InteractiveFly: GeneBrief

Ubiquitin-specific protease 7: Biological Overview | References

Meiosis is a form of cell division that is essential to sexually reproducing organisms and is therefore highly regulated. Each event of meiosis must occur at the correct developmental stage to ensure that chromosomes are segregated properly during both meiotic divisions. One unique meiosis-specific structure that is tightly regulated in terms of timing of assembly and disassembly is the complex (SC). While the mechanism(s) for assembly and disassembly of the SC are poorly understood in Drosophila melanogaster, posttranslational modifications, including ubiquitination and phosphorylation, are known to play a role. This study identified a role for the deubiquitinase Ubiquitin-specific protease 7 Usp7 in the maintenance of the SC in early prophase and shows that its function in SC maintenance is independent of the meiotic recombination process. Using two usp7 shRNA constructs that result in different knockdown levels, this study has shown that the presence of SC through early/mid-pachytene is critical for normal levels and placement of crossovers (Lake, 2024).

The meiotic cell cycle is highly regulated from its entry to its exit through the regulation of cyclin-dependent kinases. In addition, specific events throughout meiosis are tightly regulated in both time and space. For example, the SC, a key meiosis-specific structure, which is required for meiotic processes including DSB formation, meiotic recombination, and chromosome segregation [all of which are also regulated posttranslationally, is regulated both in its formation and disassembly. This study uncovered a layer of regulation of the SC in Drosophila by the deubiquitinase Usp7. Using RNAi knockdown usp7 was shown to be required for SC maintenance, a function that is independent of the meiotic recombination process, and likely affecting the SC structure directly. These studies allowed a study of the importance of the SC at specific time points during meiotic prophase and provided strong evidence that maintaining the SC through early/mid-pachytene was essential for the normal level and placement of crossovers (Lake, 2024).

While small posttranslational protein modifiers like ubiquitination and sumoylation are known to be important for SC formation and meiotic recombination, the process of deubiquitination has not been extensively studied as a potential regulator of meiotic prophase. In general, Usp7 is one of the most well-studied deubiquitinases due to its diverse cellular functions and its role in regulating cellular levels of p53, a tumor suppressor protein. As a cysteine peptidase, Usp7 plays roles in transcriptional regulation, cell cycle progression, DNA damage response, DNA replication, and epigenetic regulation. Because of Usp7's p53-dependent role in tumor suppression, as well as its p53-independent role in triple-negative breast cancers, substantial research has focused on finding inhibitors of Usp7 for therapeutic use (Lake, 2024).

Several DUBs have been shown to play a role in spermatogenesis, including Usp7. In mice pachytene spermatocytes, Usp7 localizes to the XY body in a SCML2 (testes-specific polycomb protein)-specific manner which likely aids in mediating transcriptional silencing. In the absence of SCML2, Usp7 fails to load at the XY body, which indirectly leads to an upregulation of histone H2A monoubiquitination and subsequent apoptosis. This action of Usp7 is thought to directly occur through the regulation of RNF2, an E3 ubiquitin ligase (Lake, 2024).

Given the diverse roles and overall importance of Usp7, studying its function during specific cellular events can be difficult. Although usp7 is an essential gene, in Drosophila, its essential roles in growth and development were circumvented by specifically downregulating its presence using shRNA constructs driven in a tissue specific manner. Using the Gal4-UAS system to knockdown expression in the female and male germlines (nanos-Gal4), it was possible to show that when there was a greater than 50% reduction in usp7 germline expression (usp7 RNAiII), there was a significant reduction in female fertility. This same construct when assayed for male fertility led to virtual sterility, indicating a role for Usp7 in spermatogenesis. From our data, it is concluded that usp7 likely has a diverse set of functions in both the female and male germlines (Lake, 2024).

The SC maintenance phenotype observed in usp7 RNAi oocytes is separable from the effects on fertility in both sexes. Drosophila males do not form SC, nor recombine. Thus, the defects observed in males cannot be due to effects on SC assembly or structure. In addition, in delaying the knockdown of usp7 from starting in the germarium (where the SC and crossovers are established) to beginning in the vitellarium (matα-Gal4), showed that the decrease in fertility is due to a postpachytene role of usp7 in the vitellarium stages of oogenesis or in early embryos. Further studies will be required to identify whether Usp7 is involved in transcriptional silencing or apoptosis in the Drosophila male germline or in the vitellarium in the female germline (Lake, 2024).

