Ttk69 proteins colocalize on polytene chromosomes, indicating that the dSmt3-conjugated Ttk69 species can bind at sites of Ttk69 action in vivo. Altogether, these data indicate a high conservation of the Smt3 conjugation pathway and further suggest that this mechanism may play a role in the transcriptional regulation of cell differentiation in Drosophila flies (Lehembre, 2000).

The identification of the transcriptional repressor Ttk69 as a substrate of the dSmt3 conjugation pathway suggests that this mode of posttranslational modification may play a direct role in the modulation of transcriptional regulation. Supporting this possibility, the localization of dSmt3 at particular chromosomal sites shows that the dSmt3 modification can be chromosome associated. Its partial colocalization with Ttk69 and the ability of the dSmt3-modified Ttk69 protein to bind Ttk69 sites are also consistent with the binding of modified Ttk69 to a subset of Ttk69 recognition elements. Although Ttk69 is the first transcription factor shown to be modified by the SUMO-1/Smt3 homologs, it seems likely that SUMO-1 also modifies several transcription factors in mammalian cells, as suggested by the observed interaction in a two-hybrid assay of Ubc9 with E1A, IB, WT1, Jun, p53, ATF2, ETS-1, the glucocorticoid receptor, and other nuclear proteins and thus may perform a more general role in transcriptional regulation. These data also indicate that the pattern of covalent modification of Ttk69 may be more complex. In particular, Ttk69 can be phosphorylated as well as conjugated with dSmt3. Notably, general inhibition of serine/threonine phosphorylation prevents dSmt3 conjugation, although it is uncertain whether this is a consequence of a reduction in substrate availability or conjugating activity (Lehembre, 2000 and references therein).

The biological role and consequences of the conjugation of dSmt3 to Ttk69 are unclear. Among several possibilities would be effects on the targeting of the repressor to specific chromosomal sites or on its interaction with specific protein partners. Another attractive hypothesis is that dSmt3 modification might antagonize the degradation of Ttk69 by a proteasome-dependent pathway. Indeed, it has recently been suggested that in human cells, SUMO-1 modification of IB might serve to block signal-induced ubiquitination and thus degradation of IB. In this context it is intriguing that Sina interacts directly with and destabilizes the other isoform of Ttk, Ttk88, but that no comparable interaction of Sina and Ttk69 was observed in a two-hybrid assay. Nevertheless, Ttk69 levels are stabilized in SL2 cells by MG132, an inhibitor of proteasome-mediated proteolysis. It is therefore suggested that dSmt3 modification might provide a mechanism for the differential stabilization of splicing isoforms, such as Ttk69 and Ttk88, that are transcribed from the same promoter. Genetic analysis of dSmt3 mutants in Drosophila should hopefully lead to a better understanding of the role of dSmt3 modification in the transcriptional regulation of sense organ development (Lehembre, 2000).

Regulation of Nuclear Import

The maternal transcript of the anterior segmentation gene bicoid (bcd) is localized at the anterior pole of the Drosophila egg and translated to form a gradient in the nuclei of the syncytial blastoderm embryo after fertilization. The nuclear gradient of Bcd protein (a transcription factor) leads to differential expression of zygotic segmentation genes. The rapid nuclear division in the early zygote requires that Bcd quickly enters the nuclei after each mitosis using an active nuclear import system. Nuclear transport depends on the asymmetrical distribution of two forms of the small GTPase Ran: Ran-GTP that is concentrated in the nucleus and Ran-GDP in the cytoplasm. Ran requires RanGTPase-activating protein-1 (RanGAP1) on the cytoplasmic side of nuclear pore complexes to convert Ran-GTP to Ran-GDP. In vitro studies with vertebrate proteins demonstrate that the RanGAP1 associated with the nuclear pore complex is modified with small ubiquitin related modifier-1 (SUMO-1) by a ubiquitin-conjugating enzyme (E2 enzyme). Mutation of the Drosophila semushi (semi) gene, which encodes an E2 enzyme, blocks nuclear import of Bcd during early embryogenesis and results in misregulation of the segmentation genes that are Bcd targets. Consequently, semi embryos have multiple defects in anterior segmentation. This study demonstrates that an E2 enzyme is required for nuclear transport during Drosophila embryogenesis. semi could be responsible for modification of other proteins essential for Bcd nuclear transport. Nevertheless, these results indicate the possible connection of the function of an E2 enzyme of the Ubc9 family to nuclear import in Drosophila. Hunchback is accumulated in the nucleus in a normal fashion in semi mutants. In semi mutants, posterior segmentation genes function correctly (Epps, 1998).

