InteractiveFly: GeneBrief

lesswright: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - lesswright

Synonyms - Ubc9, dUbc9, semushi

Cytological map position - 21E2

Function - enzyme

Keywords - hematopoiesis, mesoderm, Toll pathway, nuclear transport, meiosis, protein stabilization

Symbol - lwr

FlyBase ID: FBgn0010602

Genetic map position - 2L

Classification - SUMO E2-conjugating enzyme

Cellular location - cytoplasmic and nuclear



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Ryu, T., Spatola, B., Delabaere, L., Bowlin, K., Hopp, H., Kunitake, R., Karpen, G.H. and Chiolo, I. (2015). Heterochromatic breaks move to the nuclear periphery to continue recombinational repair. Nat Cell Biol [Epub ahead of print]. PubMed ID: 26502056
Summary:
Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. This study shows that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/Smc6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote.

Schwartz, S., Truglio, M., Scott, M.J. and Fitzsimons, H.L. (2016). Long-term memory in Drosophila is influenced by the histone deacetylase HDAC4 interacting with the SUMO-conjugating enzyme Ubc9. Genetics [Epub ahead of print]. PubMed ID: 27182943
Summary:
HDAC4 is a potent memory repressor with overexpression of wild-type or a nuclear-restricted mutant resulting in memory deficits. Interestingly, reduction of HDAC4 also impairs memory via an as yet unknown mechanism. Although histone deacetylase family members are important mediators of epigenetic mechanisms in neurons, HDAC4 is predominantly cytoplasmic in the brain and there is increasing evidence for interactions with non-histone proteins, suggesting HDAC4 has roles beyond transcriptional regulation. To that end, this study performed a genetic interaction screen in Drosophila and identified twenty-six genes that interact with HDAC4, including Ubc9, the sole SUMO E2-conjugating enzyme. RNAi-induced reduction of Ubc9 in the adult brain impairs long-term memory in the courtship suppression assay, a Drosophila model of associative memory. It was also demonstrated that HDAC4 and Ubc9 interact genetically during memory formation, opening new avenues for investigating the mechanisms through which HDAC4 regulates memory formation and other neurological processes.
BIOLOGICAL OVERVIEW

The lesswright (lwr) or semushi gene encodes an enzyme, Ubc9, that conjugates a small ubiquitin-related modifier (SUMO). Since the conjugation of SUMO occurs in many different proteins, a variety of cellular processes probably require lwr function. This study demonstrates that lwr function regulates the production of blood cells (hemocytes) in Drosophila larvae. lwr mutant larvae develop many melanotic tumors in the hemolymph at the third instar stage. The formation of melanotic tumors is due to a large number of circulating hemocytes, which is approximately 10 times higher than those of wild type. This overproduction of hemocytes is attributed to the loss of lwr function primarily in hemocytes and the lymph glands, a hematopoietic organ in Drosophila larvae. High incidences of Dorsal (Dl) protein in the nucleus were observed in lwr mutant hemocytes, and the dl and Dorsal-related immunity factor (Dif) mutations were found to be suppressors of the lwr mutation. Therefore, the lwr mutation leads to the activation of these Rel-related proteins, key transcription factors in hematopoiesis. Also demonstrate was the fact that dl and Dif play different roles in hematopoiesis. dl primarily stimulates plasmatocyte production, but Dif controls both plasmatocyte and lamellocyte production (Huang, 2005). Similar results have been reported by Chiu (2005). Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene Drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Although both studies are clear in concluding that Ubc9/Lesswright appears to function upstream of Cactus and Dorsal/Dif, the specific target is not yet known. Wild-type Ubc9/Lesswright hold the Toll pathway in check, and could target Cactus, Dorsal/Dif or another upstream component in the pathway (Huang, 2005; Chiu, 2005).

What is a possible molecular mechanism for the regulation of the Toll pathway? The Lwr protein can regulate activities of its target proteins by conjugating SUMO molecules. For example, the human Cact homologue, IκBα, is known to be sumoylated, and the sumoylated IκBα molecules have been shown to be resistant to ubiquitination because the ubiquitination sites on IκBα are occupied by SUMO molecules (Desterro, 1998). In other words, sumoylation stabilizes IκBα, which in turn represses NF-κB activity. Alternatively, their physical associations alone can control physiological functions of Lwr's targets. Recently, the Paired-like homeobox protein Vsx-1 was reported to physically interact with Ubc9 (the vertebrate Lwr homologue) and to require Ubc9 function for Vsx-1's nuclear localization. However, sumoylation of the Vsx-1 protein was not detected (Kurtzman, 2001). In this case, physical association with Ubc9 itself is a key for Vsx-1 function (Huang, 2005).

The Lwr protein may regulate activities of its target proteins by conjugating SUMO molecules or simply by physical association with its target. In the Tl (or Tl-like) pathway of Drosophila, either of these mechanisms are possible. The Dorsal (Dl) protein was shown to physically interact with Lwr and to be sumoylated in a transfection experiment using Schneider L2 cells. Sumoylated Dl proteins dissociated from Cactus (Cact) and entered the nucleus, where they exhibited an increased level of transactivation (Bhaskar, 2000; Bhaskar, 2002). However, this model does not fit observations of higher incidence of Dl nuclear localization in lwr mutant hemocytes than in wild type hemocytes (Huang, 2005).

Another possible target protein is Cact. As in IκBα, the Cact protein was shown to physically associate with Lwr (Bhaskar, 2000). Unlike IκBα, sumoylated Cact proteins were not detected in the same study. However, physical association of Lwr and Cact might stabilize Cact proteins in the cytoplasm by counteracting ubiquitination or phosphorylation, a prerequisite for ubiquitination of Cact. In this scenario, the loss of lwr function would make Cact be susceptible to degradation, which results in migration of Dl and Dif to the nucleus. Other components in the Tl pathway could be regulated similarly, although they have not been reported to interact with Lwr (Huang, 2005).

The innate immune system is evolutionarily ancient and common among most eukaryotes and consists of humoral and cellular components. In Drosophila, the humoral response primarily represents the production of antimicrobial peptides in the fat body. The cellular innate immunity is handled by circulating blood cells capable of recognizing and neutralizing foreign objects. These cells also work as scavengers of apoptotic cells in normal development. Because the balance of these functions is so important, the proliferation and differentiation of hemocytes must be tightly regulated (Huang, 2005).

The Drosophila larval hemocyte population consists of several cell types, some of which reside only in a blood-cell-forming organ, the lymph gland. Three cell types, plasmatocytes, crystal cells, and lamellocytes (the subject of this study), are present in hemolymph. The majority are plasmatocytes, which are able to phagocytose microbes and apoptotic cells. A small fraction (<5%) are crystal cells, which are involved in melanization reactions. Rarely found in circulation in normal circumstances, lamellocytes are key defensive players when larvae are infested by parasitoid wasp eggs. In addition to these cells, prohemocytes (hematopoietic stem cells) and secretory cells are found in the lymph glands. A small number of prohemocytes circulate in hemolymph. The population structure of hemocytes changes as larvae develop and undergo metamorphosis. Several genes have been found to direct differentiation of specific hemocytes. A Drosophila GATA homologue, serpent, is required for the expression of two lineage-specific genes, glial cell missing (gcm) and lozenge (lz). The gcm gene encodes a transcription factor and is required for the plasmatocyte lineage. A Drosophila acute myeloid leukemia-1 homologue, lz, and the Notch pathway are required for the crystal cell lineage. Recently, yantar and collier were found to direct lamellocyte differentiation. However, precise mechanisms of the proliferation and differentiation of hemocytes still remain elusive (Huang, 2005).

Three signal transduction pathways are known to influence the number of hemocytes in circulation. They are the Ras, JAK/STAT, and Tl (dealt with in this study) pathways. Mutations that activate these pathways increase the total hemocyte numbers 10- to 100-fold and often stimulate lamellocyte production in the absence of parasitoid wasp infestation. Ras functions in the MAP kinase pathway and stimulates cell division. It is one of the most common oncogenes found in many different human cancers. When the Drosophila ras1 gene is overexpressed in the lymph gland and hemocytes, total hemocyte counts increase nearly 100-fold. The JAK-STAT pathway is a conserved signal transduction pathway and plays a variety of roles in Drosophila and many other eukaryotes including humans. A dominant mutation of JAK, hopscotchTum-l, causes a drastic increase in plasmatocytes as well as lamellocytes in a temperature-dependent manner. Similarly, dominant mutations of Toll, Tl10B, and Tl3, produce a large number of hemocytes. In this case, the lamellocyte population increases to 10%-20% of the entire hemocyte population. Several genes in the Tl signaling pathway also exhibit a similar defect when mutated. Although it is not clear if these signal transduction pathways possess lineage specific functions, they play some general role in Drosophila hematopoiesis (Huang, 2005).

The studies of the NF-κB pathway, discovered in 1986, have made a huge contribution to the understanding of mammalian immune systems, particularly in acquired (or adaptive) immunity. NF-κB is a dimer of members of the Rel-related transcription factors. A unique aspect in the NF-κB pathway is that Inhibitors of κB, IκBs, keep the transcription factor NF-κB in the cytoplasm from its action in the nucleus. Ubiquitination, thus degradation, of IκB is required for the activation of the NF-κB pathway. A Drosophila Rel-related gene, dl, was discovered to be an important component in the establishment of the embryonic dorsal-ventral axis and was found to be a part of Tl signaling. The activation of Tl signaling starts from the cell surface receptor Tl and ends at the entrance of the Dl transcription factor into the nucleus. The function of Tl signaling in innate immunity was later discovered indirectly. Sequence analysis of several antimicrobial peptide genes spotted κB sites, binding sites for NF-κB and Dl, in their 5′ regulatory regions, thjat connected the Tl pathway to humoral immunity. Discovery and subsequent studies of other Rel-related genes, Dif and Relish, further support a key role of the Tl pathway in Drosophila immunity. Furthermore, the homology between mammalian IκBs and Cactus (Cact) demonstrates that the parallelism extends to the regulatory mechanism of mammalian and Drosophila NF-κB activity. The presence of multiple Rel-related proteins and Tl-like receptors in Drosophila as well as in other eukaryotes makes the system more complex, but versatile in innate immune responses and hematopoiesis. In the last decade, remarkable progress has been made toward the understanding of Drosophila humoral immunity including the role of the Tl pathway. In contrast, the regulatory mechanisms of cellular immunity are much less understood (Huang, 2005).

Recently, ubiquitin's small cousins, SUMO molecules, were discovered to add another layer of complexity in NF-κB signaling. SUMO conjugation (sumoylation) regulates a variety of cellular functions (Melchior, 2000; Muller, 2001; Yeh, 2000). Proteins subjected to sumoylation are involved in oncogenesis, transcriptional regulation, nuclear transport, and others. Ubiquitin and SUMO molecules are biochemically quite similar to each other. Furthermore, they require a set of similar modifying enzymes, activating (E1), conjugating (E2), and ligating (E3) enzymes, all of which show high conservation among most eukaryotes. The similarity between ubiquitination and sumoylation creates an interesting situation where the same lysine residues on a given protein can be modified by both ubiquitin and SUMO. Thus, ubiquitination and sumoylation can counteract each other. This scenario is seen in the regulation of IκBα degradation (Desterro, 1998). In addition to NF-κB signaling, the activity of the JAK/STAT pathway might be regulated in part by sumoylation since a Protein inhibitor of activated STAT (Pias) possesses a RING finger motif, an E3 SUMO ligase signature. However, these possibilities have not yet been explored in Drosophila hematopoiesis (Huang, 2005 and references therein).

This study reports that the Lwr protein, a SUMO conjugase, plays an important role in regulation of larval hematopoiesis in Drosophila melanogaster. Recessive as well as dominant negative mutations of the lwr gene result in the overproliferation of hemocytes in larvae. Loss of lwr function led to the accumulation of the Rel-related protein Dl in the nuclei of circulating hemocytes, and dl and Dif mutations are suppressors of the lwr mutation. Taken together, these results indicate that the lwr function is inhibitory to dl and Dif activities. Furthermore, it is demonstrated that dl and Dif possess different properties in hematopoiesis, particularly in lamellocyte production (Huang, 2005).

Thus mutation of the lwr gene, which encodes a SUMO-conjugating enzyme, causes the activation of Rel-related proteins, Dl and Dif, and leads to overproduction of larval hemocytes. Immunochemical and genetic analyses indicate that the lwr mutation results in the activation of Dl and Dif primarily in plasmatocyte and prohemocytes, leading to overproliferation of plasmatocytes and lamellocytes in larvae. Dl nuclear localization in the lwr mutant hemocytes was observed as frequently as those in the Tl10B mutant hemocytes. The high hemocyte counts of the lwr mutant larvae are almost completely suppressed by removing both dl and Dif functions from the lwr mutant background. Therefore, it is proposed that the wild type lwr function negatively regulates the activity of the Tl pathway and/or the pathways of Tl-like genes (Huang, 2005).

It has been demonstrated that lwr function is required for the Ran-dependent nuclear transport system (Epps, 1998). This raises a question of how efficiently Dl and Dif proteins are transported to the nucleus in lwr mutant hemocytes. Nuclear import of Dl and Dif was not overly impaired. These contrary observations can be explained by functional redundancy of lwr with 25 other Drosophila E2 enzymes. Since functions of most E2 enzymes have not been clearly defined, some of them may be SUMO conjugases. This possibility was also suggested in the embryo, particularly in the posterior half of the embryo (Epps, 1998). Alternatively, the residual lwr function may be sufficient to mediate nuclear import of Dl proteins because hypomorphic lwr alleles were used in this study. In any case, a possible negative effect on nuclear transport appeared minimal in lwr mutant hemocytes (Huang, 2005).

The loss of dl function in the lwr mutant background resulted in a reduction of plasmatocyte, not lamellocyte counts, and the overexprssion of dl with the CgGAL4 driver stimulated plasmatocyte production only. Therefore, the role of dl in hematopoiesis is limited to plasmatocyte production. Although dl plays a role in plasmatocyte production in the wild type background, the contribution of dl to plasmatocyte production seems minimal when Dif is overexpressed. In other words, Dif can replace dl function in Drosophila hematopoiesis (Huang, 2005).

