The ubiquitin-proteasome system in circadian regulation

Monoubiquitination, the attachment of a single ubiquitin moiety to a protein, typically regulates protein function, localization, and activity rather than signaling degradation (Lange et al., 2022; Liao et al., 2024). Core histones are key targets, with H2A monoubiquitination essential for neurodevelopment and linked to related disorders (Srivastava et al., 2017; Scheuermann et al., 2012). Similarly, H2B monoubiquitination alters chromatin structure, increasing accessibility for transcription and DNA repair, thereby promoting gene activation (Minsky et al., 2008; Fierz et al., 2011; Espinosa, 2008).

In circadian regulation, histone monoubiquitination has emerged as a dynamic and reversible mechanism that plays a key role in the rhythmic control of gene expression. Specifically, oscillations in H2B monoubiquitination have been associated with the transcriptional cycling of circadian genes, such as PER1 and PER2, indicating that chromatin remodeling through histone ubiquitination is closely aligned with the molecular clock (Tamayo et al., 2015). These modifications may act downstream of core circadian regulators or be orchestrated by ubiquitin ligases and deubiquitinating enzymes that themselves may be under circadian control.

Polyubiquitination is central to circadian regulation by controlling how quickly clock proteins accumulate or are degraded, thus influencing the pace and progression of the circadian cycle. K48-linked polyubiquitination, a signal for proteasomal degradation, ensures timely turnover of core clock components. For example, in Drosophila, the E3 ligase Cul3 promotes K48-linked ubiquitination of tim at the day–night transition, facilitating its degradation and phase progression (Szabó et al., 2018). In mammals, HRD1 mediates K48-linked degradation of BMAL1, regulating transcriptional activity of downstream circadian genes (Guo et al., 2020). These rhythms in protein turnover ensure the timely clearance of clock proteins, stabilizing period length and phase transitions.

Other polyubiquitin linkages, like K29 and K11, may modulate clock function without directly inducing degradation or indirectly via influencing pathways involved. For example, K63-linked chains affect protein interactions and chromatin recruitment (Chen et al., 2018a,Wang et al., 2017), while K11 chains may fine-tune timing and subcellular localization of clock protein degradation (Hirano et al., 2013). Together, these modifications form a dynamic ubiquitin code that regulates circadian timing and sleep-related processes by controlling the tempo of the feedback loop.

E3 ubiquitin ligases, though structurally built on a limited set of catalytic domains, achieve functional diversity through specialized substrate recognition and regulatory elements. This modularity enables them to selectively regulate a wide array of proteins in response to cellular signals (Zheng and Shabek, 2017). With over 600 E3 ligases in humans–compared to only 2 E1s and 38 E2s–they represent the most specific step in the ubiquitin-proteasome system, playing essential roles in processes such as protein degradation, intracellular signaling, and transcriptional regulation (Zhao and Sun, 2013; Leestemaker and Ovaa, 2017; Bachiller et al., 2020). Recent evidence highlights their involvement in circadian regulation, where they modulate the stability, localization, and activity of core clock proteins (Table 1; Abdalla et al., 2022; Yang et al., 2012; Yang et al., 2014). The next several sections summarize the current status of knowledge in the field regarding E3 ligases and their roles in circadian regulation. The ligases are listed alphabetically by gene name.

ARF-BP1, also known as HUWE1 in Drosophila, and PAM, also known as highwire in Drosophila, are critical E3 ubiquitin ligases that regulate the stability of REV-ERBα, a component of the circadian clock mentioned above that is responsible for repressing genes like BMAL1 (Yin et al., 2010). These E3 ligases associate with REV-ERBα and mediate its ubiquitination, targeting it for proteasomal degradation following circadian cues such as lithium treatment or serum shock, both of which synchronize circadian oscillations in mammalian cells (Stojkovic et al., 2014; Yin et al., 2010). Knockdown of ARF-BP1 or PAM in mouse hepatoma cells stabilizes REV-ERBα protein preventing its timely degradation, which results in sustained repression of BMAL1 and disruption of oscillations in multiple other clock genes (Yin et al., 2010).

