InteractiveFly: GeneBrief

degringolade: Biological Overview | References


Gene name - degringolade

Synonyms - CG10981

Cytological map position- 83C4-83C4

Function - enzyme

Keywords - ubiquitin ligase, sex determination, Notch pathway, negative regulator of the repressor Hairy and its corepressor Groucho, segmentation, neurogenesis

Symbol - dgrn

FlyBase ID: FBgn0037384

Genetic map position - 3R:1,650,327..1,652,298 [+]

Classification - RING finger/E3 ubiquitin ligase, STUbL family

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature
Ryu, T., Bonner, M. and Chiolo, I. (2016). Cervantes and Quijote protect heterochromatin from aberrant recombination and lead the way to the nuclear periphery. Nucleus [Epub ahead of print] PubMed ID: 27673416
Summary:
Repairing double-strand breaks (DSBs) is particularly challenging in heterochromatin, where the abundance of repeated sequences exacerbates the risk of ectopic recombination and chromosome rearrangements. In Drosophila cells, faithful homologous recombination (HR) repair of heterochromatic DSBs relies on a specialized pathway that relocalizes repair sites to the nuclear periphery before Rad51 recruitment. This study shows that HR progression is initially blocked inside the heterochromatin domain by SUMOylation and the coordinated activity of two distinct Nse2 SUMO E3 ligases: Quijote (Qjt) and Cervantes (Cerv). In addition, the SUMO-targeted ubiquitin ligase (STUbL) Dgrn, but not its partner dRad60, is recruited to heterochromatic DSBs at early stages of repair and mediates relocalization. However, Dgrn is not required to prevent Rad51 recruitment inside the heterochromatin domain, suggesting that the block to HR progression inside the domain and relocalization to the nuclear periphery are genetically separable pathways. Further, SUMOylation defects affect relocalization without blocking heterochromatin expansion, revealing that expansion is not required for relocalization. Finally, nuclear pores and inner nuclear membrane proteins (INMPs) anchor STUbL/RENi components and repair sites to the nuclear periphery, where repair continues. Together, these studies reveal a critical role of SUMOylation and nuclear architecture in the spatial and temporal regulation of heterochromatin repair and the protection of genome integrity
Koltun, B., Shackelford, E., Bonnay, F., Matt, N., Reichhart, J. M. and Orian, A. (2017). The SUMO-targeted ubiquitin ligase, Dgrn, is essential for Drosophila innate immunity. Int J Dev Biol 61(3-4-5): 319-327. PubMed ID: 28621429
Summary:
This paper reports that Degringolade (Dgrn), a SUMO-targeted ubiquitin ligase connecting the two pathways, is essential for the innate immunity response in Drosophila. dgrnDK null and heterozygous mutant adult flies are severely immune-compromised and succumb rapidly to both pathogenic bacteria and fungi infections. The sensitivity to infection stems from the inability to produce multiple anti-microbial peptides, and transcriptional analyses suggest that the overexpression of Dgrn enhances the transcriptional output of the NF-κB related Toll and immune deficiency (IMD)-pathways. Moreover, expression of Dgrn alleviated the inhibitory impact of the cytoplasmic NF-κB inhibitor Cactus and the nuclear co-repressor Groucho/TLE (Gro). Additionally, Dgrn was found to be required for the local regenerative response of the midgut following infection. Upon oral infection, dgrn mutant flies fail to activate the Delta-Notch pathway in stem cells and enteroblasts, and are unable to regenerate and replace the damaged and dying enterocytes. Interestingly, the ubiquitin-specific protease CG8334 (dUSP32/dUSP11) antagonizes Dgrn activity in the gut, and halving the dose of CG8334 restores Delta-Notch signaling and rescues the lethality observed in dgrn mutants. Collectively, these data suggest that Dgrn is essential for both systemic and local tissue response to infection.
BIOLOGICAL OVERVIEW