In contrast, Dif is capable of stimulating the production of both plasmatocytes and lamellocytes. Because the loss of Dif function in the lwr mutant background led the lamellocyte population to a level very close to those of wild type strains, Dif is most likely to play an essential and sole role in lamellocyte production when the Tl signaling is activated by the lwr mutation. Furthermore, when Dif was overexpressed by the CgGAL4, the levels of plasmatocytes and lamellocytes were similar to those reached when Tl10B was overexpressed by the same driver. Taken together, it is concluded that Dif is necessary and sufficient to promote the production of plasmatocytes and lamellocytes in larvae in the Tl (Tl-like) pathway. This also means that Dl-responsive genes can be regulated by Dif (Huang, 2005).

It is hypothesized that the genes for plasmatocyte production can be divided into at least two classes. The first class consists of genes regulated primarily by Dl. The genes in this class can be stimulated by Dif in the absence of Dl. It is known that Dl proteins bind to more strictly defined κB sites than Dif, and that Dif can bind more broadly to κB consensus sequences including Dl-binding sites. Several humoral immunity genes were found to belong to this class. Thus, it is possible that such genes exist among genes essential for plasmatocyte production. The other class includes genes regulated only by Dif. In these genes, consensus κB sites are probably responsible for their expression via the Dif protein (Huang, 2005).

The Dl protein can function as a repressor in a context-dependent manner. A good example in the Drosophila immunity is the Cecropin gene, whose expression depends heavily on Dif. Co-expression of Dl expression along with Dif strongly represses the expression of the Cecropin gene. However, no significant dominant negative effect of dl on plasmatocyte production was observed in overexpression experiments. At the same time, no synergistic effect on plasmatocyte production was observed when both dl and Dif were co-expressed. Therefore, regulatory mechanisms of plasmatocyte-specific genes are not so complex (Huang, 2005).

The collagen IV (Cg25C) gene, whose promoter was used for the CgGAL4 driver, is expressed in embryonic and larval hemocytes (Yasothornsrikul, 1997). These larval hemocytes are most likely plasmatocytes, although the authors of the study did not distinguish plasmatocytes and prohemocytes. Nonetheless, the current results show that these Cg25C-expressing hemocytes are capable of proliferating and differentiating into plasmatocytes when Dl and Dif is activated (Huang, 2005).

It was surprising that the CgGAL4 driver induced a relatively large number of lamellocytes because this driver is not capable of inducing GAL4 in lamellocytes and because the collagen IV gene is expressed primarily in plasmatocytes. This can be interpreted in two different ways. The first one is that the Cg25C gene is expressed in lamellocyte precursors in the lymph gland, where these precursor cells can divide. In addition to different cell types distinguished by their morphologies, the cells in the lymph gland can be classified based on expression of different genes. For example, the Posterior Signaling Center, which is important for crystal cell differentiation, was defined as Serrate-expressing cells in the first lobe of the lymph gland. Thus, it is conceivable that the Cg25C gene might be expressed in the cells that have not been clearly defined in the lymph gland. The second interpretation is that plasmatocytes can differentiate into lamellocytes when Dif are highly activated. This interpretation supports a classical view of lamellocyte differentiation. Further investigations are necessary to answer these questions (Huang, 2005).

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).


REGULATION

Protein Interactions

A nuclear ubiquitin-conjugating enzyme, DmUbc9, interacts with small heat shock proteins

In a two hybrid screen designed to identify proteins that interact with small heat shock proteins (sHsps), a Drosophila melanogaster homologue of yeast and human ubc9 (Dmubc9) was found to interact with Drosophila Hsp23. Further, two-hybrid system analysis reveals DmUbc9 interaction with Drosophila and mammalian Hsp27. In situ hybridization localizes Dmubc9 as a doublet at locus 21D on chromosome 2L, and genomic cloning of the gene reveals a single open reading frame without introns. The predicted Dmubc9 protein sequence shares a very high level of homology with mouse (85.4%) and human (> or = 82.9%) Ubc9. Genetic complementation analysis show that Dmubc9 functionally rescues a temperature-sensitive S. cerevisiae ubc9ts mutant. Co-immunoprecipitation with antibody raised against DmUbc9 confirms the interaction with Drosophila Hsp23 and Hsp26 and preferentially with Hsp27 (Joanisse, 1998).

The Drosophila semushi mutation blocks nuclear import of Bicoid during embryogenesis

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).

A SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II

Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library. At least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 in vivo. The interactions observed in the two-hybrid screen may therefore reflect catalytic events. To understand the role of SUMO conjugation in the brain, a characterization of the system was undertaken. The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches. Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9. DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system. Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond. Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates. High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality. Expression in the developing eye led to a rough eye phenotype with retinal degeneration. These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function (Long, 2000).

A functional interaction between Dorsal and components of the Smt3 conjugation machinery

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). 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

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, 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).

DmUba2/DmAos1 and DmUbc9 are the activating and conjugating enzymes, respectively, for DmSmt3, and they form a complex

The septins are a family of proteins involved in cytokinesis and other aspects of cell-cortex organization. In a two-hybrid screen designed to identify septin-interacting proteins in Drosophila, several genes were isolated, including homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9. Yeast Uba2p and Ubc9p are involved in the activation and conjugation, respectively, of the ubiquitin-like protein Smt3p/SUMO, which becomes conjugated to a variety of proteins through this pathway. Uba2p functions together with a second protein, Aos1p. The Drosophila homologues of AOS1 (Aos1/Dmaos1) and SMT3 (Dmsmt3) were also cloned and characterized. Biochemical data suggest that DmUba2/DmAos1 and DmUbc9 indeed act as activating and conjugating enzymes for DmSmt3, implying that this protein-conjugation pathway is well conserved in Drosophila. Immunofluorescence studies showed that DmUba2 shuttles between the embryonic cortex and nuclei during the syncytial blastoderm stage. In older embryos, DmUba2 and DmSmt3 are both concentrated in the nuclei during interphase but dispersed throughout the cells during mitosis, with DmSmt3 also enriched on the chromosomes during mitosis. These data suggest that DmSmt3 could modify target proteins both inside and outside the nuclei. No concentration was observed of DmUba2 at sites where the septins are concentrated, and no DmSmt3 modification was detected of the three Drosophila septins tested. However, DmSmt3 localization to the midbody during cytokinesis was observed both in tissue-culture cells and in embryonic mitotic domains, suggesting that DmSmt3 modification of septins and/or other midzone proteins occurs during cytokinesis in Drosophila (Shih, 2002).

To identify proteins interacting with the Drosophila septins, two-hybrid screens were conducted using Pnut, Sep1, and Sep2 as baits. These screens identified 27 positive clones that proved to represent eight genes. Among these were the other septins, as expected from other data indicating that septins interact with one another. In addition, the screens identified Drosophila homologues (Dmuba2 and Dmubc9) of yeast UBA2 and UBC9, whose products are involved in the activation and conjugation of the ubiquitin-like molecule Smt3p/SUMO. These screening results and the recent discovery of Smt3p modification of septins in S. cerevisiae (Johnson, 1999; Takahashi, 1999) stimulated a study the Smt3p/SUMO conjugation machinery in Drosophila (Shih, 2002).

In further two-hybrid analyses, the C-terminal portion of DmUba2 interacted strongly with full-length Sep1 and Sep2 and with the N-terminal portion of Sep1. Interactions were also detected with the N-terminal portions of Pnut and Sep2 and with the C-terminal portions of Sep1 and Sep2. In contrast, a full-length AD-DmUba2 fusion showed none of these interactions, although other studies indicated that this fusion was functional for other interactions. Interestingly, full-length AD-DmUbc9 showed a pattern of interactions very similar to those seen with the C-terminal portion of DmUba2 (Shih, 2002).

The other genes identified in the screens had not been described previously; they were designated sip1-sip4 (for septin-interacting protein). sip1 (Accession No. AF221101; Drosophila genome annotation No. CG7238) encodes a protein with predicted P-loop and coiled-coil domains; it appeared to interact specifically with the C-terminal portion of Pnut. sip2- sip4 encode proteins without obviously informative motifs. sip2 (CG9188) encodes a protein that interacted with full-length Sep1 and Sep2 but not with Pnut. sip3 (CG1937) encodes a protein that appeared to interact specifically with the C-terminal portion of Sep2. sip4 was identified independently as dip2 (Dorsal interacting protein 2) (Bhaskar, 2000); it encodes a protein that interacts with all of the Sep1 and Sep2 fusions and (weakly) with the N-terminal portion of Pnut (Shih, 2002).

Sequencing of a full-length clone of Dmuba2 showed that the predicted DmUba2 contains 700 amino acids and has 29% sequence identity to yeast Uba2p and 48% identity to human hUba2, as observed also by others (Bhaskar, 2000; Long, 2000; Donaghue, 2000). Like its homologues and other E1-type enzymes, DmUba2 contains an ATP-binding motif (amino acids 26-31) and the consensus Cys (C175) corresponding to those essential for thiolester bond formation in other E1-type enzymes. The original two-hybrid clone of Dmubc9 appeared to be full length by comparison to yeast UBC9, and the sequence for Dmubc9 agreed with that reported by Joanisse (1998). Antibodies were raised to DmUba2. The affinity-purified antibodies recognized mainly one polypeptide of apparent molecular weight ~97 kDa, which is presumably DmUba2 (predicted molecular weight, 77.5 kDa). Support for this conclusion was obtained by expressing Dmuba2 under GAL-promoter control in yeast. When cells were grown under inducing conditions, the antibodies recognized primarily a polypeptide of apparent molecular weight ~97 kDa that was absent when cells were grown under repressing conditions. Similarly anomalous low mobility on SDS-PAGE has been noted for both Uba2p and hUba2 (Shih, 2002).

In S. cerevisiae, the E1 enzyme for ubiquitin activation is the 1024 amino-acid Uba1p (see Drosophila Uba1). In contrast, Smt3p is activated by a heterodimer of the 636 amino-acid Uba2p, which is related to the C-terminal part of Uba1p, and the 347 amino-acid Aos1p, which is related to the N-terminal part of Uba1p. Similarly, the 700 amino-acid DmUba2 is related in sequence (~40% identity over the ~225 amino acids of the three similarity boxes defined for other Uba1- type and Uba2-type enzymes to the C-terminal part of the putative Drosophila ubiquitin-activating enzyme DmUba1. Therefore, a Drosophila homologue of yeast AOS1 was identified among the ESTs from the BDGP (Shih, 2002).

Sequencing (Accession No. AF193554) showed that Dmaos1 encodes a polypeptide of 337 amino acids that has 28% sequence identity to yeast Aos1p and 40% identity to the human Aos1p homologue Sua1, as also observed by others (Bhaskar, 2000; Long, 2000). As expected, DmAos1 is related in sequence to the N-terminal part of DmUba1 (~37% identity over the ~202 amino acids of the similarity boxes as defined previously, suggesting that a heterodimer of DmUba2 and DmAos1 is the Drosophila Smt3/SUMO-activating enzyme. In support of this hypothesis, an interaction was detected between full-length DmUba2 and full-length DmAos1 in the two-hybrid system. An attempt to use the two-hybrid system to delimit the region of DmUba2 responsible for its interaction with DmAos1 was unsuccessful (Shih, 2002).

A homolog of SMT3/SUMO-1 was identified among the BDGP EST clones. The predicted DmSmt3 contains 90 amino acids with 48% identity to yeast Smt3p and 54% identity to human SUMO-1, as also observed by others (Bhaskar, 2000; Huang, 1998; Lehembre, 2000). Polyclonal antibodies were generated and immunoblots were performed on fly extracts, expecting to detect both free DmSmt3 and DmSmt3- modified proteins. Because yeast and mammalian cells contain Smt3p/SUMO-1-specific isopeptidases, which remove Smt3/SUMO from modified proteins, extracts were prepared both with and without N-ethylmaleimide (NEM), an isopeptidase inhibitor. In both extracts, the purified antibodies recognized both a polypeptide of ~16 kDa (presumably free DmSmt3) and many polypeptides of higher molecular weight (presumably DmSmt3-conjugated proteins). As expected, the higher molecular-weight species were both less abundant and of lower average molecular weight when extracts were prepared without NEM (Shih, 2002).

To test the hypothesis that DmUba2/DmAos1 and DmUbc9 are activating and conjugating enzymes for DmSmt3, but not for ubiquitin (DmUb), in vitro protein-binding assays were used to investigate the interactions among these proteins. Because ubiquitin and ubiquitin-like proteins undergo proteolytic cleavage of their C-termini to leave the sequence Gly-Gly, which is essential for both activation and conjugation, DmSmt3 and DmUb were cloned such that they terminated with Gly88 (DmSmt3) or Gly76 (DmUb) and were tagged with His6 at their N-terminal ends. Then incubated purified His6DmSmt3 and purified His6DmUb were incubated with fly extracts. The His6-tagged proteins were isolated using Ni-NTA beads, and the associated proteins were analyzed. As expected, both DmUba2 and DmUbc9 associated only with His6DmSmt3 and not with His6DmUb. The anti-DmUba2 antibodies detected not only the free form of DmUba2 (~97 kDa) but also species whose lower mobilities suggested that they might represent DmUba2 conjugated to one, two, or three molecules of DmSmt3 (and/or some other ubiquitin-like molecule). To ask if the interactions of DmUba2 and DmUbc9 with DmSmt3 involved thiolester bonds, the experiments were repeated but ß-mercaptoethanol (which reduces thiolester bonds) was omitted during sample preparation. As expected, the most abundant species now observed with the anti-DmUba2 antibodies had an apparent molecular weight of ~116 kDa, consistent with its being DmUba2 with a single His6DmSmt3 linked by a thiolester bond. Similarly, the anti-DmUbc9 antibodies now revealed an additional species with an apparent molecular weight of ~30 kDa, presumably representing DmUbc9 with a single His6DmSmt3 linked by a thiolester bond (Shih, 2002).

