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stuxnet: Biological Overview | References

BIOLOGICAL OVERVIEW Polycomb-group (PcG) proteins function to ensure correct deployment of developmental programs by epigenetically repressing target gene expression. Despite the importance, few studies have been focused on the regulation of PcG activity itself. This study reports a Drosophila gene, stuxnet (stx), that controls Pc protein stability. Heightened stx activity leads to homeotic transformation, reduced Pc activity, and de-repression of PcG targets. Conversely, stx mutants, which can be rescued by decreased Pc expression, display developmental defects resembling hyperactivation of Pc. Biochemical analyses provide a mechanistic basis for the interaction between stx and Pc; stx facilitates Pc degradation in the proteasome, independent of ubiquitin modification. Furthermore, this mode of regulation is conserved in vertebrates. Mouse stx promotes degradation of Cbx4, an orthologous Pc protein, in vertebrate cells and induces homeotic transformation in Drosophila. These results highlight an evolutionarily conserved mechanism of regulated protein degradation on PcG homeostasis and epigenetic activity (Du, 2016).

Polycomb-group (PcG) genes were first identified in Drosophila for their roles in maintaining correct expression patterns of homeotic genes. PcG-mediated transcription silencing was later proved to be a well-conserved regulatory mechanism throughout metazoans. Classical PcG targets, such as Hox genes, play important roles in biological processes ranging from stem cell maintenance to genomic imprinting. Recent genome-wide studies unveiled additional PcG targets, many of which encode transcription factors and cell-signaling proteins that regulate a diverse array of downstream effectors. Thus, PcG may act in a much broader spectrum of cellular processes than previously anticipated (Du, 2016).

PcG silencing depends primarily on the activities of two Polycomb repressive complexes (PRC). In Drosophila, PRC1 is composed of Pc (Polycomb), Ph (Polyhomeotic), Psc (Posterior sex combs), and Sce (Sex combs extra). The main subunits of the PRC2 include Esc (Extra sex combs), E(z) (Enhancer of zeste), Su(z)12 (Suppressor of zeste 12) and Caf1 (Chromatin assembly factor 1). Relying on the presence of a conserved enzymatic SET domain in E(z), PRC2 catalyzes tri-methylation of histone H3 at Lys 27 (H3K27me3). Pc then employs its chromo domain to recognize H3K27me3 mark, resulting in recruitment of PRC1 to PcG targets. Mechanisms utilized by PRC1 to silence target genes include histone H2A mono-ubiquitination, chromatin compaction, and direct interaction with the general transcription machinery (Du, 2016).

While intensive studies have been focused on uncovering mechanisms by which PcG proteins epigenetically repress target gene expression, few are devoted to define how the PcG activities are regulated. Nevertheless, several transcription factors and microRNAs are known to directly modulate PcG expression. Feedback regulatory loops may also be important to maintain proper expression of PcG, which themselves are subject to epigenetic repression. Furthermore, post-translational modifications on several PcG proteins have been reported, and the importance of such modifications has only been revealed recently. For example, SUMOylation is shown to modulate PcG activity by affecting chromatin targeting of the Pc protein, and O-GlcNAcylation has been demonstrated to prevent aggregation of PRC1 subunit Ph in Drosophila (Du, 2016).

This report describe that a Drosophila gene CG32676, which was named stuxnet (stx), functions through ubiquitin-independent degradation (UID) to control Pc protein stability and thereby PcG-mediated epigenetic repression. This study shows further that vertebrate Stx regulates orthologous Pc protein in the same fashion. Together, these results highlight a conserved regulatory mechanism for Pc, the founding member of the PcG family of proteins (Du, 2016).