Transcriptional cofactors are essential for proper embryonic development. One such cofactor in Drosophila, Degringolade (Dgrn), encodes a RING finger/E3 ubiquitin ligase. Dgrn and its mammalian ortholog RNF4 are SUMO-targeted ubiquitin ligases (STUbLs; see Model for SUMO-directed ubiquitination by the conserved STUbL family). STUbLs bind to SUMOylated proteins (see Smt3/SUMO) via their SUMO interaction motif (SIM) domains and facilitate substrate ubiquitylation. This study shows that Dgrn is a negative regulator of the repressor Hairy and its corepressor Groucho [Gro/transducin-like enhancer (TLE)] during embryonic segmentation and neurogenesis, as dgrn heterozygosity suppresses Hairy mutant phenotypes and embryonic lethality. Mechanistically Dgrn functions as a molecular selector: it targets Hairy for SUMO-independent ubiquitylation that inhibits the recruitment of its corepressor Gro, without affecting the recruitment of its other cofactors or the stability of Hairy. Concomitantly, Dgrn specifically targets SUMOylated Gro for sequestration and antagonizes Gro functions in vivo. These findings suggest that by targeting SUMOylated Gro, Dgrn serves as a molecular switch that regulates cofactor recruitment and function during development. As Gro/TLE proteins are conserved universal corepressors, this may be a general paradigm used to regulate the Gro/TLE corepressors in other developmental processes (Abed, 2011).

Transcriptional cofactors are essential for the function of sequence-specific transcription factors and are part of the machinery required to execute temporally coordinated gene expression programs. Regulation of cofactor recruitment and activity is emerging as a major level of gene expression regulation. For example, Hairy/Enhancer of split/Deadpan (HES) family repressors are the primary transducers of the Notch signalling pathway that has a central role in patterning, stem cell development, and is misregulated in cancers. A well-studied case is the Drosophila repressor Hairy, a typical HES family member, which encodes a basic helix-loop-helix (bHLH) Orange repressor required for embryonic segmentation and adult peripheral nervous system (PNS) specification. Hairy-mediated repression is dependent on its ability to recruit cofactors. For example, Hairy recruits the corepressor Groucho (Gro) through it C-terminal WRPW domain, an interaction that is essential for periodic repression of fushi tarazu (ftz). In addition, Hairy recruits dCtBP and dSir2 through its PLSLV and basic domains, respectively. While these cofactors are required for Hairy-mediated repression, they exhibit context-dependent recruitment and function (Bianchi-Frias, 2004). Interestingly, some cofactors enhance Hairy-mediated repression (e.g., Gro and dSir2), whereas others are required to refine Hairy's function (e.g., dCtBP and dTopors; Phippen, 2000; Secombe, 2004). Consistent with this, it was found that most of the genomic loci bound by Hairy in the context of Kc cells exhibit corecruitment of dSir2 and dCtBP, but are not co-bound by Gro (Bianchi-Frias, 2004). However, the mechanisms that regulate context-selective cofactor association with Hairy or that may regulate cofactor activities are largely unknown (Abed, 2011).

A possible mechanism is that post-translational modification of Hairy regulates its association with a given cofactor and determines its overall function. One such modification is ubiquitylation that in many cases regulates the stability of transcription factors. However, ubiquitylation can also serve as a regulatory modification that does not lead to degradation, but affects protein-protein interaction or intracellular localization (Ikeda, 2008). Similarly, SUMOylation is a post-transcriptional modification that is involved in the regulation of gene expression and is mediated by the SUMO-specific E1-, E2-, and E3-SUMO ligase enzymes. Both ubiquitin and SUMO modifications are highly regulated. These two modifications can also be connected through proteins collectively termed SUMO-targeted ubiquitin ligases (STUbLs; Sun, 2007; Geoffroy, 2009). STUbLs are RING proteins that bind non-covalently to the SUMO moiety of SUMOylated proteins via their N-terminal SUMO interaction motif (SIM) domains, and subsequently target the SUMOylated protein for ubiquitylation via their RING domain. Thus, STUbLs are able to 'sense' SUMOylated targets and modify them by ubiquitylation. The observation that STUbLs are associated with transcription complexes suggests that their function is directly linked to regulation of gene expression. For example, the STUbL protein RNF4 was found to be a positive regulator of steroid hormone transcription (Poukka, 2000). Importantly, STUbLs are structurally and functionally conserved, as the mouse and human RNF4 proteins can substitute for their yeast orthologs in functional assays (Prudden, 2007). STUbLs are required for the correct assembly of kinetochores, for the cell's ability to cope with genotoxic stress, and for genome stability (Kosoy, 2007; Prudden, 2007; Nagai, 2008; Rouse, 2009; Mukhopadhyay, 2010). RNF4 is highly expressed in the stem cell compartment of the developing gonads and brain, and its expression is enriched in progenitor cells, likely representing its role in 'stemness' (Galili, 2000; Ramalho-Santos, 2002). Recently, RNF4 was shown to regulate the SUMO- and ubiquitin-mediated degradation of PML and PML-RAR (Lallemand-Breitenbach, 2008; Tatham, 2008; Geoffroy, 2009). However, the role of STUbL proteins in transcription during development of higher eukaryotes is largely unknown (Abed, 2011).

