InteractiveFly: GeneBrief

smt3: Biological Overview | References

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).

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).

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 Citation: 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 Citation: 19101520

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

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 Citation: 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 Citation: 11756545

Capelson, M. and Corces, V. G. (2006). SUMO conjugation attenuates the activity of the gypsy chromatin insulator. Embo J. 25: 1906-1914. PubMed Citation: 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 Citation: 9822580

Geiss-Friedlander, R. and Melchior, F. (2007). Concepts in sumoylation: a decade on. Nat. Rev. Mol. Cell. Biol. 8: 947-956. PubMed Citation: 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 Citation: 19474794

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

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 Citation: 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 Citation: 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 Citation: 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 Citation: 10629064

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 Citation: 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 Citation: 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 Citation: 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 Citation: 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 Citation: 19529778

Nowak, M. and Hammerschmidt, M. (2006). Ubc9 regulates mitosis and cell survival during zebrafish development. Mol. Biol. Cell 17: 5324-5336. PubMed Citation: 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 Citation: 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 Citation: 15990876

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 Citation: 15788563

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 Citation: 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 Citation: 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 Citation: 17762864

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

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

date revised: 29 August 2011

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