Using immunofluorescence analyses, this study showed that Usp7 is required for the maintenance of the SC during prophase I. Although these studies cannot discern the precise knockdown level of usp7 at each stage of pachytene in the germarium, the overall differences between the two shRNA constructs were shown to correlate with the differences in the timing of the SC defect observed. Regardless of whether the overall decrease in germline expression was >50% (in usp7 RNAiII) or ~10% (in usp7 RNAiIII), the following observations were the same: 1) tripartite SC structure is able to form; 2) the SC progressively falls apart as the pro-oocytes proceeds though pachytene in the germarium; 3) the resulting SC phenotype in the oocyte by mid-pachytene was indistinguishable; 4) homologous centromeres are paired; and 5) DSB induction was normal. This study also showed that the meiotic recombination process was not required to observe the SC phenotype found when usp7 was reduced. However, knockdown of usp7 starting in the germarium (nanos-Gal4) was required, as there was no SC maintenance phenotype when knockdown began in the vitellarium (matα-Gal4) (Lake, 2024).

Because the initiation of the meiotic recombination process was not altered nor required for the usp7 RNAi phenotype, and centromere pairing remained intact even in the stronger of the two shRNA lines, the failure to maintain the SC structure was not a result of gross effects on cohesion. For that reason, whether Usp7 might directly be affecting an SC component required for the formation and/or maintenance of the SC was investigated by analyzing whether reduced usp7 expression altered the ability to form large SC PCs in a sina mutant, which could point to a more direct role in SC formation and/or maintenance. When usp7 expression is reduced, sina induced PC form and appear organized, but they are not able to form the elaborate structures normally found in this genetic background, like cones and complex shapes. While the substate for either the ubiquitin ligase Sina or the deubiquitinase Usp7 regarding their roles in SC formation and maintenance is not known, an intriguing possibility is that they act upon the same substrate(s) to modulate their activity. Future studies will need to be done to address this possibility (Lake, 2024).

Taken together with the finding that normally structured SC forms prior to it coming apart in most usp7 RNAi germaria, it is speculated that Usp7 may be required to stabilize, through the action of deubiquitination, a component of the SC or its connection to the lateral element. The possibility cannot be excluded that the effect could be along the lateral elements of the SC, as alterations in sina PC structure has also been observed in a c(2)M mutant, albeit in length not width. The idea the Usp7 is stabilizing a component of the SC is further strengthened by the finding that 83% of the sina induced PC in usp7 RNAi oocytes were associated with one or more CID foci (39/47 PC analyzed), indicating that the SC at the centromeres might be less affected than the SC along the euchromatic arms. Which component is the direct target of Usp7 remains to be determined, as many of the known components of the SC and lateral elements in Drosophila melanogaster, [C(3)G], Cona, Corolla, and C(2)M, all contain multiple motifs for Usp7 docking based on eukaryotic linear motif (ELM) prediction. Nonetheless, these studies show that when Usp7 is reduced, the SC structure either between homologs or in aberrant PC structures is not able to form and/or maintain long extensions of SC (Lake, 2024).

Although the reduction of usp7 expression by usp7 RNAiII reduced fecundity, fertility was not so affected that it prohibited the analysis of offspring in some genotypes. Through analysis of the genetic outcomes, this study found that usp7 RNAiII females show high levels of ND and reduced recombination and altered crossover placement on the X chromosome. These studies analyzing the rates of ND when females have achiasmate X chromosomes, in addition to the inability of usp7 RNAiII oocytes to biorient their homologs properly at metaphase I, indicate a global reduction in the ability to form crossovers. Supporting this finding is the result that offspring from usp7 RNAiII females resulted in meiosis II exceptions, which speculate are from preceding meiosis I errors. These combined studies suggest that the failure to maintain the SC throughout the time DSBs are initiated (early pachytene) is important for both recombination processes and proper disjunction of homologs. On the other hand, usp7 RNAiIII females showed only a modest increase in ND, reduced recombination but normal distribution of crossovers on the X chromosome, and normal levels of crossovers that are shifted toward the centromeres on one of the autosomes, the 2nd chromosome. These females were also able to biorient all their homologs, which suggests that recombination rates on the autosomes are likely also not significantly affected. These results show that the ability to maintain the SC throughout early/mid-pachytene is critical for normal levels of crossovers, specifically on the X chromosome, and placement of crossovers on the autosomes (Lake, 2024).