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

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

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

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

As an ubiquitin ligase, Dgrn catalyses the formation of mixed poly-ubiquitin chains on Hairy. This ubiquitylation does not map to Hairy's basic region, its putative SUMOylation sites, or to a single Lys residue. Importantly, this poly-site ubiquitylation does not affect Hairy protein stability or integrity, but rather selectively inhibits Gro binding to Hairy. Furthermore, in cells in which Dgrn protein levels are reduced via RNAi, Hairy protein levels are also decreased compared with control cells, suggesting that Dgrn is likely required for Hairy expression. This is different from dTopors, a Hairy-associated PHD-RING finger protein, which catalyses Lys48-linked chains and regulates Hairy turnover (Secombe, 2004). Further work will be required to determine the exact molecular events and the role that specific ubiquitin chain linkage has in Dgrn's ability to inhibit Gro from binding to Hairy in vivo (Abed, 2011).

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

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

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

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

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

In vivo, it was found that Dgrn antagonism of Gro is highly relevant for embryonic segmentation, PNS development, and sex determination, processes that are regulated by Gro (Barry, 2011). Indeed, Dgrn can suppress the gain-of-function phenotypes of Gro, as well as rescue the phenotypes associated with tissue-specific inactivation of Gro using RNAi transgenes. The genomic targets of Gro and Dgrn are distinct from that of dCtBP or dSir2, and that 38% of Gro direct targets are shared with Dgrn. Thus, it is predicted that Dgrn will be involved in other HES-independent, but Gro-regulated, processes as well. It is likely that both proteins have unique regulatory roles during early development. This notion stems from observations that each of the factors has exclusive, non-overlapping, genomic binding sites, and that neither of the two genes can functionally rescue the embryonic lethality associated with mutants of the other protein (i.e., Gro cannot rescue the female sterility associated with dgrn null females, and reducing the dose of Dgrn does not rescue the lethality associated with the groE48 mutant) (Abed, 2011).

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

The Drosophila STUbL protein Degringolade limits HES functions during embryogenesis

Degringolade encodes a Drosophila SUMO-targeted ubiquitin ligase (STUbL) protein similar to that of mammalian RNF4. Dgrn facilitates the ubiquitylation of the HES protein Hairy, which disrupts the repressive activity of Hairy by inhibiting the recruitment of its cofactor Groucho. This study shows that Hey and all HES family members, except Her, interact with Dgrn and are substrates for its E3 ubiquitin ligase activity. Dgrn displays dynamic subcellular localization, accumulates in the nucleus at times when HES family members are active and limits Hey and HES family activity during sex determination, segmentation and neurogenesis. Dgrn interacts with the Notch signaling pathway by antagonizing the activity of E(spl)-C proteins. dgrn null mutants are female sterile, producing embryos that arrest development after two or three nuclear divisions. These mutant embryos exhibit fragmented or decondensed nuclei and accumulate higher levels of SUMO-conjugated proteins, suggesting a role for Dgrn in genome stability (Barry, 2011).