Immunoprecipitation was used to ask whether DmUba2 and DmUbc9 interact in vivo. When immunoprecipitates were prepared from embryo extracts using antibodies to either protein, the other protein was detected by immunoblotting. Taken together, the results of in vitro binding assays and coimmunoprecipitation suggest that DmUba2/DmAos1 and DmUbc9 are indeed the activating and conjugating enzymes, respectively, for DmSmt3. They may form a complex containing both the E1-type and E2-type enzymes (Shih, 2002).

To begin investigating the roles of the DmSmt3-conjugation pathway in Drosophila, immunofluorescence and confocal microscopy was used to characterize the intracellular localization of DmUba2. Yeast Uba2p is concentrated in the nucleus, but the localization of the homologous enzyme has not been examined in multicellular organisms. Interestingly, DmUba2 is not exclusively nuclear during early embryogenesis. Before migration of the nuclei to the embryo cortex after nuclear division 9, DmUba2 was found largely in the cortex, and its distribution there appeared homogenous. During the interphase preceding nuclear division 10, DmUba2 gradually became organized into a cap corresponding approximately to the cortical actin cap that forms over each nucleus. DmUba2 was also found in the deeper cytoplasm and gradually moved into the nuclei. During mitosis, DmUba2 was dispersed in the cortex and in the cytoplasm near the cortex. During the three subsequent nuclear cycles, the cap-like localization, progressive nuclear accumulation, and dispersion during mitosis of DmUba2 remained evident. However, during the successive cycles, the cap-like cortical localization became more organized and the degree of nuclear enrichment became more pronounced. By cycle 13, although some DmUba2 still localized to the cortex during interphase, it was predominantly nuclear (Shih, 2002).

Because DmUba2 was identified by its two-hybrid interaction with Sep1 and Sep2, whether DmUba2 colocalized with the septins either at the cellularization front or in cleavage furrows in mitotic domains after gastrulation was examined carefully. During cellularization, most DmUba2 localized to nuclei, accumulating preferentially at their apical ends. No DmUba2 was detected at the cellularization front. However, some DmUba2 remained at the cortex, where diffuse septin staining was also observed. During mitosis in older embryos, DmUba2 spread throughout the cell but did not become detectably concentrated in cleavage furrows; it then moved back into the nuclei after mitosis, with no detectable concentration at the midbody. Thus, no substantial colocalization of DmUba2 and septins was detected in early embryogenesis. However, it remains possible that DmUba2 could interact with septins at the cortex in syncytial-blastoderm embryos, during cellularization, or in mitotic cells. DmUba2 was concentrated in nuclei of nondividing cells and dispersed throughout the cell during mitosis throughout embryonic stages 6 to 15. This was particularly striking in the CNS, where the septins, in contrast, are enriched in axons (Shih, 2002).

DmUba2 localization was also examined during oogenesis. DmUba2 localized to the nuclei of both germ cells and somatic follicle cells in the germarium. After encapsulation of the germ-line cells by the follicle cells, DmUba2 remained localized to follicle cell nuclei. DmUba2 localization to nurse cells decreased as egg chamber development progressed, but it remained enriched in the oocyte nucleus. In contrast, Pnut localizes primarily to the basal surface of the follicle cells and is excluded from nuclei (Shih, 2002).

The two-hybrid interactions between the septins and DmUba2 and DmUbc9 might reflect a physiologically significant but transient interaction, such as might occur if Drosophila septins, like yeast septins, are Smt3 modified. To explore this possibility, immunofluorescence was used to examine DmSmt3 localization in cultured cells and in cellularizing and older embryos. DmSmt3 did not colocalize detectably with the septins at the cellularization front. Instead, DmSmt3 localized to nuclei, with a particular enrichment at their apical ends, as did DmUba2. In cultured cells and in cells of postgastrulation embryos, DmSmt3 was concentrated in nuclei throughout interphase. During mitosis, DmSmt3 initially appeared to spread throughout the cell. However, during metaphase, DmSmt3 appeared to concentrate in the region of the chromosomes; this was confirmed by localizing DmSmt3 relative to the mitotic spindle. Strikingly, DmSmt3 was also found concentrated in a spot at cleavage furrows and midbodies both in cultured cells and in dividing embryonic cells; this spot overlapped, but did not appear to coincide fully, with the concentration of the septins in these furrows. DmSmt3 was not enriched at the cleavage furrows early in furrow formation, but only during later stages, and it remained concentrated in the midbody after most DmSmt3, and essentially all of the DmUba2, had reaccumulated in nuclei (Shih, 2002).

DmSmt3 localization was also examined during other stages of embryogenesis. During the syncytial cell cycles, DmSmt3 was concentrated in the nuclei during interphase and appeared to localize to the chromosomes during mitosis. Like DmUba2, DmSmt3 localized primarily to the nuclei of non-mitotic cells throughout the rest of embryogenesis, including in the CNS (where the septins, in contrast, localized to axons). The concentration of DmSmt3 in late cleavage furrows and midbodies suggested that one or more of the Drosophila septins might be modified by DmSmt3. Various experimental approaches were tried to look for DmSmt3-modified septins. None of these approaches detected DmSmt3 modification of Pnut, Sep1, or Sep2. These negative results may mean that the septins are not Smt3 modified. However, immunofluorescence studies detected colocalization of Smt3 with the septins only for a brief period at the end of mitosis, and even at this stage the overlap was not complete. Thus even if septins are Smt3 modified during this period, they would probably comprise only a very small fraction of the total septins in the embryo and thus might have escaped detection (Shih, 2002).

The Drosophila septins appear to be essential for cytokinesis in at least some cell types, and it is likely that they have a variety of non-cytokinesis roles as well. Because studies in yeast suggest that a primary function of the septins is to serve as a matrix or template for the organization of other proteins at the cell surface, the identification of septin-interacting proteins should be critical to the elucidation of septin function in Drosophila. This study began with an attempt to identify such proteins using the yeast two-hybrid system. Of 27 positive clones identified with three septin baits, 17 contained fragments of the septin genes themselves. Because other evidence suggests strongly that the septins interact with each other in vivo, this result suggests that the baits used were good and that the screen was otherwise of high fidelity. Thus, it seems likely that at least some of the other positive clones represent genes whose products really interact with septins in vivo (Shih, 2002).

In conclusion several lines of evidence suggest that the two-hybrid interactions observed between DmUba2, DmUbc9, and the septins reflect physiologically significant interactions. (1) Among the 10 positive clones that did not encode septins, four contained either Dmuba2 or Dmubc9, and Dmuba2 fragments were isolated with two different septin baits. (2) Because DmUba2 and DmUbc9 also interact with each other, the identification of both genes independently in screens using septin baits suggests that the interactions are relevant. (3) While these studies were in progress, it became clear that yeast septins are extensively modified by Smt3p, although the functional significance of that remains uncertain (Johnson, 1999; Takahashi, 1999; Takahashi, 2001; Johnson, 2001). (4) Several other groups also isolated Uba2 and/or Ubc9 in two-hybrid screens using other protein baits. Several of these interactors, including Drosophila calcium/calmodulin-dependent kinase II (CAM-kinase II; Long, 2000) and Dorsal (Bhaskar, 2000), were subsequently shown to be Smt3 modified (Shih, 2002).

Other genetic data may also reflect an interaction between the septins and the DmSmt3 system. The pnut septin mutation was originally identified as an enhancer of a sina mutation that affects R7 photoreceptor development. Although the significance of this genetic interaction remains unclear, it may reflect the recently discovered crosstalk between Smt3/SUMO modification and ubiquitination. Mammalian SUMO-1 is conjugated to the protein Mdm2, a RING-finger E3 ubiquitin ligase involved in p53 degradation. Modification by SUMO-1 appears to regulate Mdm2 activity and hence the level of p53, probably by regulating the ubiquitination and degradation of Mdm2 itself. Other RING-finger proteins, including Drosophila Sina, interact with Ubc9 family proteins and/or are modified by Smt3/SUMO (Duprez, 1999; Hu, 1997). Thus, it seems possible that DmSmt3 modification regulates the activities of Sina, such as its role in downregulating the transcriptional repressor Tramtrack (one of whose isoforms is itself DmSmt3 modified) (Lehembre, 2000; Li, 1997; Neufeld, 1998; Tang, 1997), and that Pnut plays a role in mediating the requisite interactions. Finally, in several types of dividing Drosophila cells, it was found that DmSmt3 colocalizes with septins in the cleavage furrows and/or midbodies during cytokinesis. Interestingly, DmSmt3 is not enriched in the furrow during the early stages of furrow formation but only later, at a time when most DmUba2 has moved back into the nuclei. Although DmSmt3 modification of Sep1, Sep2, or Pnut could not be detected (despite considerable effort), it remains possible that one or more of these proteins is modified at low levels or that DmSmt3 is conjugated to Sep4 or Sep5. However, it is also possible that the DmSmt3 in the midzone is conjugated to other proteins, such as the 'chromosomal passenger proteins,' which are associated with the chromosomes and then relocate to the spindle midzone in mitosis. The involvement of such proteins in chromosome segregation as well as in cytokinesis might help to explain the observations suggesting that the Smt3/SUMO system is involved in chromosome segregation (Apionishev, 2001; Meluh, 1995; Tanaka, 1999; Shih, 2002 and references therein).

It also remains unclear whether the Drosophila septins ever serve as a matrix/template for the localization of the DmSmt3 conjugation system. Although immunofluorescence studies show that DmUba2 and the septins are sometimes in the same compartment, so that interaction would be possible, no persuasive colocalization of the proteins was detected. Thus, elucidation of the possible interactions between the septins and the Smt3 system in Drosophila, and of their functional significance, will need to await further studies using other approaches (Shih, 2002).

Biochemical and two-hybrid studies indicate that there are multiple DmSmt3-modified proteins, that DmSmt3-specific isopeptidases probably exist, that DmUba2 and DmAos1 interact with each other, and that both DmUba2 and DmUbc9 become conjugated to DmSmt3, but not to DmUbiquitin, by thiolester bonds. While these studies were in progress, related findings were also made by other investigators who were led by other routes to the Smt3/SUMO system in Drosophila. In particular, several other proteins were shown to interact with DmUba2 and DmUbc9 using the two-hybrid system, and multiple DmSmt3-modified proteins were observed (Bhaskar, 2000; Donaghue, 2001; Joanisse, 1998; Lehembre, 2000; Long, 2000; Ohsako, 1999), supporting the hypothesis that DmSmt3 is indeed conjugated to a variety of proteins in vivo. In addition, the DmUba2/DmAos1 interaction and the conjugation of DmSmt3 to DmUba2 and DmUbc9 by thiolester bonds were also observed using other methods (Lehembre, 2000; Long, 2000). Finally, it was shown that Dmubc9 can functionally complement a yeast ubc9 mutation (Joanisse, 1998; Ohsako, 1999). Taken together, these results make clear that the Smt3/SUMO conjugation system is closely conserved between Drosophila, yeast, and mammals. The data also show that DmUba2 and DmUbc9 form a complex in vivo, suggesting that the conjugation machinery may act in a concerted fashion (Shih, 2002).

Studies in other laboratories also provided clues to possible functions of modification by DmSmt3. In particular, the semushi (Epps, 1998) and lesswright (Apionishev, 2001) mutations are both in Dmubc9, suggesting (from their mutant phenotypes) that DmUbc9 has roles in the nuclear import of the transcription factor Bicoid and in meiotic chromosome segregation. In addition, two other transcriptional regulators, Dorsal and Tramtrack, as well as CAM-kinase II, have also been shown to be modified by DmSmt3 (Bhaskar, 2000; Lehembre, 2000; Long, 2000). In the case of Dorsal, as with Bicoid, DmSmt3 conjugation appears to promote nuclear localization, whereas Tramtrack modification may help to regulate its activity and/or its degradation by the proteasome, as discussed above. The modification of CAM-kinase II may regulate its activity (Shih, 2002).

Although most suggested functions of the Smt3/SUMO system in Drosophila, as well as in yeast and mammalian cells, center on nuclear proteins, immunofluorescence and two-hybrid data support the hypothesis that there are also cytoplasmic targets. (1) It remains likely that in Drosophila, as in yeast, there is an interaction between the Smt3/SUMO system and the septins, which appear to be exclusively cytoplasmic proteins. (2) Some DmSmt3- modified proteins appear to remain both at the embryo cortex during cellularization and in the midbodies that remain after the nuclear envelopes have reformed at the end of cytokinesis. (3) Although DmUba2 and DmUbc9 (Joanisse, 1998; Lehembre, 2000) are found primarily in nuclei, considerable DmUba2 is also found in the cytoplasm and at the cortex during the syncytial-blastoderm stage, suggesting that DmSmt3 modification of cortical and/or cytoplasmic proteins could occur. (4) The cell-cycle-regulated translocation of DmUba2 between cytoplasm and nucleus both in syncytial blastoderm and in post-cellularization embryos may suggest that the partitioning of the DmSmt3-conjugation system between the cytoplasm and the nucleus is important and thus well regulated during embryonic development (Shih, 2002).

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>cactusRNAi) 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 (spzrm7/spzrm7) 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).

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).


DEVELOPMENTAL BIOLOGY

The DmUbc9 protein, which localizes primarily to the nucleus in Drosophila S2 cells, is found at high levels in embryos but is also present at lower levels throughout development (Joanisse, 1998).

Drosophila Sex lethal gene initiates female development in germline progenitors

Sex determination in the Drosophila germ line is regulated by both the sex of the surrounding soma and cell-autonomous cues. How primordial germ cells (PGCs) initiate sexual development via cell-autonomous mechanisms is unclear. This study demonstrates that, in Drosophila, the Sex lethal (Sxl) gene acts autonomously in PGCs to induce female development. Sxl is transiently expressed in PGCs during their migration to the gonads; this expression, which was detected only in XX PGCs, is necessary for PGCs to assume a female fate. Ectopic expression of Sxl in XY PGCs was sufficient to induce them to enter oogenesis and produce functional eggs when transplanted into an XX host. These data provide powerful evidence that Sxl initiates female germline fate during sexual development (Hashiyama, 2011).