Taking advantage of genetic tools available in Drosophila, the function of a UBL-domain-containing protein, Stx, was examined, and its unexpected role of regulated Pc protein degradation in epigenetic repression. These analyses on classical PcG targets demonstrate that Stx functions as a Pc-specific regulator that negatively modulates the PcG activity. Importantly, this mode of regulation was found to be conserved from flies to vertebrates (Du, 2016).

stx activity is essential for Drosophila development. The fact that pupal lethal phenotype associated with loss-of-function stx mutations can be rescued by removing 50% of Pc activity strongly supports that modulating the Pc expression is the major developmental process regulated by stx. Stx might not be a constitutive component of the canonical PRC1. However, the ability of Stx to reduce Pc recruitment to target gene loci argues that Stx may act as a gatekeeper for control of Pc availability to form highly dynamic PRC complexes on target chromatin. As stx activity is necessary for PcG target expression, Stx could function in an intrinsic machinery to regulate Pc protein homeostasis. Stx directly binds Pc through a serine-rich PcB domain and interacts with the proteasome through the UBL domain. As Pc protein degradation does not rely on ubiquitination, the UBL domain in Stx, upon interaction with Pc, could serve as a recognition signal that marks Pc protein for degradation in the proteasome. Thus, a model is proposed in which Stx acts first as an adapter and then a chaperone-like protein to facilitate proteasomal degradation of Pc, resulting in altered PcG activity in animal development. Intriguingly, upon inspection of modENCODE database, multiple binding sites were found for PcG components, including Pc, Psc, Sce, and Pho, and Ubx, which is itself a PcG target, thus pointing to the existence of a potential feedback loop between Stx and PcG activity (Du, 2016).

Altered Pc protein abundance has been noted in several biological processes. In the Sce mutant fly embryos, the bulk level of Pc protein is significantly reduced, but Ph and Psc are not affected. Similar results have been reported in mouse ES cells for RING1B and Cbx4, mammalian orthologs of Sce and Pc. However, the significance of such regulation was not understood. It is suspected that binding with Sce might stabilize Pc, which is crucial for PRC1 assembly. It is interesting to note that the level of Pc changes rapidly in the cell cycle. The oscillation of Pc protein during the cell cycle is thought to be important for establishment and maintenance of cellular epigenetic memory. The observation of the reciprocal expression pattern of Pc and Stx as well as the ability of Stx to control Pc abundance in cell cycle are in favor of a notion that regulated Pc protein stability may be one way to dynamically control Pc activity in physiological contexts. How Stx participates in such regulation is an interesting question that awaits further exploration (Du, 2016).

The PRC1 is composed of four core subunits, each of which has unique molecular activities non-exchangeable among each other. However, the loss-of-function phenotypes of individual PRC1 subunits in Drosophila only partially overlap, revealing the complexity of PRC1 regulation in various cellular processes. The differential requirement of PRC1 subunits in development might be due to the presence of distinct PRC complexes in a temporal and tissue-specific manner. This view is further complicated in vertebrates by partially redundant orthologous PRC1 proteins and the formation of multiple non-canonical complexes. Thus, it will be necessary to explore the regulatory machineries utilized by individual PRC1 components to better understand how PRC complexes exert versatile functions in vivo. This study has shown that Stx targets Pc for proteasomal degradation, but whether parallel regulators exist for other PRC1 components is still unknown (Du, 2016).

This study of Stx regulation on Pc stability reveals that the activity of Pc protein, the founding member of the PRC complexes, can be controlled through regulated protein degradation. Surprisingly, it was found that fly Pc protein is largely regulated by UID. The list of substrates that undergo UID has expanded rapidly in recent years. Intriguingly, many UID substrates are localized to the nucleus, including transcription factors and chromatin remodeling factors. The addition of Pc, a key epigenetic regulator, to this list leads to the belief that UID in the nucleus may participate in the control of gene expression (Du, 2016).

Consistent with a role of Stx on Pc stability in Drosophila development, proteasomal degradation has been reported to affect the stability of several PcG components in cultured vertebrate cells, including three PRC1 proteins BMI1, RING1B, and PHC, and one PRC2 protein EZH2. It is thus highly likely that protein degradation may play a general role in regulating PcG activity (Du, 2016).

Appropriate PcG activity is essential for stem cell maintenance and lineage specification in vertebrates. Altered PcG activity is associated with malignant human diseases, including cancer. Furthermore, dysregulated stx expression and Stx mutations are reported in several forms of cancer in the COSMIC database. Consistently, genes co-expressed with stx shown in COXPRESdb are clustered into pathways in cancer as well as Notch and MAPK signaling pathways. Very recently, Stx mutations were found in patients with autism spectrum disorders (ASD) by whole-exome sequencing. Given the strong connection between PcG and ASD, Stx may play a role in ASD through its regulation of PcG activity. Thus, the identification of regulators of PcG activity, such as Stx, may provide additional therapeutic targets for relevant diseases (Du, 2016).