A common theme among DNA-bound transcriptional regulators is the recruitment of co-activators and/or co-repressors to carry out their function. An important aspect of all HES family regulation is their recruitment of the co-repressor Groucho. Abed (2011) has shown that the ubiquitylation of Hairy does not lead to its degradation, but rather interferes with the ability of Hairy to recruit Gro, thereby antagonizing Hairy's repressive activity. This study found that dgrn mutant embryos show defects in segmentation. It is suggested that, similar to Dgrn's interaction with Hairy in segmentation, the ubiquitylation of other HES family members or Hey leads to their inability to recruit the cofactor Gro and thus antagonizes the repressor activity of this protein family. Consistent with this, it was found that loss of Dgrn function affects known Hey and HES family early functions, including sex determination and nervous system development (Barry, 2011).

It was surprising that Dgrn is female sterile rather than exhibiting zygotic lethality. Another, as yet unidentified, STUbL protein might function redundantly to Dgrn postzygotically. As the early Drosophila embryo develops essentially as a closed system running on maternally provided mRNA and proteins, the early syncytial embryo relies heavily on translational and post-translational modifications to control protein activity. Both Dgrn and Gro are maternally contributed and ubiquitously distributed. Thus, Dgrn might be recruited to the nucleus at different times during these early developmental stages to attenuate Gro's ability to be a potent co-repressor in the absence of active transcription, thereby modulating Hey and HES family activity (Barry, 2011).

Dgrn's human homolog is the transcriptional cofactor and STUbL protein RNF4. Indeed, human RNF4 acts as a functional homolog of Dgrn. RNF4 has also been shown to be a functional ortholog of the Rfp1/Rfp2-Slx8 heterodimer (from now on referred to as Rfp-Slx8) in S. pombe (Kosoy, 2007; Prudden, 2007) and the Slx5-Slx8 heterodimer in S. cerevisiae (Uzunova, 2007). RNF4 and the yeast Rfp-Slx8 and Slx5-Slx8 heterodimers have been shown to be important for DNA repair, kinetochore assembly and genome stability, with the loss of these proteins leading to fragmented chromosomes, elongated nuclei, asymmetric positioning of the nuclei and an accumulation of SUMOylated proteins (Burgess, 2007; Kosoy, 2007; Prudden, 2007; Sun, 2007; Mullen, 2008; Cook, 2009; Mukhopadhyay, 2010; Barry, 2011 and references therein).

The budding yeast Slx5-Sx8 proteins were identified as a complex of proteins required for the viability of SGS1 (a gene encoding the only RecQ helicase involved in genomic integrity in S. cerevisiae) mutant cells (Mullen, 2001). In Drosophila, loss of RecQ5 function leads to the loss of synchronous divisions in the syncytial embryo, an increased number of double strand breaks and a slight increase in the number of abnormal nuclei falling from the surface of the embryo (Nakayama, 2009). Mutations of the RecQ family member DmBlm (mus309 -- FlyBase; the Drosophila ortholog of human BLM, which leads to the human disorder Bloom Syndrome when mutated) are female sterile with severe defects in embryogenesis: syncytial embryos frequently include anaphase bridges, gaps in the normally uniform monolayer of nuclei and asynchronous mitoses (McVey, 2007; Barry, 2011 and references therein).

Recently, smt3 (SUMO) mutant embryos were shown to display embryonic nuclear cycle defects, including irregular size and distribution of nuclei, chromosome clustering, chromosome bridges, fragmentation and reduced number of nuclei in relation to the centrosome pairs (Nie, 2009). Several cell cycle factors were identified that are substrates for SUMOylation, it was and proposed that SUMOylation of these factors is important for controlling the cell cycle. The fragmented and de-condensed nuclei observed in the early arrest phenotypes of dgrnDK null embryos are reminiscent of the Slx8-Slx5 mutant phenotypes, fly RecQ mutant phenotypes and smt3 mutant phenotypes, suggesting that the role of STUbL proteins in genome stability and DNA repair might be a conserved function (Barry, 2011).