Primordial germ cells (PGCs) are able to differentiate into eggs or sperm. It is thought that PGCs do not assume a sexual fate until they reach the gonads, where sexual dimorphism is imposed by both the sex of the surrounding soma and cell-autonomous cues. In Drosophila, pole cells or PGCs differentiate to a male fate in response to JAK/STAT signaling from the gonadal soma. The method by which female sexual development is initiated in pole cells, however, has not been elucidated. To clarify the mechanism that initiates a female fate in pole cells, a female-specific marker for this cell type was identified. Although several sex-specific markers, including mgm-1, disc proliferation abnormal, and minichromosome maintenance 5, have been reported, they are all expressed only in male pole cells after gonad formation (stage 15), based on signals from the male gonadal soma. lesswright (lwr), a gene that regulates posttranslational modification of proteins by small ubiquitin-related modifiers, is expressed in pole cells during embryogenesis. lwr is not characterized by sex-specific expression. When a dominant-negative form of lwr (lwrDN) was expressed in the pole cells of either sex, however, apoptosis was induced only in female (XX) pole cells during migration to the gonads. This effect caused a significant reduction in the number of XX pole cells in the gonads. Introduction of female-specific germline apoptosis induced by a dominant-negative form of lwr (f-gal) provides a previously uncharacterized marker of female sexual identity in migrating pole cells (Hashiyama, 2011).

Sex determination is controlled by the Sex lethal (Sxl) gene, which is first expressed at the blastodermal stage in the embryonic soma. Sxl encodes an RNA binding protein involved in alternative splicing and translation. In the soma of XX embryos, it functions through transformer (tra) and transformer-2 (tra-2), which in turn regulate alternative splicing of the doublesex (dsx) gene to produce a female-specific form of Dsx. In male (XY) embryos, this pathway is turned off, and a male-specific form of Dsx is produced by default. These Dsx proteins determine the sexual identity of somatic tissues. Previous reports, however, suggested that Sxl does not induce female sexual development in the germ line, as it does in the soma. Although Sxl is autonomously required for female sexual development, constitutive mutations in Sxl (SxlM) that cause XY animals to undergo sexual transformation from male to female do not necessarily interfere with male germline development. Moreover, tra, tra-2, and dsx are not required for female germline development. Finally, female-specific Sxl expression has been detected later in gametogenesis, but not in early germline development (Hashiyama, 2011).

Contrary to previous observations, this study found that Sxl was expressed in XX but not XY pole cells during their migration to the gonads. In the soma, Sxl transcripts are first expressed from the establishment promoter (Sxl-Pe) in a female-specific manner. Using a probe specific to the early transcript derived from Sxl-Pe, in situ hybridization signals were detected in migrating XX pole cells at around stage 9/10. Transgenic embryos, which expressed enhanced green fluorescent protein (EGFP) under the control of the Sxl-Pe promoter, were used to further confirm this female-specific Sxl-Pe activation. RTPE and sequencing analyses in pole cells were used to detect early Sxl transcripts that had the same sequence as the transcripts expressed in the soma (Hashiyama, 2011).

Next, it was determined whether Sxl feminize early pole cells using f-gal as a marker for female identity. It was found that the loss-of-function mutation SxlfP7B0 represses f-gal in XX pole cells. This repression is unlikely to result from sexual transformation of the soma, because an amorphic tra-2 mutation, which alters somatic sex, did not affect f-gal. Conversely, when the expression of Sxl together with lwrDN is forced in pole cells from stage 9 onward by using nanos-Gal4 and UAS-Sxl, f-gal is ectopically observed in XY pole cells. Sxl alone does not induce apoptosis or developmental defects in pole cells. These observations suggest that female sexual identity of migrating pole cells is regulated cell-autonomously by Sxl (Hashiyama, 2011).

It was then determined whether Sxl induces female development in XY pole cells. Because XY soma produces signals that direct XX germline cells to a male fate, XY pole cells expressing Sxl were transplanted into XX females, and their developmental fate was examined. Even in the presence of a gain-of-function Sxl mutation (SxlM1) that causes XY soma to transform from male to female, XY (or XO) pole cells enter the spermatogenic pathway when transplanted into XX females. These results suggest that Sxl is not sufficient to activate female germline development. SxlM1 mutations, however, do not affect transcription from the Sxl-Pe promoter, but instead structurally alter the late transcript from the Sxl maintenance promoter (Sxl-Pm), which allows Sxl protein production in both males and females. Consistent with this observation, Sxl transcripts derived from Sxl-Pe were detected in the pole cells of only female SxlM1 embryos. Thus, the SxlM1 mutation does not result in Sxl expression in XY pole cells as early as in XX pole cells (Hashiyama, 2011).

Instead, nanos-Gal4 and UAS-Sxl were used to induce Sxl expression in XY pole cells. Three types of XY pole cells were transplanted, each characterized by a different duration of Sxl expression: (1) XY pole cells in which Sxl was expressed from stage 9 until stage 16/17 using maternal nanos-Gal4 (XY-mSxl), (2) XY pole cells in which Sxl was expressed from stage 15/16 onward using zygotic nanos-Gal4 (XY-zSxl), and (3) XY pole cells in which Sxl was expressed from stage 9 onward using both maternal and zygotic nanos-Gal4 (XY-mzSxl). XY-mzSxl and XY-mSxl pole cells entered the oogenic pathway and produced mature oocytes in XX females. These oocytes contributed to progeny production. Thus, the XY pole cells produced functional eggs, even though oogenesis and egg production were reduced compared with XX pole cells. In contrast, XY-zSxl pole cells did not enter the oogenic pathway in almost all (92.3%) of the XX female hosts and instead were characterized by a tumorous phenotype, an indication of XY germline cells that have maintained male characteristics. Control XY pole cells from the embryos expressing Sxl only in the soma (XY-nullo-Sxl) showed a similar phenotype to that of XY-zSxl pole cells. These observations demonstrate that Sxl expression in XY pole cells during embryogenesis induces functional egg differentiation in the female soma (Hashiyama, 2011).

Sxl-specific double-stranded RNA (UAS-SxlRNAi) under the control of maternal nanos-Gal4 was used to reduce Sxl activity in XX pole cells during embryogenesis. Introducing UAS-SxlRNAi resulted in tumorous and agametic phenotypes in female adults, indicating that the XX germ line lost female characteristics. Taken together, these results show that Sxl acts as a master gene necessary and sufficient to induce female development in pole cells (Hashiyama, 2011).

XY-mzSxl pole cells adopted a male fate and executed spermatogenesis when they developed in an XY male soma. This observation suggests that the male soma plays a dominant role in determining the male germline fate, overriding the feminizing effect of Sxl. Another possibility is that the XX female soma plays a critical role in maintaining the Sxl-initiated female germline fate. Indeed, an XX germ line in the male soma shows a male gene-expression profile, whereas an XY germ line in the female soma exhibits a female expression profile, although these germ lines does not execute gametogenesis. Thus, female germline development requires interactions between the germline and somatic cells, in addition to germline-autonomous mechanisms involving Sxl (Hashiyama, 2011).

In mice, germline sexual identity is also regulated by both germline-autonomous and somatic signals. In the coelenterate Hydra, the germline sex is not influenced by the surrounding soma, and the germ line determines the phenotypic sex of the polyp. Thus, germline-autonomous regulation of sex has probably been present throughout the evolution of animals, and somatic control may have evolved with the emergence of mesodermal tissues, including gonadal soma. Sxl does not appear to play a key role in sex determination in non-drosophilid animals. Nevertheless, future studies should determine whether Sxl homologs are expressed in the germ line of non-drosophilids. Moreover, it would be of particular interest to identify downstream targets of Sxl in the Drosophila germ line and to test whether these genes have a widespread role in germline sex determination (Hashiyama, 2011).


EFFECTS OF MUTATION

lesswright mutation activates Rel-related proteins, leading to overproduction of larval hemocytes in Drosophila

To investigate why lwr mutant larvae develop melanotic tumors, the number of circulating hemocytes of lwr mutants was measured. Total hemocyte counts of wild type and heterozygous controls varied from 2.1 × 106 to 4.1 × 106 per ml of hemolymph. The number of hemocytes of lwr mutant larvae was 23.0 × 106 per ml of hemolymph, which was statistically higher than that of the heterozygous control animals. This high hemocyte count was very similar to that of Tl10B mutants (20.1 × 106 per ml of hemolymph). Therefore, an excess number of circulating hemocytes probably causes the development of melanotic tumors in lwr mutant larvae (Huang, 2005).

Since three different hemocytes are known to be in the hemolymph, how hemocyte populations were affected in these mutant larvae was examined. Crystal cells were omitted from this study because they usually burst within 1 min or less after bleeding, leading to inaccurate estimates of crystal cells. The proportion of lamellocytes in the total circulating hemocytes was estimated as a parameter to represent differences in the hemocyte population. Lamellocytes were distinguished by using a lamellocyte specific marker, msn-lacZ, or by their characteristic morphology. The percentage of lamellocytes in the total hemocyte pool significantly increased in lwr and Tl10B mutant larvae. It was also noted that most aggregated hemocytes in the hemolymph consisted of lamellocytes, and that these aggregated masses were partially to fully melanized in most cases. These observations strongly suggest that lamellocytes are heavily involved in the formation of melanotic tumors in lwr mutant larvae (Huang, 2005).

It was also of interest to discover whether the lwr mutant hemocytes divide in circulation. The mitotic indexes of lwr mutant hemocytes were measured as well as those of Tl10B and Canton-S hemocytes as control. Cells in mitosis were identified using anti-phospho-Histone H3 antibodies. The mitotic index of lwr mutants was 10.4% in a total of 3109 cells from five larvae, which was slightly higher than that of Tl10B (7.1% in a total of 2515 cells from five larvae). In contrast, no dividing hemocytes were observed in Canton-S (a total of 1344 cells from ten larvae). Based on their sizes and morphologies, these dividing cells in lwr and Tl10B mutants were plasmatocytes and prohemocytes, not lamellocytes. Therefore, some hemocytes do indeed divide in circulation in lwr mutants. However, the excess number of mature lamellocytes in circulation is due to proliferation of lamellocyte precursors, presumably in the lymph gland. Taken together, it is concluded that the effects of the lwr mutation on hemocytes are similar to those of other known hematopoietic mutants in the Tl pathway such as cact and Tl10B (Huang, 2005).

To determine whether the overproduction of hemocytes can be attributed to the loss of lwr function in hematopoietic tissues, a dominant negative form of lwr (lwrDN) was expressed in the lymph gland and hemocytes. Since the lwr mutation exhibits pleiotropic effects, it is possible that the increased number of hemocytes in lwr mutant larvae is due to a secondary effect that might stimulate hemocyte production. For this purpose, the GAL4/UAS system was used. Two GAL4 drivers, CgGAL4 and e33CGAL4, were chosen for this experiment because they are known to induce expression of GAL4 in the lymph glands and hemocytes. Although these drivers induce GAL4 expression in multiple tissues, the common cell types expressing GAL4 with these two drivers are hemocytes in the lymph gland and in circulation. The e33CGAL4 driver induces a UAS-GFP transgene in most cells in the lymph gland at different expression levels, while the CgGAL4 promotes GFP expression in a subset of the cells in the anterior lobes at a relatively consistent level. The overall expression level of GFP by the e33CGAL4 is lower than that of the CgGAL4 driver. Nevertheless, these GAL4 drivers exhibited similar effects on the total hemocyte counts (Huang, 2005).

The CgGAL4 driver effectively increased the number of hemocytes to a level that was higher than those of lwr and Tl10B. The total hemocyte count was 25.0 × 106 per ml of hemolymph, which was statistically higher than that of the corresponding control. The e33CGAL4 driver also induced many hemocytes at 28°C, and this level was higher than that of the corresponding heterozygous control. Since hemocytes in the lymph gland and in circulation are the only common cell types that express GAL4 with these drivers, it is most likely that the loss of lwr function in hematopoietic tissues is responsible for high hemocyte counts in lwr mutants. This conclusion is also supported by the fact that a fat body specific driver, Lsp2GAL4, does not show any significant effect on hemocyte counts when the lwrDN allele is induced by this driver (Huang, 2005).

In addition to the total hemocyte counts, the UAS-lwrDN/CgGAL4 combination promoted lamellocyte production. Lamellocyte levels rose to 15.5%. This value was lower than that of lwr mutants, but higher than that of Tl10B mutants. Furthermore, most hemocytes expressing lwrDN showed nuclear localization of Dl protein, which is characteristic of the lwr mutant hemocytes. These observations strongly suggest that this lwrDN allele is very effective with the CgGAL4 in mimicking effects of the lwr mutation on hemocyte production in larvae. Thus, it is concluded that the increase in the total hemocyte counts in the lwr mutant background is attributed to the loss of lwr function in the hematopoietic tissues such as the lymph gland (Huang, 2005).

How does the lwr mutation affect hemocyte production? Because the hematopoietic defects of lwr mutants are similar to those of dominant Tl mutations, Tl10B and Tl3, possible interactions between lwr and the Tl signal transduction pathway were investigated. Moreover, IκBα, a human Cact homologue, is subject to sumoylation (Desterro, 1998), which suggests that the Cact protein may interact with the Lwr protein. Since both dl and Dif are expressed in the hematopoietic tissues, the entrance of Dl proteins were used as an indicator for Tl signaling activity. At this moment, it is not yet clear whether other Tl-like receptors are involved in hematopoiesis. Hereby, the term Tl signaling in this report does not exclude possible signaling inputs from other Tl-like receptors (Huang, 2005).

Nuclear localization of Dl protein was observed in many lwr mutant hemocytes as well as in Tl10B hemocytes. In lwr mutants, nearly half of hemocytes showed accumulation of Dl proteins in the nuclei, which was comparable to that of Tl10B mutants . In contrast, nuclear localization of Dl protein was observed in only a few control heterozygous and wild type hemocytes, e.g., 3.6% in a total of 1291 cells from 10 Canton-S larvae. These observations indicate that the activation of the Tl pathway is a consequence of the loss of lwr function, which is manifested as abnormal production of larval hemocytes. Although some small lamellocytes were positive for Dl nuclear staining, little nuclear localization of Dl protein was observed in large matured lamellocytes of lwr and Tl10B mutants. Thus, there is not a mechanism to allow entrance of Dl proteins to the nucleus in mature lamellocytes (Huang, 2005).