Search PubMed for articles about Drosophila Sumo

Abed, M., et al. (2011). Degringolade, a SUMO-targeted ubiquitin ligase, inhibits Hairy/Groucho-mediated repression. EMBO J. 30(7): 1289-301. PubMed ID: 21343912

Ahn, J. W., Lee, Y. A., Ahn, J. H. and Choi, C. Y. (2009). Covalent conjugation of Groucho with SUMO-1 modulates its corepressor activity. Biochem. Biophys. Res. Commun. 379: 160-165. PubMed ID: 19101520

Barry, K. C., et al. (2011). The Drosophila STUbL protein Degringolade limits HES functions during embryogenesis. Development 138(9): 1759-69. PubMed ID: 21486924

Berdnik, D., Favaloro, V. and Luo, L. (2012). The SUMO protease Verloren regulates dendrite and axon targeting in olfactory projection neurons. J Neurosci 32: 8331-8340. PubMed ID: 22699913

Bhaskar, V., Valentine, S. A. and Courey, A. J. (2000). A functional interaction between dorsal and components of the Smt3 conjugation machinery. J. Biol. Chem. 275: 4033-4040. PubMed ID: 10660560

Bhaskar, V., Smith, M. and Courey, A. J. (2002). Conjugation of Smt3 to dorsal may potentiate the Drosophila immune response. Mol. Cell. Biol. 22: 492-504. PubMed ID: 11756545

Bielska, K., Seliga, J., Wieczorek, E., Kedracka-Krok, S., Niedenthal, R. and Ozyhar, A. (2012). Alternative sumoylation sites in the Drosophila nuclear receptor Usp. J Steroid Biochem Mol Biol 132: 227-238. PubMed ID: 22676916

Capelson, M. and Corces, V. G. (2006). SUMO conjugation attenuates the activity of the gypsy chromatin insulator. Embo J. 25: 1906-1914. PubMed ID: 16628226

Epps, J. L. and Tanda, S. (1998). The Drosophila semushi mutation blocks nuclear import of bicoid during embryogenesis. Curr. Biol. 8: 1277-1280. PubMed ID: 9822580

Geiss-Friedlander, R. and Melchior, F. (2007). Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell. Biol. 8: 947-956. PubMed ID: 18000527

Geoffroy, M. C. and Hay, R. T. (2009). An additional role for SUMO in ubiquitin-mediated proteolysis. Nat. Rev. Mol. Cell Biol. 8: 564-568. PubMed ID: 19474794

Golovnin, A., Volkov, I. and Georgiev, P. (2012). SUMO conjugation is required for the assembly of Drosophila Su(Hw) and Mod(mdg4) into insulator bodies that facilitate insulator complex formation. J. Cell Sci. 125(Pt 8): 2064-74. PubMed ID: 22375064

Grönholm, J., et al. (2010). Sumoylation of Drosophila transcription factor STAT92E. J. Innate Immun. 2(6): 618-24. PubMed ID: 20616536

Gonzalez, I., Mateos-Langerak, J., Thomas, A., Cheutin, T. and Cavalli, G. (2014). Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen. Mol Cell 54: 485-499. PubMed ID: 24703951

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: 1334-1348. PubMed ID: 11390354

Hashiyama, K., Shigenobu, S. and Kobayashi, S. (2009). Expression of genes involved in sumoylation in the Drosophila germline. Gene Expr. Patterns 9: 50-53. PubMed ID: 18755298

Huang, H. W., Tsoi, S. C., Sun, Y. H. and Li, S. S. (1998). Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster. Biochem. Mol. Biol. Int. 46: 775-785. PubMed ID: 9844739