Alternatively, mutations in actin cytoskeleton and cell cycle checkpoint components in Drosophila have also been shown to exhibit nuclear arrest phenotypes. Defects in cell cycle checkpoint proteins, including Pan Gu, Plutonium and Giant Nuclei affect the S-phase checkpoint in the early embryo such that mutation of any of these genes leads to unregulated S-phase, resulting in giant polyploid nuclei. Disruption of the actin cytoskeleton can also lead to nuclear division abnormalities of cortical nuclei. For example, the scrambled and nuclear fallout mutants exhibit severe abnormalities in the appearance and localization of cortical nuclei. Further experiments will be needed to determine the molecular mechanism(s) underlying Dgrn's early arrest phenotype. However, regardless of the mechanism, this represents a new function for Hey or HES family proteins or a function for Dgrn that is not HES-dependent (Barry, 2011).

Interestingly, nuclear cycles during which Dgrn accumulates in the nucleus correspond to times when HES family members are active, which would be necessary for Dgrn to interact physically with HES proteins and subsequently affect their functions. One exception to this is Dgrn nuclear localization at nuclear cycle 9. There are no known HES family activities at nuclear cycle 9; however, several HES family members are yet to be characterized molecularly and genetically. Dgrn also exhibits a novel accumulation pattern during the gastrulation stages where it prefigures morphogenetic furrows, suggesting a possible role for Hey or HES family members in morphogenesis. Chromatin profiling experiments identifying direct transcriptional targets of Hairy identified a number of targets important for morphogenesis, suggesting that Hairy might play a role in morphogenesis (Bianchi-Frias, 2004). Consistent with this, a new hairy allele (h674) has been reported to affect the early stages of salivary gland morphogenesis. Thus, although Dgrn might work with Hey, Hairy and/or other HES family members during these times, it also remains possible that these Dgrn activities are Hey- and HES-independent (Barry, 2011).

Dpn is a negative regulator of Sxl. dpn mutants have a modest effect on Sxl in males leading to ectopic expression from the Sxl-Pe promoter that is sufficient to induce the inappropriate female fate in some cells. The Hey and HES family co-repressor Gro has also been shown to act as a negative regulator of Sxl; the loss of maternal Gro results in severe misexpression of Sxl in males leading to female fate. The relatively mild effect of Dpn on Sxl regulation compared with Gro led Lu (2008) to search for additional HES family proteins involved in Sxl regulation. Hey was identified as a maternal repressor of Sxl-Pe, albeit in a spatially variable pattern in males. Unlike the mammalian homologs of Hey, which are unable to bind Gro presumably owing to its C-terminal YRPW domain, this study found that Drosophila Hey binds Gro in GST pulldown assays. The data suggests that Dgrn is an important player in sex determination where it interferes with the repressive activities of Dpn and Gro; both Dpn and Hey are substrates for Dgrn's E3 ubiquitin ligase activity. In addition, Sxl protein staining and in vitro transcription assays demonstrate that Dgrn antagonizes the repression of Sxl. As proposed for the interaction of Dgrn with Hairy during segmentation (Abed, 2011), this study found that Dgrn provides a new level of control over the activity of Hey and the HES family members in sex determination. This control is mediated by ubiquitylation that probably disrupts the ability of these repressors to recruit Gro, thereby antagonizing their ability to repress transcription of Sxl-Pe in males (Barry, 2011).

Erickson (2007) has proposed that sex in Drosophila is not determined by the ratio of X-chromosomes to sets of autosomes (X:A ratio), but rather by X chromosome dose. It was speculated that a feedback mechanism in females is caused by the acetylation of chromatin, which inhibits Gro-mediated repression. Interestingly, the finding that Dgrn antagonizes Gro activity via the ubiquitylation of Hey and HES family repressors and targets SUMOylated Gro for sequestration provides an alternate scenario for this feedback mechanism in females (Barry, 2011).