To further investigate the relationship between the lwr mutation and Tl signaling, genetic interactions were examined of lwr with dl and Dif single mutants as well as with dl Dif double mutants. The rationale here is that genetic interactions between lwr and dl as well as Dif would be clearly detected if lwr function modulates signaling from Tl receptor and possibly from other Tl-like receptors. The results of these three combinations were examined separately because the dl and the Dif mutations showed different effects on the hematopoietic defects of the lwr mutation. Nonetheless, the results below indicate that the majority of lwr's mutant effects on larval hematopoiesis are manifested through the Tl signal transduction pathway, which agrees with the results of the immunohistochemical study (Huang, 2005).

A double mutant combination of lwr with dl [lwr4-3 dl1/lwr5 Df(2L)J4] was created. The total hemocyte counts and lamellocyte percentages were measured. The total hemocyte counts were significantly reduced in the lwr dl double mutant background. The numbers of plasmatocytes and lamellocytes were then calculated based on the total hemocyte counts and lamellocyte percentages. It was noticed that plasmatocyte counts were reduced by approximately 50% in the double mutant combination. Interestingly, the production of lamellocytes was not overly affected in the same mutant background. which indicates that dl plays a minor role in lamellocyte production (Huang, 2005).

Although the production of plasmatocytes was suppressed by the complete loss of dl function, this suppression was not absolute, suggesting that Dif may contribute equally to larval plasmatocyte production. If Dif only played a minor role in this process, the lwr mutation would affect plasmatocyte production via another pathway besides the Tl pathway. Note that the latter hypothesis was proven to be very unlikely (Huang, 2005).

To examine the effect of Dif in the lwr mutant background, a double mutant combination of lwr and Dif [lwr4-3 Dif2/lwr5 Df(2L)J4] were created and the total hemocyte counts and lamellocyte percentages was measured. The loss of Dif function in the lwr background significantly reduces total hemocyte counts, and the effects were observed on both plasmatocyte and lamellocyte levels. Plasmatocyte population was reduced to half the level caused by the lwr mutation, while the number of lamellocytes was similar to that found in wild type larvae (Huang, 2005).

Similar to what was observed in the lwr dl double mutants, the reduction of the plasmatocyte population due to the loss of Dif function in the lwr background was approximately 50%. This result strongly suggests, in conjunction with the results of the lwr dl double mutants, that the increase of hemocytes observed in the lwr mutation is mediated by the functions of both dl and Dif (Huang, 2005).

In contrast to the lwr dl double mutant, the lamellocyte population was considerably affected by the introduction of the Dif mutation into the lwr mutant background, and the number of lamellocytes observed in the lwr Dif double mutants was almost identical to those in wild type larvae. Therefore, lamellocyte production is primarily controlled by Dif function when the Tl pathway is activated. This in turn suggests that dl and Dif play different roles in hematopoiesis when it is stimulated by the lwr mutation (Huang, 2005).

To obtain a more conclusive answer as to whether the effects of the lwr mutation are observed mostly through Rel-related proteins, Dl and Dif, lwr dl Dif triple mutants [lwr4-3 Df(2L)J4/lwr5 Df(2L)TW119] were examined. The loss of both gene functions almost completely cancels the effects of the lwr mutation on hematopoiesis (Huang, 2005).

The functions of dl and Dif were eliminated by combining the deficiencies Df(2L)J4 and Df(2L)TW119. Total hemocyte counts of lwr5 Df(2L)J4/lwr4-3 Df(2L)TW119 heterozygotes were significantly reduced from those of the lwr single mutants and were indistinguishable from those in wild type larvae as well as Df(2L)J4/ Df(2L)TW119 heterozygotes. The number of lamellocytes was almost identical to those of wild type larvae. These results indicate that the hematopoietic defects of the lwr mutation are manifested through dl and Dif function (Huang, 2005).

The loss of both dl and Dif functions with and without the lwr mutation did not lead to complete loss of hemocytes. This observation agrees with the fact that loss-of-function mutations of the Tl gene do not eliminate all hemocytes completely. Low levels of hemocytes in these mutant backgrounds can be explained by hemocyte production using other pathways such as JAK/STAT and Ras, which are known to be involved in hematopoiesis. Therefore, these observations do not rule out the importance of dl and Dif functions in hematopoiesis (Huang, 2005).

In order to obtain additional evidence that supports different but overlapping roles of dl and Dif in hematopoiesis, UAS-dl and UAS-Dif transgenes were overexpressed in the lymph glands and hemocytes using the CgGAL4 driver. Dif promote lamellocyte production more effectively than do dl, which shows good agreement with genetic analysis (Huang, 2005).

Since the CgGAL4 driver expresses the GAL4 transcription factor in the fat body, the Lsp2GAL4 driver, which is fat body specific, was used to express the UAS constructs used in this study. The combinations of all UAS transgenes with the Lsp2GAL4 serve as a control to assess any possible additional effect of the CgGAL4 driver. In all cases examined, while the differences between controls and experimental sets were statistically significant in some cases, total hemocyte counts fell in the range of wild type, Oregon-R and Canton-S. Therefore, the results with the CgGAL4 driver, which are described below, are most likely to represent the effects of these genes in the hematopoietic tissues, the lymph gland and hemocytes (Huang, 2005).

Both dl and Dif exhibited significant increases in total hemocyte counts when they were overexpressed by the CgGAL4 driver. Interestingly, overexpression of Dif produced more hemocytes than dl. After dividing the total hemocyte population into plasmatocytes and lamellocytes, it was observed that plasmatocyte counts increased to levels similar to those of lwr and Tl10B mutants in both UAS-dl/CgGAL4 and UAS-Dif/CgGAL4 combinations. Unlike the plasmatocyte population, lamellocytes responded differently. Overexpression of dl showed no effect on lamellocyte production. In contrast, overexpression of Dif promoted lamellocyte production and its effect was similar to that of a Tl10Btransgene. These results indicate that dl and Dif share a similar function in plasmatocyte production, and that Dif is likely to be a sole factor for lamellocyte production in the Tl pathway (Huang, 2005).

To investigate how coordinately dl and Dif function in hematopoiesis, effects on hemocyte production were examined by overexpressing Tl10B and both dl and Dif simultaneously. It was found that dl may be dispensable in the production of both plasmatocytes and lamellocytes when there were enough Dif proteins around. Furthermore, the activation of Tl signaling by Tl10B, a constitutively active form of the Tl receptor, showed an additional effect on plasmatocyte production compared to those by the simultaneous expression of dl and Dif (Huang, 2005).

dl and Dif were overexpressed simultaneously with the CgGAL4 driver and the total hemocyte number and the proportion of lamellocytes in the total hemocyte population was estimated. Even though dl and Dif showed significant effects on hemocyte production when they were individually overexpressed, they did not show any synergistic effect when they were induced at the same time. When both genes were expressed together, the total hemocyte counts did not differ from those when Dif was overexpressed by the CgGAL4 driver, but were statistically higher than those when dl was overexpressed by the same driver. Thus, Dif is sufficient to represent the effect of the dl and Dif double expression combination, and dl did not suppress the effect of Dif (Huang, 2005).

As far as lamellocyte production is concerned, the combination of dl and Dif showed the highest lamellocyte estimate among all constructs tested including UAS-Tl10B. Although a statistical test could not be applied, the differences among the constructs seemed to be marginal, indicating that the effect of dl on lamellocyte production may be small, if there is any. Taken together, dl only plays a minimal role in hematopoiesis when both dl and Dif are highly induced (Huang, 2005).

In order to verify whether the dl-Dif double combination represents the activation of the Tl pathway, a UAS-Tl10B transgene was overexpressed with the CgGAL4 driver. While lamellocyte production appeared to be very similar to that of a dl–Dif double combination, the overexpression of Tl10B exhibited the most pronounced effect on plasmatocyte production among all the combinations used in this study. The results indicate that there are abundant Dl and Dif proteins in the CgGAL4 expressing cells and that these transcription factors can fully respond to the activation of the Tl pathway, i.e., the overexpression of Tl10B. It is also possible that the activation of the Tl receptor may stimulate plasmatocyte production, in part bypassing the Dl and Dif transcription factors. This possible bypass indicates that the Tl receptor might use a different set of transducers and transcription factors to control plasmatocyte production. Alternatively, the differences might be due to the different levels of UAS transgene expression (Huang, 2005).

dUbc9 negatively regulates the Toll-NF-kappaB pathways in larval hematopoiesis and drosomycin activation in Drosophila

Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. Mutations in the Drosophila Ubc9 (dUbc9) gene have been used to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. Also, an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae, are observed. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-κB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IκB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene Drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, these results support a role for dUbc9 in the negative regulation of the Drosophila NF-κB signaling pathways in larval hematopoiesis and humoral immunity (Chiu, 2005).

Ubc9 was discovered in Saccharomyces cerevisiae based on its sequence similarity to other known ubiquitin-conjugating enzymes (Seufert, 1995). A loss of function of Ubc9 causes an increase in the S- and M (B type)-phase cyclins, resulting in an arrest of the cell cycle at the G2 or early M phase. This cessation of the cell cycle causes an accumulation of large budded cells with a single nucleus and replicated DNA. Ubc9 was also identified in a screen for DNA damage checkpoint control in S. pombe (Al-Khodairy, 1995; Tanaka, 1999). Although not directly involved in the checkpoint control, Ubc9 (encoded by hus5) is required for the efficient recovery from DNA damage or S-phase arrest, and for chromosome segregation. Yeast mutants display severe impairment in growth and exhibit a high frequency of failed mitosis. Further studies in yeast revealed that unlike other E2 ubiquitin-conjugating enzymes, Ubc9 is unable to form a thioester bond with ubiquitin; instead it conjugates the ubiquitin-like protein SUMO/Smt3 to specific targets in a yeast extract (Johnson, 1997; Chiu, 2005 and references therein).

Vertebrate homologs of Ubc9 were subsequently identified in many laboratories, showing that Ubc9 may interact with a wide variety of cellular proteins, regulating cellular processes such as cell division, protein trafficking, signal transduction, and transcriptional regulation (Pichler, 2002; Muller, 2000). Studies designed to identify biochemical targets of Ubc9 highlighted a role for sumoylation in the regulation of chromatin organization, gene expression, and genome surveillance (Melchoir, 2002; Muller, 2004; Zhao, 2004; Chiu, 2005 and references therein).

While SUMO-1 is structurally similar to ubiquitin and sumoylation and ubiquitination are enzymatically similar processes, the conjugation of SUMO-1 or ubiquitin to the same protein can have opposite effects. For example, ubiquitin or SUMO-1 can be directly conjugated to lysine residues 21 and 22 of mammalian IκBα in reactions catalyzed by activating enzymes Ubch5 and Ubc9, respectively (Scherer, 1995; Desterro, 1998). IκBα is a cytoplasmic inhibitor of the transcription factor NF-κB. In unstimulated cells, cytoplasmic NF-κB, complexed with IκBα, remains inactive. Activation of NF-κB is achieved by ubiquitination and proteasome-mediated degradation of IκBα, allowing NF-κB translocation to the nucleus. However, when the same lysine residues in IκBα are conjugated to SUMO-1/Smt3, ubiquitination is blocked, thereby stabilizing the cytoplasmic pool of this protein. This increase in stabilization of IκB sequesters NF-κB in the cytoplasm, leading to a downregulation of the NF-κB pathway (Tashiro, 1997; Desterro, 1998; Hay, 1999). Thus, while ubiquitination targets proteins for degradation, SUMO-1 modification acts antagonistically to render proteins resistant to degradation. Given that SUMO modification alters the ability of proteins to interact with their partners, alters their subcellular localization, and controls their stability, understanding the role of sumoylation in different cellular processes is of fundamental importance in normal and diseased cells (Chiu, 2005).

Drosophila has been used as a model system to understand the functions of Ubc9 in vivo. An alignment of the Drosophila Ubc9 (dUbc9) with its counterparts in yeast, C. elegans, and humans shows that dUbc9 shares a higher level of structural similarity with the human Ubc9 (84% identical) than with either the C. elegans (76% identical) or yeast (35% identity) proteins. Strikingly, expression of either human or Drosophila Ubc9 can rescue an S. cerevisiae Ubc9ts mutant (Yasugi, 1996; Joanisse, 1998). dUbc9, also called semushi and lesswright, (lwr), has many biological functions. For example, mutation in dUbc9 disrupts anterior segmentation in embryogenesis by interfering with the nuclear uptake of the homeodomain transcription factor Bicoid (Epps, 1998). Mutants in dUbc9 also suppress the nodDTW (dominant antimorphic allele of no distributive disjunction) phenotype, implying a role in chromosome segregation during meiosis in females (Apionishev, 2001). Biochemically, the SUMO-1/Ubc9 pathways are conserved between flies and humans: (1) dSmt3 and dUbc9 are coexpressed during development, (2) dSmt3 can be processed and conjugated in human cells, and (3) human transcription factor PML can be modified by dSmt3 in Drosophila SL2 or human HeLa cells. Like their human counterparts, dSmt3 and dUbc9 colocalize in nuclear foci (Lehembre, 2000; Chiu, 2005 and references therein).

Drosophila Ubc9 was also identified in a yeast two-hybrid screen for Dorsal-interacting proteins (Bhaskar, 2000). Dorsal is one of three Drosophila Rel/NF-κB-family proteins. The nuclear localization of Dorsal is controlled by the Toll receptor. Toll activation leads to signal transduction via Tube and Pelle, as well as the phosphorylation and degradation of Cactus, the Drosophila IκB protein. Components of the Toll-Dorsal pathway were first identified as maternal-effect genes controlling the development of the embryonic dorsal-ventral axis. Although the precise mechanism underlying Cactus degradation in the embryo is still unclear, in vivo studies suggest that, like mammalian IκBα, Cactus degradation is regulated by Toll signal-dependent phosphorylation. dUbc9 conjugates a Drosophila SUMO/Smt3 to lysine 382 of Dorsal (Bhaskar, 2002). Whether Cactus also serves as a sumoylation target is not known (Chiu, 2005 and references therein).