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. This study identified an epigenetic regulatory cascade (Stuxnet-Polycomb-Octβ2R) that modulates the octopamine receptor in Drosophila. stuxnet positively regulates Octβ2R through repression of Polycomb in the ellipsoid body of the adult fly brain and Octβ2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octβ2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octβ2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octβ2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis (Zhao, 2021).

Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep. Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118 (Zhao, 2021).

In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep. The dorsal FSB also integrates some sleep inhibiting signals. Both dorsal FSB and EB ring 2 are important in sleep homeostasis. Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2, indicating that these EB and FSB are connected (Zhao, 2021).

Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation. However, a direct link between epigenetic regulation and sleep homeostasis is not yet established (Zhao, 2021).

Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep. Activation of OA signaling inhibits sleep homeostasis, while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed. Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neuronsproject to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling. However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons. Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant, arguing that another receptor or circuit may participate in this process (Zhao, 2021).

Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R. Although the expression pattern of OA is identified, the endogenous expression profile of these receptors is lacking. A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA. Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation. How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown (Zhao, 2021).

A previous study showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation, the role of stx in adult physiological process is unknown (Zhao, 2021).

This study identified the role of the epigenetic regulator stx in sleep regulation. stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, the role of various Octβ receptors was systematically identified in sleep regulation. Octβ2R was found to be one of the receptors that mediates OA function in sleep homeostasis. More interestingly, it was found that stx was OA-responsive depending on the Octβ1R. Based on these data, it is proposed that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount (Zhao, 2021).

This study highlights the importance of epigenetic regulation on sleep. Although epigenetic regulation was intensively studied in adult pathological processes such as cancer, epigenetic factors have been far less studied in other physiological processes such as sleep. This study provides an example of the maintenance role of PcG complex in sleep regulation. Although the core PcG complex component Pc is ubiquitously expressed, its regulator stx is tissue specifically distributed, and this distribution may keep appropriate activity of Pc as well as the PcG complex in a tissue-specific manner. The factors regulating the tissue specificity of stx expression need to be further investigated (Zhao, 2021).

A previous study found that mutation of Octβ2R does not have an obvious sleep phenotype. The current data were compared with the published Octβ2R^f05679 mutant data. Although Octβ2R^f05679 mutant was shown not significantly affected total sleep, this study found that the Octβ2R^f05679 has mild effect on sleep. Other Octβ2R mutants were tested, and it was found that the male flies from these mutations indeed have sleep phenotype (Zhao, 2021).

Published studies have shown that the sleep phenotype of octopamine pathway mutants is different between video-based method and DAM-based method. For example, based on DAM data, the TβH mutant resulted in increased sleep per day, while the same mutant showed decreased sleep based on video data. This study used the video-based method to repeat the TβH mutant phenotype. The results showed that compared with the control flies, the TβH mutant got significantly less sleep. This result is consistent with the previously published data. Through close observation of TβH mutant and control flies, this study found that this mutant has much more frequent grooming behavior than the controls. The TβH mutant and control flies were video recorded for 10 min between ZT3.5 and ZT4.5. The results showed a statistically significant increase of the total number of grooming case. The difference between video-based method and DAM-based method is that these grooming behaviors can be detected in video-based methods, but not in DAM-based methods. Multiple studies have established a positive correlation between octopamine treatment and grooming behavior. Theoretically, TβH allele should result in a decrease in octopamine synthesis. The opposite phenotype may be caused by increased tyramine in TβH mutant or by other feedback regulation. The alleles for Octβ2R receptor used in this study show a similar grooming behavior as the control flies. The previously published octβ2R knockout allele should be a stronger one. The difference of sleep phenotypes between video-based and DAM-based methods may be due to the grooming behavior induced by the massive decrease of octopamine detection. Or other unrelated effects caused by the compensation effect previously reported. One hypothesis is that the significant change of grooming behavior probably masks the sleep behavior. The relationship between grooming and sleep needs to be further clarified. The detection of the sleep phenotype without significant changes in grooming phenotype may be a better strategy to get reliable sleep phenotype. If the increase of grooming in TβH mutant is a side effect caused by the increased tyramine, the identification of the phenotypes of octopamine treatment or collective phenotype of octopamine receptors may be more reliable ways to draw conclusions on the function of octopamine. Furthermore, whether grooming is epistatic to sleep is a problem worthy of further study (Zhao, 2021).