Previous studies indicate temporal events that occur between early, early/mid, and mid-pachytene could explain the differences observed in the requirements of the SC at these stages. For instance, DSB induction occurs immediately after SC formation in flies, at early pachytene, and the largest number of DSBs are visualized at this stage when using an antibody to the phosphorylation event that occurs immediately following DSB formation (γH2Av). However, DSB formation and repair is very dynamic, and even in wild-type conditions only a subset of the DSBs that form are ever visualized. This indicates that DSB repair is occurring at the same time DSBs are being formed. By the time the pro-oocytes reach early/mid-pachytene, the number of DSBs has been reduced to the number of crossover events, as they colocalize with the recombination nodule components Vilya and Narya. it would be predicted that a failure of the SC to be maintained at this stage would have significant global effects on crossover and noncrossover formation. Unfortunately, to date, the only proteins in flies that have been physically visualized at sites of recombination are themselves also found at DSB sites in both the future oocyte and pro-oocytes that are disassembling their SC as they back out of meiosis. Therefore, it is not certain that it is possible to distinguish sites for recombination from DSBs that have failed to undergo repair when the SC is fragmented, as it is in usp7 RNAi females (Lake, 2024).

A recent study also highlights the importance of maintaining the SC throughout pachytene in Drosophila and shows that disruptions of the SC at critical time points affect the X chromosome differently than the autosomes. Using small deletions in the major transverse filament protein, C(3)G, that differ in when they affect SC structure, it was shown that there are critical times during which you need to maintain the SC. Using these mutants, a timeline was generated for when the SC is necessary to maintain pairing and support the recombination landscape on each of the chromosomes. These mutants suggest that the X chromosome requires the SC earlier than the autosome for pairing and crossing over and failure to maintain the SC in early/mid-pachytene alters only the recombination landscape on the autosomes. This study is consistent with this timeline of full-length SC requirements. However, what has yet to be determined is whether any of these proteins, the C(3)G mutants or Usp7, directly affect the shift in crossovers toward the centromere on the autosomes. The hypothesis is favored that mutations that affect both the level and placement of X chromosome crossovers and only alter the distribution of crossovers on the autosomes may reflect an interchromosomal effect, rather than directly altering the position of the crossovers toward the centromere (Lake, 2024).

Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy

Ubiquitination-mediated protein degradation is the selective degradation of diverse forms of damaged proteins that are tagged with ubiquitin, while deubiquitinating enzymes reverse ubiquitination-mediated protein degradation by removing the ubiquitin chain from the target protein. The interactions of ubiquitinating and deubiquitinating enzymes are required to maintain protein homeostasis. The ubiquitin-specific protease USP7 is a deubiquitinating enzyme that indirectly plays a role in repairing DNA damage and development. However, the mechanism of its participation in aging has not been fully explored. Regarding this issue, USP7 was found to be necessary to maintain the normal lifespan of Drosophila melanogaster, and knockdown of dusp7 shortened the lifespan and reduced the ability of Drosophila to cope with starvation, oxidative stress and heat stress. Furthermore, the ability of USP7 to regulate aging depends on the autophagy and ubiquitin signaling pathways. Furthermore, 2,5-dimethyl-celecoxib (DMC), a derivative of celecoxib, can partially restore the shortened lifespan and aberrant phenotypes caused by dusp7 knockdown. These results suggest that USP7 is an important factor involved in the regulation of aging, and related components in this regulatory pathway may become new targets for anti-aging treatments (Cui, 2020).

Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie

The Hippo pathway plays an important role in organ development and adult tissue homeostasis, and its deregulation has been implicated in many cancers. The Hippo signaling relies on a core kinase cascade culminating in phosphorylation of the transcription coactivator Yorkie (Yki). Although Yki is the key effector of Hippo pathway, the regulation of its protein stability is still unclear. This study shows that Hippo pathway attenuates the binding of a ubiquitin-specific protease Usp7 to Yki, which regulates Hippo signaling through deubiquitinating Yki. Furthermore, the mammalian homolog of Usp7, HAUSP plays a conserved role in regulating Hippo pathway by modulating Yap ubiquitination and degradation. Finally, we find that the expression of HAUSP is positively correlated with that of Yap, both showing upregulated levels in clinical hepatocellular carcinoma (HCC) specimens. In summary, our findings demonstrate that Yki/Yap is stabilized by Usp7/HAUSP, and provide HAUSP as a potential therapeutic target for HCC (Sun, 2019).

Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling

Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. This study shows that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-Cul3 E3 ligases. Furthermore, Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, it was shown that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. These findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers (Zhao, 2015).

GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7

The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. This study shows that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). USP7 was purified from embryo nuclear extracts as a stable heteromeric complex with 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation (van der Knaap, 2005).