In Drosophila, the NF-κB pathway regulates many biological processes at different developmental stages. The Toll-Dorsal/Dif pathway activates transcription of antifungal and antibacterial (Gram-positive) peptide genes in the larval and adult fat body. Dif (Dorsal-related immunity factor) belongs to the NF-κB family . The regulation of genes encoding antibacterial peptides that kill Gram-negative bacteria (e.g., diptericin) is under the control of Relish, the third Drosophila Rel/NF-κB protein similar to the mammalian p100/p105 proteins. Relish activation is Toll-independent. Instead, the intracellular Relish phosphorylation and activation are regulated by activities of Immune deficiency (Imd) pathway including proteins of the IKK complex and the Dredd caspase. This pathway is negatively regulated by the ubiquitin proteasome system (Chiu, 2005 and refenreces therein).

The Toll-IκB pathway also contributes to proliferation of blood cells (hemocytes) during normal larval hematopoiesis and during the hematopoietic proliferation that accompanies immune challenge. Unchallenged Drosophila larvae have two hemocyte types in circulation: the plasmatocyte and the crystal cell, both of which are specified and formed during embryonic stages. More than 90% of all hemocytes in circulation are plasmatocytes. Plasmatocytes are phagocytic cells, ridding the larvae of microbial infections. The remaining hemocytes in circulation (5% or less), the crystal cells, carry prominent crystalline inclusions and, when activated, lyse and release their contents, melanizing target cells. Mutations that upregulate the Toll pathway (loss-of-function mutations in cactus, gain-of-function mutations in Toll, or overexpression of Dorsal in larval hemocytes) result in overproliferation of hemocytes, whereas mutations that downregulate the pathway (loss-of-function in Toll, tube, or pelle) lead to a reduction in the number of circulating hemocytes. Changes in hemocyte counts in mutations affecting either dorsal or Dif are unremarkable (Chiu, 2005 and references therein).

A third kind of hemocyte, the lamellocyte, appears in the larval hemolymph only in response to infections by naturally occurring endoparasitoid wasps and other foreign bodies. The parasitoids constitute a major threat to the Drosophila population in nature, as they hijack the larval body for their own development. Lamellocytes are adhesive, and rapidly aggregate around a parasite egg to form a cellular capsule. Parasite-induced lamellocyte differentiation in the lymph gland is accompanied by a modest increase in the number of plasmatocytes and crystal cells. The encapsulated wasp egg is melanized. Lamellocyte precursors are normally quiescent. However, mutations that lead to the overproliferation of hemocytes (loss-of-function cactus alleles, gain-of-function Toll alleles) also result in constitutive differentiation of lamellocytes, resulting in the encapsulation of self-tissue in the absence of wasp infection (innate autoimmune response). Because of their dark appearance, these capsules are called melanotic tumors (Chiu, 2005 and references therein).

This study reports that larvae carrying loss-of-function mutations in dUbc9 show strong hematopoietic proliferation and differentiation defects. Furthermore, the antifungal gene drosomycin is constitutively active in developmentally delayed dUbc9 mutants. Both constitutive humoral and cellular immune defects are rescued by mutations in dorsal and Dif. These results suggest that dUbc9 contributes to the regulation of both humoral immunity and hemocyte proliferation by acting as a negative regulator of the Toll pathway (Chiu, 2005).

Genetic experiments in Drosophila emphasize the central regulatory role for Ubc9 function and sumoylation in different cells and during different life cycle stages. lwr adults exhibit defects in eye, wing, and leg morphogenesis: mutant males die and the few surviving females are sterile, revealing roles for dUbc9 in cell division and differentiation, in tissue patterning, and in oogenesis. This paper describes additional defects in dUbc9 mutants, based on studies of mutant larvae. Phenotypes are described that point to the involvement of dUbc9 in larval metamorphosis, proliferation, and differentiation of hematopoietic precursors, in antimicrobial gene expression, and in NF-κB signal transduction. These observations show that diverse biological processes share a common regulatory mechanism involving sumoylation and the variety of the phenotypes observed suggest the possibility of multiple biochemical targets in vivo. Drosophila is an excellent model for the identification of cell- and tissue-specific sumoylation targets of Ubc9, and it is very likely that their conserved mammalian counterparts will be similarly modified and regulated (Chiu, 2005).

Like many larval lethals (e.g., cact mutants), mutations in dUbc9 result in a prolonged third instar period followed by larval death. Hematopoietic defects are observed as early as 4 days after egg lay, whereas the immune defects are evident 6 days after egg lay. Defects of both types become severe as mutant animals persist in larval stages even 10 days after their birth. Indeed, constitutive expression of antimicrobial genes during larval stages continues through lwr4-3/lwr5 pupae and adults. How dUbc9 affects the rate of development is currently not known. Epistasis experiments between lwr and mutants of the ecdysone genetic hierarchy may reveal a role for dUbc9 sumoylation during development and metamorphosis. Also not known is whether hemocyte survival or their apoptosis is affected in lwr mutants. One study of salivary gland apoptosis in Drosophila during pupariation provides evidence that the Rel family members are not required for salivary gland cell death during metamorphosis. The current results provide a link between lwr negative regulation of dl and Dif in the larval fat body and hematopoietic tissues. In any case, the hematopoietic and immune defects in lwr mutants may be tied to abnormalities in larval development as these defects are most pronounced in older animals. The rescue of the immune defects by expression of the dUbc9 protein in the fat body suggests that the misregulation of drom expression in lwr mutants is due to a specific reduction or an absence of the Ubc9 function. Analysis of mutant clones in the fat body or lymph gland will reveal if the requirement of this enzyme is cell autonomous or not (Chiu, 2005).

Four distinct hematopoietic defects affecting hemocyte abundance, differentiation, and morphology are observed in both the lymph gland and circulating hemocyte populations in 4-day-old lwr larvae. The mean CHC values in mutants are significantly higher than those of the pooled control class of larvae; in two of the three lwr genetic combinations studied, this increase in abundance correlates with an increase in cyclin-B-positive hemocytes. Such increase in cyclin B-positive hemocyte population is reminiscent of increase in the B type cyclins in mutant yeast lacking Ubc9 (Seufert, 1995). The current observations suggest that dUbc9 negatively regulates the rate at which hematopoietic cells divide. The prehemocyte classes influenced by the dUbc9 mutation are not known, however, it is interesting that while there is an increase in circulating plasmatocyte and lamellocyte percentages, there is a clear reduction in the number of crystal cells. The opposite effect of lwr on crystal cell numbers indicates a distinct role for dUbc9 function in this hemocyte lineage. Prohemocytes following the crystal cell fate require a combination of signals from the transcription factors Serpent (Srp; human GATA-2) and Lozenge (lz; human AML-1), along with permissive signals from Ser/Notch. Perhaps dUbc9 asserts a role in crystal cell development by propagating the above required signals or alleviating suppression of this cellular fate exerted by the combinatorial interaction of SrpNC (Srp isoform containing both N- and C-terminal Zinc finger domains) and the U-shaped protein. These observations suggest multiple requirements for dUbc9 in the hematopoietic tissue. Furthermore, as mitotic defects are observed in both lymph gland hemocytes and circulating hemocytes, and since these groups of hemocytes originate independently, it is likely that dUbc9 function is independently required in each hemocyte population (Chiu, 2005).

The presence of large numbers of lamellocytes accounts for a significant fraction (10%-20%) of the increase of CHC in lwr mutants. The coincident expansion of the plasmatocyte population and constitutive differentiation of lamellocytes are hallmarks of melanotic tumor mutants in which affected genes are not necessarily related by either structure or function. Yet, hemocyte proliferation and differentiation have distinct genetic requirements. For example, while the Drosophila kinase Hopscotch and transcription factor STAT92E are required for lamellocyte differentiation, Toll and Tube proteins are not, even though upregulation of either the JAK/STAT or the Toll/NF-κB pathways results in the production of melanotic tumors. One explanation for the simultaneous expansion of the plasmatocyte and lamellocyte populations in melanotic tumor mutants such as lwr, cact, and Toll10b is that these mutations affect proliferation of precursors in the lymph gland (or in circulation) that differentiate as plasmatocytes or lamellocytes (Chiu, 2005).

The aberrant nuclear morphologies observed in lwr4-3/lwr14 and lwr4-3/lwr5 hemocytes are variable in appearance and fall into four categories: aneuploidy, abnormal nuclear shapes, presence of multiple nuclei, and presence of fragmented nuclear material (additional smaller Hoechst-positive structures). Genetic evidence suggests that dUbc9 is required for the proper disjunction of homologous chromosomes in meiosis I (Apionishev, 2001). It is thus possible that dUbc9 is also involved in chromosome segregation in mitotic divisions. Indeed, defects in lwr hemocytes are strikingly similar to proliferation defects in cultured chicken cells conditionally depleted of Ubc9 protein, in which cells with multiple or fragmented nuclei are also observed (Hayashi, 2002). The defects in chicken cells arise due to chromosomal loss during chromosome segregation. In both Drosophila and chicken, the frequency of cells with multiple or fragmented nuclei increases with age (Hayashi, 2002). Thus, it is possible that the biochemical targets of Ubc9 in chromosome segregation in Drosophila and chicken cells (and possibly other vertebrates) are conserved and that chromosomal damage accumulates in Ubc9-depleted cells because of similar molecular processes. The proportion of multinucleate hemocytes (in lwr4-3 larvae) is reduced in lwr4-3 dl as well as lwr4-3 Dif dl mutant larvae. The identity of the extra chromosomes and extrachromosomal DNA in dUbc9 mutant hemocytes is not known nor the specificity of this phenotype to lwr allele 4-3. The presence of multinucleate hemocytes in circulation within hemocyte overproliferation mutants is unique to lwr and, to date, has not been reported in other mutants where similar overproliferation and lamellocyte differentiation defects have been documented (e.g., hopTum-l; Toll10b/+; cactus), reflecting the multiplicity of effects of dUbc9 on the cell cycle (Chiu, 2005).

The genetic interaction and immunohistochemical studies presented in this study constitute the first clear evidence of a role for Dorsal and Dif during hemocyte proliferation in Drosophila larvae, even though these functions were predicted from previous experiments. Like Cactus, the cellular function of dUbc9 is to regulate the nuclear localization of NF-κB proteins. Interestingly, Dorsal and Dif appear to have somewhat redundant functions as suppression of lwr phenotypes is stronger in triple mutants than in double mutants and suppression appears earlier in development in triple mutants than in double mutants. Functional redundancy may also explain why clear hematopoietic phenotypes (e.g., lowered hemocyte concentration or reduced encapsulation of wasp egg) have not been observed in dl or Dif single mutants. Similar redundancy in Dorsal and Dif function has been reported for antimicrobial peptide gene activation in the larval fat body (Chiu, 2005).

Genetic experiments here support a model for negative regulation by dUbc9 of antifungal peptide-encoding genes drosomycin and Cecropin, making it the second negative regulator of the Toll pathway to be identified so far. In general, the effects of lwr mutants on drosomycin expression are stronger than on that of Cecropin. These effects are evident by characterization using promoter-driven GFP reporters representative of the Toll and Imd downstream antimicrobial genes and subsequent confirmation by Northern analysis in whole animals. Perhaps the expression pattern of Cecropin A observed in lwr mutants is partially independent of the Toll pathway. cecropin A has been shown to be regulated by both Toll and Imd immunity pathways. Indeed, dynamic expression patterns of the two variant Cec A1 and A2 transcripts are detected in adult flies, after specific microbial infection regiments, further delineating their expression into the two immune pathways. A detailed analysis of Cecropin A regulation in lwr mutant larvae in combination with loss of function alleles of the Imd pathway may elucidate control of this gene further (Chiu, 2005).

The constitutive activation of drosomycin and Cecropin in lwr mutants is also dependent on Dorsal and Dif, whose roles and functional redundancy in the larval fat body are already recognized. Furthermore, genetic epistasis experiments described here place dUbc9 function upstream of Dorsal/Dif and Cactus. These observations are largely consistent with previous biochemical experiments on these proteins (Bhaskar, 2000; Bhaskar, 2002) and provide additional support for a model in which Dorsal, Dif, Cactus and dUbc9 exist in a complex that is activated by a Toll-dependent signal. Significantly, however, the results suggest that dUbc9 blocks the nuclear localization of Dorsal and Dif and differ from observations made in Drosophila S2 cell cultures, in which dUbc9 facilitates the nuclear localization of Dorsal-GFP (Bhaskar, 2000; Bhaskar, 2002). This divergence in experimental results is likely to be due to different experimental models used in the two studies. The genetic results are consistent with Ubc9's role in IκB sumoylation and downregulation of the mammalian NF-κB pathway (Hay, 1999). Consistent with this model of mammalian Ubc9 function, it is likely that sumoylation of Cactus by dUbc9 protects it from phosphorylation, assisting the retention of Dorsal/Dif in the cytoplasm. This model requires biochemical support as Cactus sumoylation has not been demonstrated (Chiu, 2005).

The intracellular Toll-Dorsal/Dif pathways in both the fat body and in hemocytes include Toll, Tube, Pelle, Ubc9, Dorsal, and Dif, and in both cases, dUbc9 appears to function upstream of Cactus and Dorsal/Dif. These observations with GFP reporter constructs suggest that the effect of dUbc9 is restricted to the Toll pathway and it is possible that the Imd signaling cascade is not regulated by sumoylation. Indeed, a ubiquitin proteasome pathway involving function of SkpA and Slimb has been identified and shown to repress the Imd pathway. Thus, sumoylation and ubiquitination of specific targets appear to have parallel but specific effects in downregulating these pathways (Chiu, 2005).

In conclusion, strong evidence is presented that dUbc9 is a negative regulator of the Toll-NF-κB pathways that control both the humoral and cellular aspects of immune responses in Drosophila. Spatzle activates the Toll pathway in the fat body; however, a role for Spatzle function in hemocyte proliferation or differentiation has not been demonstrated, and therefore the mechanism of Toll activation in the hemocytes is not known. Similarly, target genes of the NF-κB pathway in hemocytes (besides the antimicrobial peptide genes) have not yet been identified. The differences in phenotypes observed in lwr and cact mutants are likely to arise from differences in gene expression programs in the two mutants. These differences provide a unique opportunity to resolve issues of biological specificity in the regulation of NF-κB activation and gene expression in vivo (Chiu, 2005).