Notch (N), through the E(spl) proteins (its downstream targets), heads one of the major developmental signaling pathways that functions in progenitor cell fate determination and differentiation. Recently, Sxl has been shown to inhibit Notch RNA translation and to negatively regulate the Notch signaling pathway in females (Penn, 2007). The notched wing phenotype of N was shown to be sensitive to Sxl, such that reducing the dose of Sxl suppressed the lethal effects of N hypomorphic alleles. This study found that reducing the dose of dgrn can also partially rescue the lethal effects of N hypomorphic alleles suggesting that Dgrn antagonizes N signaling. More specifically, it is hypothesized that the rescue of N1 male lethality is due to a decrease in Sxl expression. Dgrn heterozygosity also suppresses the vein patterning phenotype associated with NAX1682, suggesting that it is required for N signaling in this context also. Interestingly, Dgrn could be antagonizing N by two distinct mechanisms (or a combination of the two): the first an indirect antagonization of N signaling through Dgrn's control of Sxl expression, and the second by direct inhibition of the repressor activities of the E(spl)-C protein by ubiquitylation, thus blocking the repressive arm of the N pathway. The second mechanism has implications in regulating crosstalk between N and EGFR signaling pathways. Further studies will be required to determine the role of Dgrn's STUbL activity and whether Dgrn's activity on E(spl)-C proteins is redundant to EGFR signaling or whether both of these activities are required to antagonize Notch signaling (Barry, 2011).


REFERENCES

Search PubMed for articles about Drosophila Degringolade

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

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

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

Bianchi-Frias, D., et al. (2004). Hairy-mediated transcriptional repression and cofactor recruitment in Drosophila. PloS Biol. 2(7): E178. PubMed ID: 15252443

Burgess R. C., et al. (2007). The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination. Mol. Cell. Biol. 27: 6153-6162. PubMed ID: 17591698

Cook, C. E., Hochstrasser M. and Kerscher O. (2009). The SUMO-targeted ubiquitin ligase subunit Slx5 resides in nuclear foci and at sites of DNA breaks. Cell Cycle 8: 1080-1089. PubMed ID: 19270524

Erickson, J. W. and Quintero J. J. (2007). Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila. PLoS Biol. 5: e332. PubMed ID: 18162044

Galili, N., Nayak, S., Epstein, J. A. and Buck, C. A. (2000). RNF4, a RING protein expressed in the developing nervous and reproductive systems interacts with Gscl, a gene within the DiGeorge critical region. Dev. Dyn. 218: 102-111. PubMed ID: 10822263

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

Ikeda, F. and Dikic, I. (2008). Atypical ubiquitin chains: new molecular signals. ‘Protein Modifications: Beyond the Usual Suspects’ review series. EMBO Rep. 9: 536-542. PubMed ID: 18516089

Kosoy, A., et al. (2007). Fission yeast Rnf4 homologs are required for DNA repair. J. Biol. Chem. 282: 20388-20394. PubMed ID: 17502373

Lallemand-Breitenbach, V., et al. (2008) Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway. Nat. Cell Biol. 10: 547-555. PubMed ID: 18408733

Lu, H., et al. (2008). Maternal Groucho and bHLH repressors amplify the dose-sensitive X chromosome signal in Drosophila sex determination. Dev. Biol. 323: 248-260. PubMed ID: 18773886

McVey, M., Andersen S. L., Broze Y. and Sekelsky, J. (2007). Multiple functions of Drosophila BLM helicase in maintenance of genome stability. Genetics 176: 1979-1992. PubMed ID: 17507683

Mukhopadhyay, D., Arnaoutov, A. and Dasso, M. (2010). The SUMO protease SENP6 is essential for inner kinetochore assembly. J. Cell Biol. 188: 681-692. PubMed ID: 20212317

Mullen, J. R., Kaliraman V., Ibrahim S. S. and Brill S. J. (2001). Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae. Genetics 157: 103-118. PubMed ID: 11139495

Mullen J. R. and Brill S. J. (2008). Activation of the Slx5-Slx8 ubiquitin ligase by poly-small ubiquitin-like modifier conjugates. J. Biol. Chem. 283: 19912-19921. PubMed ID: 18499666

Nagai, S. et al. (2008). Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 322: 597-602. PubMed ID: 18948542

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Nie M., et al. (2009). Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation. PLoS One 4: e5905. PubMed ID: 19529778

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Biological Overview

date revised: 15 May 2011

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