Lehembre, F., Badenhorst, P., Muller, S., Travers, A., Schweisguth, F., et al. (2000). Covalent modification of the transcriptional repressor tramtrack by the ubiquitin-related protein Smt3 in Drosophila flies. Mol. Cell. Biol. 20: 1072-1082. PubMed ID: 10629064

Lima, C. D. and Reverter, D. (2008). Structure of the human SENP7 catalytic domain and poly-SUMO deconjugation activities for SENP6 and SENP7. J Biol Chem 283: 32045-32055. PubMed ID: 18799455

Long, X. and Griffith, L. C. (2000). Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster. J. Biol. Chem. 275: 40765-40776. PubMed ID: 10995744

Martin, S., Wilkinson, K. A., Nishimune, A. amd Henley, J. M. (2007). Emerging extranuclear roles of protein SUMOylation in neuronal function and dysfunction. Nat. Rev. Neurosci. 8: 948-959. PubMed ID: 17987030

Miles, W. O., et al. (2008). Medea SUMOylation restricts the signaling range of the Dpp morphogen in the Drosophila embryo. Genes Dev. 22: 2578-2590. PubMed ID: 18794353

Nacerddine, K., et al. (2005). The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice. Dev. Cell 9: 769-779. PubMed ID: 16326389

Nie, M., Xie, Y., Loo, J. A. and Courey, A. J. (2009). Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation. PLoS One 4(6): e5905. PubMed ID: 19529778

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

Nowak, M. and Hammerschmidt, M. (2006). Ubc9 regulates mitosis and cell survival during zebrafish development. Mol. Biol. Cell 17: 5324-5336. PubMed ID: 16326389

Paddibhatla, I., Lee, M. J., Kalamarz, M. E., Ferrarese, R. and Govind, S. (2010). Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae. PLoS Pathog. 6(12): e1001234. PubMed ID: 21203476

Poulin, G., et al. (2005). Chromatin regulation and sumoylation in the inhibition of Ras-induced vulval development in Caenorhabditis elegans. Embo J. 24: 2613-2623. PubMed ID: 15990876

Powell, L. M., et al. (2012). The SUMO pathway promotes bHLH proneural factor activity via a direct effect on the Zn finger protein, Senseless. Mol. Cell. Biol. 32(14): 2849-60. PubMed ID: 22586269

Ryu, T., Spatola, B., Delabaere, L., Bowlin, K., Hopp, H., Kunitake, R., Karpen, G.H. and Chiolo, I. (2015). Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nat Cell Biol 17(11):1401-11. PubMed ID: 26502056

Savare, J., Bonneaud, N. and Girard, F. (2005). SUMO represses transcriptional activity of the Drosophila SoxNeuro and human Sox3 central nervous system-specific transcription factors. Mol, Biol, Cell 16: 2660-2669. PubMed ID: 15788563

Shen, L. N., Geoffroy, M. C., Jaffray, E. G. and Hay, R. T. (2009). Characterization of SENP7, a SUMO-2/3-specific isopeptidase. Biochem J 421: 223-230. PubMed ID: 19392659

Smith, M., Bhaskar, V., Fernandez, J. and Courey, A. J. (2004). Drosophila Ulp1, a nuclear pore-associated SUMO protease, prevents accumulation of cytoplasmic SUMO conjugates. J Biol Chem 279: 43805-43814. PubMed ID: 15294908

Smith, M., Mallin, D. R., Simon, J. A. and Courey, A. J. (2011). Small ubiquitin-like modifier (SUMO) conjugation impedes transcriptional silencing by the polycomb group repressor Sex Comb on Midleg. J. Biol. Chem. 286(13): 11391-400. PubMed ID: 21278366

Sun, H., Leverson, J. D., Hunter. T. (2007). Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. EMBO J. 26: 4102-4112. PubMed ID: 17762864

Takanaka, Y. and Courey, A. J. (2005). SUMO enhances vestigial function during wing morphogenesis. Mech. Dev. 122: 1130-1137. PubMed ID: 16026969

Zhao, J. (2007). Sumoylation regulates diverse biological processes. Cell Mol. Life Sci. 64: 3017-3033. PubMed ID: 17763827

date revised: 2 December 2023

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