The Drosophila UBC9 homologue lesswright mediates the disjunction of homologues in meiosis I

In Saccharomyces cerevisiae and other organisms, the UBC9 (ubiquitin-conjugating 9) protein modifies the function of many different target proteins through covalent attachment of the ubiquitin-like protein SMT-3/SUMO. In normal female meiosis, a protein encoded by no distributive disjunction (nod) is responsible for preventing the nondisjunction of chromosome pairs that do not have an exchange. Using a second-site suppression screen of a mutation in the locus with a variable meiotic phenotype, mutations were identified in the Drosophila melanogaster UBC9 homologue, encoded by the gene lesswright (lwr). lwr mutations dominantly suppress the nondisjunction and cytological defects of female meiotic mutations that affect spindle formation. The lwr lethal phenotype is rescued by a Drosophila UBC9/lwr transgene. It is suggested that LWR mediates the dissociation of heterochromatic regions of homologues at the end of meiotic prophase I. A model proposes that when there is less LWR protein, homologues remain together longer, allowing for more normal spindle formation in mutant backgrounds and therefore more accurate meiotic chromosome segregation (Apionishev, 2001).

Genetic and cytological data show that lwr partially suppresses several mutations that affect chromosome segregation in Drosophila female meiosis. For the meiotic mutations that affect spindle formation, nod and ncd, lwr suppresses the nondisjunction of both of the chromosomes measured, the X and the 4th. In the case of two other meiotic mutations, AxsD and mei-218, lwr only suppresses 4th chromosome nondisjunction. It is not clear what the reason for this differential effect is, although it is clear that lwr suppresses nondisjunction to some extent in each meiotic mutant tested. This is a striking observation, because gene products affected by the four meiotic mutations tested act at different times and on different targets during meiosis (Apionishev, 2001).

The model for how the lwr mutation suppresses nondisjunction in several different classes of meiotic mutation is based on the hypothesis that UBC9 plays a role in freeing the 'glue' that holds chromosomes together as the spindle forms. After the homologues are aligned, that 'glue' dissolves, so that the homologues may dissociate from each other and disjoin. Thus, once the nuclear envelope breaks down and the spindle begins to form, the homologues begin to move apart. When oocytes in metaphase are observed cytologically in the wild-type background, exchange homologues are held together by chiasmata, while the nonexchange bivalents have already come apart and have moved poleward, held on the spindle by the NOD protein (Theurkauf, 1992). Thus, nonexchange chromosomes are particularly prone to nondisjunction. For example, if the spindle formation is impaired (ncd) or if there is no nod, the nonexchange chromosomes can move apart from the mass, and not end up on the major spindle (Theurkauf, 1992; Apionishev, 2001).

It is hypothesized that nonexchange homologues are more tightly associated in a lwr mutant background (the 'glue' remains longer than normal), thus delaying the separation of the nonexchange homologues from the chromosome mass. According to this model, spindle formation has a chance to 'catch up' to keep nonexchange homologues in the chromosome mass. This hypothesis would explain how lwr mutations suppress several different kinds of mutations, including those that impair spindle formation (Apionishev, 2001).

The following model is for the sequence of meiotic events. Chromosomes pair and form a synaptonemal complex, which then breaks down. All that remains are heterochromatic associations, primarily in the centromeric region, mediated by vestiges of the synaptonemal complex and/or by a distinct protein ('glue') complex specific for this function (see Dernburg, 1996). The homologues remain associated with each other, either because of chiasmata or because of the heterochromatic associations (Apionishev, 2001).

However, it is very likely that there is not enough of the material mediating the heterochromatic associations ('glue') to hold more than two pairs of nonexchange homologues together. Consistent with this model, nondisjunction rises as more chromosomes enter the nonexchange pool. Females heterozygous for a single balancer have relatively little nondisjunction (0.6%); heterozygosity for two balancers raises the rate (4%-5%), and heterozygosity for the X and both major autosomes leads to enormous rates (21%). This observation suggests that there is enough material to hold only one or two nonexchange pairs together (Apionishev, 2001).

Therefore, it is hypothesized that the proper redistribution of the 'glue' materials must occur to ensure strong associations between nonexchange homologues. The cytological manifestation of this process is the diplotene-diakinesis repulsion that is seen in many organisms as chiasmata become apparent. Although repulsion is not seen in Drosophila females, cytological evidence suggests that there is, in fact, a modification of the pairing, because euchromatic regions dissociate as prophase progresses (Dernburg, 1996), leaving the heterochromatic regions attached. Thus, such redistribution of the 'glue' materials may occur in Drosophila. In this model, LWR facilitates the redistribution of the 'glue' materials (Apionishev, 2001).

Thus it is hypothesized that reduced exchange, as in multiple balancer stocks, or recombination-defective mutants, leads to nondisjunction because the limited amount of material on each bivalent is not enough to hold them together. They separate and then behave as univalents. The genetic consequence of this premature breakdown of homologue association would be nondisjunction with an apparently normal spindle. This is exactly the phenotype that is seen for mei-218 and AxsD (Dernburg, 1996; McKim, 1993). This model resolves the long-standing issue of why decreased exchange leads to increased nondisjunction (Apionishev, 2001).

The vertebrate Cor1 synaptonemal complex protein behaves strikingly like the postulated 'glue' target of LWR. It associates with homologues as they begin to synapse and form a mature synaptonemal complex. However, as the prophase ends, Cor1 does not dissociate from the chromosomes. Instead, its distribution becomes discontinuous as it moves to heterochromatic centromeric regions. The fact that Cor1 also interacts with UBC9 in two-hybrid studies strengthens this assertion (Tarsounas, 1997). Furthermore, UBC9 localizes to the midregion of mouse bivalents in spermatocytes during the meiotic prophase (Kovalenko, 1996). Therefore, it is speculated that Cor1, which is apparently regulated by phosphorylation, is also modified by SMT-3 conjugation mediated by UBC9. This modification might promote the prompt redistribution of Cor1 to the centromere. According to this model, in lwr mutant backgrounds, the redistribution process may be delayed, allowing the homologues to stay together a little longer at the end of prophase I. Currently, experiments are being carried out to test this model of meiotic chromosome segregation and lwr function (Apionishev, 2001).

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

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

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

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

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

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

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

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


EVOLUTIONARY HOMOLOGS

Ubc9 homologs in yeast

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. The hus5+ gene has been cloned and sequenced. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control, the phenotypes were characterized of the hus5 mutants and the hus5 gene disruption. While the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. It is concluded that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation (Al-Khodairy, 1995).

At least one essential function of Smt3p, a Saccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1 (see Drosophila SUMO), involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, the second enzyme of the Smt3p conjugation pathway has been isolated and it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While the formation of a Ub approximately Ubc9p thioester was not detected, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation (Johnson, 1997).

Unlike ubiquitin, the ubiquitin-like protein modifier SUMO-1 and its budding yeast homologue Smt3p have been shown to be more important for posttranslational protein modification than for protein degradation. This study describes the identification of the SUMO-1 homologue of fission yeast that is required for a number of nuclear events including the control of telomere length and chromosome segregation. A disruption of the pmt3+ gene, the Schizosaccharomyces pombe homologue of SMT3, is not lethal, but mutant cells carrying the disrupted gene grew more slowly. The pmt3Delta cells show various phenotypes such as aberrant mitosis, sensitivity to various reagents, and high-frequency loss of minichromosomes. Interestingly, pmt3+ is required for telomere length maintenance. Loss of Pmt3p function causes a striking increase in telomere length. When Pmt3p synthesis is restored, the telomeres became gradually shorter. This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Fusion of Pmt3p to green fluorescent protein (GFP) showed that Pmt3p was predominantly localized as intense spots in the nucleus. One of the spots was shown to correspond to the spindle pole body (SPB). During prometaphase and metaphase, the bright GFP signals at the SPB disappeared. These observations suggest that Pmt3p is required for kinetochore and/or SPB functions involved in chromosome segregation. The multiple functions of Pmt3p described here suggest that several nuclear proteins are regulated by Pmt3p conjugation (Tanaka, 1999).

SMT3 of Saccharomyces cerevisiae is an essential gene encoding a ubiquitin-like protein similar to mammalian SUMO-1. When a tagged Smt3 or human SUMO-1 was expressed from GAL1 promoter, either gene rescued the lethality of the smt3 disruptant. By indirect-immunofluorescent microscopy, the HA-tagged Smt3 was detected mostly in nuclei and also at the mother-bud neck just like septin fibers. Indeed immunoprecipitation experiments revealed that Cdc3, one of septin components, was modified with Smt3. Furthermore, the protein level of the Cdc3-Smt3 conjugate was reduced and the septin rings disappeared in a ubc9-1 mutant at a restrictive temperature, where the Smt3 conjugation system should be defective. Thus, it is concluded that Smt3 is conjugated to Cdc3 in septin rings localized at the mother-bud neck. Around the time of cytokinesis, the Cdc3-Smt3 conjugate disappeared. The biological significance of this Smt3 conjugation to a septin component is discussed (Takahashi, 1999).

SUMO1/Smt3, a ubiquitin-like protein modifier, is known to be conjugated to other proteins and modulate their functions in various important processes. Similar to the ubiquitin system, SUMO1/Smt3 is activated in an ATP-dependent reaction by thioester bond formation with E1 (activating enzyme), transferred to E2 (conjugating enzyme), and passed to a substrate lysine. It remained unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in the final transfer of this modifier. This study reports a novel factor Siz1 (YDR409w) required for septin-sumoylation of budding yeast, possibly acting as E3. Siz1 is a member of a new family (Miz1, PIAS3, etc.) containing a conserved domain with a similarity to a zinc-binding RING-domain, often found in ubiquitin ligases. In the siz1 mutant, septin-sumoylation was completely abolished. A conserved cysteine residue in the domain was essential for this conjugation. Furthermore, Siz1 is localized at the mother-bud neck in the M-phase and physically binds to both E2 and the target proteins (Takahashi, 2001).

Several studies have suggested that SUMO may participate in the regulation of heterochromatin, but direct evidence is lacking. A direct link between sumoylation and heterochromatin stability is presented in this study. SUMO deletion impairs silencing at heterochromatic regions and induces histone H3 Lys4 methylation, a hallmark of active chromatin in fission yeast. The SUMO-conjugating enzyme Hus5/Ubc9 interacts with the conserved heterochromatin proteins Swi6, Chp2 (a paralog of Swi6), and Clr4 (H3 Lys9 methyltransferase). Moreover, chromatin immunoprecipitation (ChIP) revealed that Hus5 was highly enriched in heterochromatic regions in a heterochromatin-dependent manner, suggesting a direct role of Hus5 in heterochromatin formation. Swi6, Chp2, and Clr4 themselves can be sumoylated in vivo and defective sumoylation of Swi6 or Chp2 compromises silencing. These results indicate that Hus5 associates with heterochromatin through interactions with heterochromatin proteins and modifies substrates whose sumoylations are required for heterochromatin stability, including heterochromatin proteins themselves (Shin, 2005).

Covalent modification with SUMO alters protein function, intracellular localization, or protein-protein interactions. Target recognition is determined, in part, by the SUMO E2 enzyme, Ubc9, while Siz/Pias E3 ligases may facilitate select interactions by acting as substrate adaptors. A yeast conditional Ubc9P(123)L mutant is viable at 36°C yet exhibits enhanced sensitivity to DNA damage. To define functional domains in Ubc9 that dictate cellular responses to genotoxic stress versus those necessary for cell viability, a 1.75-A structure of yeast Ubc9 that demonstrates considerable conservation of backbone architecture with human Ubc9 was solved. Nevertheless, differences in side chain geometry/charge guides the design of human/yeast chimeras, where swapping domains implicated in (1) binding residues within substrates that flank canonical SUMOylation sites, (2) interactions with the RanBP2 E3 ligase, and (3) binding of the heterodimeric E1 and SUMO have distinct effects on cell growth and resistance to DNA-damaging agents. These findings establish a functional interaction between N-terminal and substrate-binding domains of Ubc9 and distinguish the activities of E3 ligases Siz1 and Siz2 in regulating cellular responses to genotoxic stress (van Waardenburg, 2006).

Ubc9 modification of transcription factors

Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. Modified IkappaBalpha has been detected, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugates SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation (Desterro, 1998).

Using a yeast two hybrid system and pull-down assays, mouse Dac (mDac) has been demonstrated to specifically bind mouse ubiquitin-conjugating enzyme mUbc9. In contrast to a direct interaction between Drosophila Dachshund and Eyes absent, mDac interaction with mEya2 could not be detected. mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner. Deletion analysis of mDac shows the intracellular localization and protein stability correlates with the binding to mUbc9. The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion. In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain. Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells. In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells (Machon, 2000).

Vsx-1 is a paired-like:CVC homeobox gene whose expression is linked to bipolar cell differentiation during zebrafish retinogenesis. The yeast two-hybrid screen was used to identify proteins interacting with Vsx-1. Ubc9, an enzyme that conjugates the small ubiquitin-like modifier SUMO-1 was isolated. Despite its interaction with Ubc9, Vsx-1 is not a substrate for SUMO-1 in COS-7 cells or in vitro. When a yeast two-hybrid assay is used, deletion analysis of the interacting domain on Vsx-1 shows that Ubc9 binds to a nuclear localization signal (NLS) at the NH(2) terminus of the homeodomain. In SW13 cells, Vsx-1 localizes to the nucleus and is excluded from nucleoli. Deletion of the NLS disrupts this nuclear localization, resulting in a diffuse cytoplasmic distribution of Vsx-1. In SW13 AK1 cells that express low levels of endogenous Ubc9, Vsx-1 accumulates in a perinuclear ring and colocalizes with an endoplasmic reticulum marker. However, NLS-tagged STAT1 protein exhibits normal nuclear localization in both SW13 and SW13 AK1 cells, suggesting that nuclear import is not globally disrupted. Cotransfection of Vsx-1 with Ubc9 restores Vsx-1 nuclear localization in SW3 AK1 cells and demonstrates that Ubc9 is required for the nuclear localization of Vsx-1. Ubc9 continues to restore nuclear localization even after a C93S active site mutation has eliminated its SUMO-1-conjugating ability. These results suggest that Ubc9 mediates the nuclear localization of Vsx-1, and possibly other proteins, through a nonenzymatic mechanism that is independent of SUMO-1 conjugation (Kurtzman, 2001).