Functions of Usp7 orthologs in other species]

Evolutionarily conserved regulators of tau identify targets for new therapies

Tauopathies are neurodegenerative diseases that involve the pathological accumulation of tau proteins; in this family are Alzheimer disease, corticobasal degeneration, and chronic traumatic encephalopathy, among others. Hypothesizing that reducing this accumulation could mitigate pathogenesis, a cross-species genetic screen was performed targeting 6,600 potentially druggable genes in human cells and Drosophila. 83 hits were found and validated in cells and 11 hits were further validated in the mouse brain. Three of these hits (USP7, RNF130, and RNF149) converge on the C terminus of Hsc70-interacting protein (CHIP) to regulate tau levels, highlighting the role of CHIP in maintaining Tau proteostasis in the brain. Knockdown of each of these three genes in adult tauopathy mice reduced tau levels and rescued the disease phenotypes. This study thus identifies several points of intervention to reduce tau levels and demonstrates that reduction of tau levels via regulation of this pathway is a viable therapeutic strategy for Alzheimer disease and other tauopathies (Kim, 2022).

USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway

An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). This study reports the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases (Zhang, 2020).

Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization

Nucleotide biosynthesis is fundamental to normal cell proliferation as well as to oncogenesis. Tumor suppressor p53 (see Drosophila p53), which prevents aberrant cell proliferation, is destabilized through ubiquitylation by MDM2. Ubiquitin-specific protease 7 (USP7) plays a dualistic role in p53 regulation and has been proposed to deubiquitylate either p53 or MDM2. This study shows that guanosine 5'-monophosphate synthase (GMPS) is required for USP7-mediated stabilization of p53. Normally, most GMPS is sequestered in the cytoplasm, separated from nuclear USP7 and p53. In response to genotoxic stress or nucleotide deprivation, GMPS becomes nuclear and facilitates p53 stabilization by promoting its transfer from MDM2 to a GMPS-USP7 deubiquitylation complex. Intriguingly, cytoplasmic sequestration of GMPS requires ubiquitylation by TRIM21, a ubiquitin ligase associated with autoimmune disease. These results implicate a classic nucleotide biosynthetic enzyme and a ubiquitin ligase, better known for its role in autoimmune disease, in p53 control (Reddy, 2014).

Search PubMed for articles about Drosophila

Cui, L., Song, W., Zeng, Y., Wu, Q., Fan, Z., Huang, T., Zeng, B., Zhang, M., Ni, Q., Li, Y., Wang, T., Li, D., Mao, X., Lian, T., Yang, D., Yang, M., Fan, X. (2020). Deubiquitinase USP7 regulates Drosophila aging through ubiquitination and autophagy. Aging (Albany NY), 12(22):23082-23095 PubMed ID: 33221768

Kim, J., de Haro, M., Al-Ramahi, I., Garaicoechea, L. L., Jeong, H. H., Sonn, J. Y., Tadros, B., Liu, Z., Botas, J. and Zoghbi, H. Y. (2022). Evolutionarily conserved regulators of tau identify targets for new therapies. Neuron. PubMed ID: 36610398

Lake, C. M., Gardner, J., Briggs, S., Yu, Z., McKown, G., Hawley, R. S. (2024). The deubiquitinase Usp7 in Drosophila melanogaster is required for synaptonemal complex maintenance. Proc Natl Acad Sci U S A, 121(36):e2409346121 PubMed ID: 39190345

Reddy, B. A., van der Knaap, J. A., Bot, A. G., Mohd-Sarip, A., Dekkers, D. H., Timmermans, M. A., Martens, J. W., Demmers, J. A. and Verrijzer, C. P. (2014). Nucleotide biosynthetic enzyme GMP synthase is a TRIM21-controlled relay of p53 stabilization. Mol Cell 53: 458-470. PubMed ID: 24462112

Sun, X., Ding, Y., Zhan, M., Li, Y., Gao, D., Wang, G., Gao, Y., Li, Y., Wu, S., Lu, L., Liu, Q., Zhou, Z. (2019). Usp7 regulates Hippo pathway through deubiquitinating the transcriptional coactivator Yorkie. Nat Commun, 10(1):411 PubMed ID: 30679505

van der Knaap, J.A., Kumar, B. R. P., Moshkin, Y. M., Langenberg, K., Krijgsveld, J., Heck, A. J. R., Karch, F. and Verrijzer, C. P. (2005). GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol. Cell 17: 695-707. 15749019

Zhang, T., Periz, G., Lu, Y. N. and Wang, J. (2020). USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway. Proc Natl Acad Sci U S A 117(45): 28114-28125. PubMed ID: 33106424

Zhou, Z., Yao, X., Li, S., Xiong, Y., Dong, X., Zhao, Y., Jiang, J. and Zhang, Q. (2015). Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling. Dev Cell 34: 58-72. PubMed ID: 26120032

date revised: 6 April 2026

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