Two aspects of sleep homeostasis need to be further studied. First, this study found that Octβ2R and stx colocalize in a subset of EB neurons. In a previously study, EB R2 neurons were found to be responsible for sleep homeostasis regulation. The relationship of these two groups of EB neurons needs further study. Second, the OA-treated Octβ2R mutant has more sleep recovery than the control. This indicates that OA induces more sleep recovery in the condition of Octβ2R downregulation. It seems that in this condition OA induces certain pathways to counteract its role in sleep homeostasis. One possibility is that Octβ2R negatively regulates Octβ3R which results in increased sleep pressure in the absence of Octβ2R. Further studies are needed to clarify the mechanism (Zhao, 2021).

The results suggest the stx-Pc-Octβ2R regulatory cascade serves as a buffering step for OA function in sleep homeostasis. Two-way regulation of OA on stx leads to reverse changes of stx-the more OA, the less stx and vice versa. Through the function of stx-Pc-Octβ2R regulatory cascade, the Octβ2R transcription is changed accordingly. Variation of Octβ2R transcription could buffer the OA response. As a result, the unfavorable effect of OA causing dramatic decrease of sleep amount and homeostasis could be compensated by its receptor (Zhao, 2021).

Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, localization of midnolin in nucleoli was not confirmed. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition it was shown that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of this study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells (Hofmeister-Brix, 2013).

Midnolin is a novel regulator of parkin expression and is associated with Parkinson's Disease

Midnolin (MIDN) was first discovered in embryonic stem cells, but its physiological and pathological roles are, to date, poorly understood. This study examined the role of MIDN in detail. In PC12 cells, a model of neuronal cells, MIDN localized primarily to the nucleus and intracellular membranes. Nerve growth factor promoted MIDN gene expression, which was attenuated by specific inhibitors of extracellular signal-regulated kinases 1/2 and 5. MIDN-deficient PC12 cells created using CRISPR/Cas9 technology displayed significantly impaired neurite outgrowth. Interestingly, a genetic approach revealed that 10.5% of patients with sporadic Parkinson's disease (PD) had a lower MIDN gene copy number whereas no copy number variation was observed in healthy people, suggesting that MIDN is involved in PD pathogenesis. Furthermore, the expression of parkin, a major causative gene in PD, was significantly reduced by CRISPR/Cas9 knockout and siRNA knockdown of MIDN. Activating transcription factor 4 (ATF4) was also down-regulated, which binds to the cAMP response element (CRE) in the parkin core promoter region. The activity of CRE was reduced following MIDN loss. Overall, these data suggests that MIDN promotes the expression of parkin E3 ubiquitin ligase, and that MIDN loss can trigger PD-related pathogenic mechanisms (Obara, 2017).

Search PubMed for articles about Drosophila Stuxnet

Du, J., Zhang, J., He, T., Li, Y., Su, Y., Tie, F., Liu, M., Harte, P. J. and Zhu, A. J. (2016). Stuxnet facilitates the degradation of polycomb protein during development. Dev Cell 37(6): 507-519. PubMed ID: 27326929

Hofmeister-Brix, A., Kollmann, K., Langer, S., Schultz, J., Lenzen, S. and Baltrusch, S. (2013). Identification of the ubiquitin-like domain of midnolin as a new glucokinase interaction partner. J Biol Chem 288(50): 35824-35839. PubMed ID: 24187134

Obara, Y., Imai, T., Sato, H., Takeda, Y., Kato, T. and Ishii, K. (2017). Midnolin is a novel regulator of parkin expression and is associated with Parkinson's Disease. Sci Rep 7(1): 5885. PubMed ID: 28724963

Zhao, Z., Zhao, X., He, T., Wu, X., Lv, P., Zhu, A. J. and Du, J. (2021). Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade. EMBO Rep: e47910. PubMed ID: 33410264

date revised: 15 December 2023

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