The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated. The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, it was found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD+-dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. These studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation (Zhao, 2005).

Sumoylation regulates the activities of several members of the ETS transcription factor family. To provide a molecular framework for understanding this regulation, the conjugation of Ets-1 with SUMO-1 was characterized. Ets-1 is modified in vivo predominantly at a consensus sumoylation motif containing Lys-15. This lysine is located within the unstructured N-terminal segment of Ets-1 preceding its PNT domain. Using NMR spectroscopy, it has been demonstrated that the Ets-1 sumoylation motif associates with the substrate binding site on the SUMO-conjugating enzyme UBC9 [K(d) approximately 400 microm] and that the PNT domain is not involved in this interaction. Ets-1 with Lys-15 mutated to an arginine still binds UBC9 with an affinity similar to the wild type protein, but is no longer sumoylated. NMR chemical shift and relaxation measurements reveal that the covalent attachment of mature SUMO-1, via its flexible C-terminal Gly-97, to Lys-15 of Ets-1 does not perturb the structure or dynamic properties of either protein. Therefore sumoylated Ets-1 behaves as 'beads-on-a-string' with the two proteins tethered by flexible polypeptide segments containing the isopeptide linkage. Accordingly, SUMO-1 may mediate interactions of Ets-1 with signaling or transcriptional regulatory macromolecules by acting as a structurally independent docking module, rather than through the induction of a conformational change in either protein upon their covalent linkage. It is also hypothesized that the flexibility of the linking polypeptide sequence may be a general feature contributing to the recognition of SUMO-modified proteins by their downstream effectors (Macauley, 2006).

Covalent modification of proteins by the small ubiquitin-related modifier SUMO regulates diverse biological functions. Sumoylation usually requires a consensus tetrapeptide, through which the binding of the SUMO-conjugating enzyme Ubc9 to the target protein is directed. However, additional specificity determinants are in many cases required. To gain insights into SUMO substrate selection, the differential sumoylation of highly similar loop structures within the DNA-binding domains of heat shock transcription factor 1 (HSF1) and HSF2 were used. Site-specific mutagenesis in combination with molecular modeling revealed that the sumoylation specificity is determined by several amino acids near the consensus site, which are likely to present the SUMO consensus motif to Ubc9. Importantly, sumoylation of the HSF2 loop impedes HSF2 DNA-binding activity, without affecting its oligomerization. Hence, SUMO modification of the HSF2 loop contributes to HSF-specific regulation of DNA binding and broadens the concept of sumoylation in the negative regulation of gene expression (Anckar, 2006)

Human p66alpha and p66beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66alpha and one in p66beta. Both p66alpha and p66beta are SUMO-modified in vivo: p66alpha at two sites (Lys-30 and Lys-487) and p66beta at one site (Lys-33). Expression of SUMO1 enhances the transcriptional repression activity of Gal-p66alpha and Gal-p66beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase results in a significant decrease of the transcriptional repression of p66alpha and p66beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 bind to both p66alpha and p66beta in vivo. Most of the interactions are not affected by the SUMO site mutations in p66alpha or p66beta, with two exceptions. HDAC1 binding to p66alpha is lost in the case of a p66alphaK30R mutant, and RbAp46 binding is reduced in the case of a p66betaK33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation (Gong, 2006).

SOX4 is a member of SOX transcriptional factor family that is crucial for many cellular processes. In this study, a yeast two-hybrid screening of human mammary cDNA library identified human ubiquitin-conjugating enzyme 9 (hUbc9) interaction with SOX4. This interaction was confirmed by GST pull-down in vitro and co-immunoprecipitation assays in vivo. Deletion mapping demonstrated that HMG-box domain of SOX4 is required to mediate the interaction with Ubc9 in yeast. Furthermore, confocal microscopy showed that Ubc9 co-localizes with SOX4 in the nucleus. Luciferase assays found that Ubc9 specifically represses SOX4 transcriptional activity in 293T cells. Ubc9 can functionally repress the transcriptional activity of endogenous SOX4 induced by progesterone in T47D cells. The C93S mutant of Ubc9, which abrogates SUMO-1 conjugation activity, does not abolish the ability to repress SOX4 activity. It shows that Ubc9 interacts with SOX4 and represses its transcriptional activity independent of its SUMO-1-conjugating activity (Pan, 2006).

KLF8 (Kruppel-like factor 8) is a member of the Kruppel transcription factor family that binds CACCC elements in DNA and activates or represses their target genes in a context-dependent manner. Sumoylation is a novel mechanism that regulates KLF8 post-translationally. KLF8 can be covalently modified by small ubiqitin-like modifier (SUMO)-1, SUMO-2, and SUMO-3 in vivo. KLF8 interacts with the PIAS family of SUMO E3 ligases PIAS1, PIASy, and PIASxalpha but not with E2 SUMO-conjugating enzyme Ubc9. Furthermore, E2 and E3 ligases enhance the sumoylation of KLF8. In addition, site-directed mutagenesis identified lysine 67 as the major sumoylation site on KLF8. Lysine 67 to arginine mutation strongly enhances activity of KLF8 as a repressor or activator to its physiological target promoters and as an inducer of the G(1) cell cycle progression. Taken together, these results demonstrate that sumoylation of KLF8 negatively regulates its transcriptional activity and cellular functions (Wei, 2006)

Miscellaneuos interactions and targets of Ubc9

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis (Kovalenko, 1996).

Ubiquitin conjugating enzymes (UBCs) comprise a family of proteins directly involved in ubiquitination of proteins. Ubiquitination is known to be involved in control of a variety of cellular processes, including cell proliferation, through the targeting of key regulatory proteins for degradation. The ubc9 gene of the yeast Saccharomyces cerevisiae (Scubc9) is an essential gene which is required for cell cycle progression and is involved in the degradation of S phase and M phase cyclins. A human homolog of Scubc9 (termed hubc9) has been identified using the two hybrid screen for proteins that interact with the human papillomavirus type 16 E1 replication protein. The hubc9 encoded protein shares a very high degree of amino acid sequence similarity with ScUBC9 and with the homologous hus5+ gene product of Schizosaccharomyces pombe. Genetic complementation experiments in a S. cerevisiae ubc9ts mutant reveal that hUBC9 can substitute for the function of ScUBC9 required for cell cycle progression (Yasugi, 1996).

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2). Ran-binding protein 2 [p340(RanBP2)] is a nuclear pore protein, and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier, and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2. This study shows that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts (Saitoh, 1998).

PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs); these complexes are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, a ubiquitin-like protein named PIC1 (now known as SUMO-1) was identified, that interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. This study shows that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, the primary PML-SUMO-1 modification site was identified as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation since only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and several other nuclear proteins have been identified that could also be targets for SUMO-1. SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. The APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. These results provide significant insight into the role of SUMO-1 modification of PML in both normal cells and the APL disease state (Duprez, 1999).

In the absence of ligands the corepressor N-CoR mediates transcriptional repression by some nuclear hormone receptors. Several protein-protein interactions of N-CoR are known; of these, mainly complex formation with histone deacetylases (HDACs) leads to the repression of target genes. In contrast, the role of posttranslational modifications in corepressor function is not well established. This study shows that N-CoR is modified by Sumo-1. SUMO-E2-conjugating enzyme Ubc9 and SUMO-E3 ligase Pias1 are novel N-CoR interaction partners. The SANT1 domain of N-CoR mediates this interaction. K152, K1117, and K1330 of N-CoR can be conjugated to SUMO and mutation of all sites is necessary to fully block SUMOylation in vitro. Because these lysine residues are located within repression domains I and III, respectively, a possible correlation between the functions of the repression domains and SUMOylation was investigated. Coexpression of Ubc9 protein results in enhanced N-CoR-dependent transcriptional repression. Studies using SUMOylation-deficient N-CoR RDI mutants suggest that SUMO modification contributes to repression by N-CoR. Mutation of K152 to R in RD1, for example, not only significantly reduce repression of a reporter gene, but also abolish the effect of Ubc9 on transcriptional repression (Tiefenbach, 2006).

Human DNA topoisomerase I has been identified as a major SUMO1 target in camptothecin-treated cells. In response to TOP1-mediated DNA damage induced by camptothecin, multiple SUMO1 molecules are conjugated to the N-terminal domain of a single TOP1 molecule. To investigate the molecular mechanism of SUMO1 conjugation to TOP1, an in vitro system using purified SAE1/2, Ubc9, SUMO1, and TOP1 peptides was developed. Consistent with results from in vivo studies, multiple SUMO1 molecules were found to be conjugated to the N-terminal domain of a single TOP1 molecule. Systematic analysis has identified a single major SUMO1 conjugation site located between amino acid residues 110 and 125 that contains a single lysine residue at 117 (Lys-117). Using a short peptide spanning this region, it was shown that a poly-SUMO1 chain is assembled in this peptide at Lys-117. Interestingly, a Ubc9-poly-SUMO1 intermediate accumulates to a high level when the sumoylation assay is performed in the absence of hTOP1 substrate, suggesting a possibility that the poly-SUMO1 chain is formed on Ubc9 first and then transferred en bloc onto hTOP1. This is the first definitive demonstration of the assembly of a poly-SUMO1 chain on protein substrate. These results offer new insight into hTOP1 polysumoylation in response to TOP1-mediated DNA damage and may have general implications in protein polysumoylation (Yang, 2006).

Mutation of Ubc9

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, a conditional UBC9 mutant of chicken DT40 cells was constructed containing the UBC9 transgene under control of a tetracycline-repressible promoter and their loss of function phenotypes were characterized. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, no accumulation was observed of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9, but an increase was observed in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment (Hayashi, 2002).

Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, mice were generated deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals (Nacerddine, 2005).

Ubc9 and meiosis

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. The two-hybrid system in the yeast Saccharomyces cerevisiae was used to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II (Tarsounas, 1997).

During meiotic prophase, homologous chromosomes engage in a complex series of interactions that ensure their proper segregation at meiosis I. A central player in these interactions is the synaptonemal complex (SC), a proteinaceous structure elaborated along the lengths of paired homologs. In mutants that fail to make SC, crossing over is decreased, and chromosomes frequently fail to recombine; consequently, many meiotic products are inviable because of aneuploidy. This study investigated the role of the small ubiquitin-like protein modifier (SUMO) in SC formation during meiosis in budding yeast. SUMO localizes specifically to synapsed regions of meiotic chromosomes and this localization depends on Zip1, a major building block of the SC. A non-null allele of the UBC9 gene, which encodes the SUMO-conjugating enzyme, impairs Zip1 polymerization along chromosomes. The Ubc9 protein localizes to meiotic chromosomes, coincident with SUMO staining. In the zip1 mutant, SUMO localizes to discrete foci on chromosomes. These foci coincide with axial associations, where proteins involved in synapsis initiation are located. These data suggest a model in which SUMO modification of chromosomal proteins promotes polymerization of Zip1 along chromosomes. The ubc9 mutant phenotype provides the first evidence for a cause-and-effect relationship between sumoylation and synapsis (Hooker, 2006).

Regulation of SUMOylation by reversible oxidation of SUMO conjugating enzymes

Posttranslational modification with small ubiquitin-related modifier (SUMO) has emerged as a central regulatory mechanism of protein function. However, little is known about the regulation of sumoylation itself. It has been reported that it is increased after exposure to various stresses including strong oxidative stress. Conversely, ROS (reactive oxygen species), at low concentrations, result in the rapid disappearance of most SUMO conjugates, including those of key transcription factors. This is due to direct and reversible inhibition of SUMO conjugating enzymes through the formation of (a) disulfide bond(s) involving the catalytic cysteines of the SUMO E1 subunit Uba2 and the E2-conjugating enzyme Ubc9. The same phenomenon is also observed in a physiological scenario of endogenous ROS production, the respiratory burst in macrophages. Thus, these findings add SUMO conjugating enzymes to the small list of specific direct effectors of H2O2 and implicate ROS as key regulators of the sumoylation-desumoylation equilibrium (Bossis, 2006).

Estrogen receptor and regulation of bcl-2 expression by Ubc9

Posttranslational modifications mediated by ubiquitin-like proteins have been implicated in regulating a variety of cellular pathways. Although small ubiquitin-like modifier (SUMO) is a new member of this family, it has caught a great deal of attention recently because of its novel and distinguished functions. Sumoylation is a multiple-step process, involving maturation, activation, conjugation and ligation. Ubc9 is an E2 conjugating enzyme essential for sumoylation. Suppression of sumoylation by a dominant negative Ubc9 mutant (Ubc9-DN) in the estrogen receptor (ER) positive MCF-7 cells is associated with alterations of tumor cell's response to anticancer drugs as well as tumor growth in a xenograft mouse carcinoma model. To dissect the underlying mechanism of Ubc9-associated alterations of drug responsiveness and tumor growth, gene expression for the cells expressing wild type Ubc9 (Ubc9-WT) and Ubc9-DN was profiled. Several tumorigenesis-related genes are downregulated in the Ubc9-DN cells. Within this group, over 10 genes are known to be regulated by ER. Experiments using the estrogen response element fused to the luciferase reporter showed that the basal level of luciferase activity is significantly reduced in the Ubc9-DN cells when compared to the vector alone or the Ubc9-WT cells. Furthermore, both the stability and the subcellular localization of steroid hormone receptor coactivator-1 (SRC-1) are altered in the Ubc9-DN cells. Together, these results suggest that Ubc9 might regulate bcl-2 expression through the ER signaling pathway, which ultimately contributes to the alterations of drug responsiveness and tumor growth (Lu, 2006).


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