FBXL3, beyond targeting CRY proteins, also indirectly influences REV-ERBα-mediated repression of BMAL1 and CRY1, as elevated REV-ERBα levels in FBXL3-mutant mice enhance transcriptional repression (Shi et al., 2013; Stojkovic et al., 2014). Deletion of REV-ERBA in the FBXL3-mutant mice rescues the mutant circadian phenotype, highlighting FBXL3's broader role in coordinating core clock transcription factors (Shi et al., 2013). Together, these findings demonstrate that ARF-BP1, PAM and FBXL3 are critical E3 ligases that regulate REV-ERBα degradation to maintain the stability and timing of circadian clock gene expression (Shi et al., 2013; Stojkovic et al., 2014; Yoo et al., 2013).

BRWD3 (Bromodomain and WD Repeat Domain Containing 3), also known as ramshackle, acts as a substrate receptor for the Cul4-RING E3 ubiquitin ligase complexes and mediates light-induced degradation of cry (Jackson and Xiong, 2009; Ozturk et al., 2013). Identified through a yeast two-hybrid assay, BRWD3 knockdown strongly attenuated light-dependent cry degradation in S2 cells (Ozturk et al., 2013). In vivo assays confirmed that BRWD3 binds cry in a light-dependent manner and, as a part of the BRWD3-Cullin4-RING Finger E3 Ligase (CRL4) complex, catalyzes cry ubiquitination and subsequent proteasomal degradation (Ozturk et al., 2013). BRWD3 also co-precipitated with other CRL4 E3 ligase components such as Damage-specific DNA binding protein 1 (DDB1), Cul4, and Regulator of cullins 1a (Roc1). These findings establish BRDW3 as a light-dependent cry receptor (Ozturk et al., 2013). Together, BRWD3 and jet appear to act cooperatively during photic resetting, where light exposure promotes the formation of a complex including tim, cry, jet, and BRWD3, enabling coordinated degradation of both tim and cry (Ozturk et al., 2013; Abdalla et al., 2022).

circadian trip (ctrip) is a HECT-type E3 ubiquitin ligase in Drosophila, with sequence homology to mammalian TRIP12 (Brunet et al., 2020). Although ctrip mRNA shows no clear circadian oscillations, its expression is highly enriched in pigment-dispersing factor (Pdf)-positive lateral ventral neurons (LN_vs), key pacemaker neurons in the fly brain (Lamaze et al., 2011; Ukita et al., 2022). Functional studies demonstrate that ctrip is a regulator of molecular behavioral rhythms. Deletion of the N-terminal exons of ctrip disrupts larval clock function, resulting in elevated levels of per, tim, and Clock in the small LN_vs and lengthened period of protein oscillations (Lamaze et al., 2011). Similarly, RNAi-mediated knockdown of ctrip in tim-expressing adult clock cells slows locomotor activity rhythms and elevates Clk protein abundance, indicating ctrip's role in promoting Clk degradation (Lamaze et al., 2011). Moreover, downregulation of ctrip leads to the persistence of phosphorylated per and tim during the subjective day, suggesting impaired degradation of these repressors as well. In per-null flies, ctrip knockdown still increases Clk protein levels but has no effect on tim, implying that ctrip may regulate Clk and per independently from tim (Lamaze et al., 2011).

In Drosophila, Cul3, a member of the Cullin E3 ligase family, plays a significant role in circadian regulation (Guo et al., 2014). Targeted knockdown of Cul3 in clock neurons disrupts morning anticipation and induces arrhythmic behavior under constant darkness (DD), while pan-neuronal knockdown causes complete lethality, highlighting its broader essential importance (Grima et al., 2012). Cul3 associates with hyperphosphorylated tim, promoting its K48-linked ubiquitination and degradation, particularly at the day–night transition (Guo et al., 2014; Szabó et al., 2018). This timing is crucial as premature degradation of tim in late night results in phase advances, whereas early night degradation leads to phase delays (Guo et al., 2014). Cul3 also mediates light-induced phase resetting in cry-deficient flies, suggesting it participates in photic signaling pathways independent of cry (Ogueta et al., 2020).

The CUL4A-DDB1-CDT2 E3 ubiquitin ligase complex is an important regulator of the mammalian circadian clock, acting through both protein turnover and chromatin modifications. This complex promotes the ubiquitin-mediated degradation of CRY1, specifically targeting lysine 585, thereby limiting CRY1 accumulation and constraining the amplitude of circadian oscillations (Tong et al., 2015; Tong et al., 2017). In parallel, CLOCK-BMAL1 recruits CUL4A-DDB1 to circadian target genes such as PER1, PER2, and CRY1, where it catalyzes rhythmic histone H2B monoubiquitination at E-box sites (Tamayo et al., 2015). This chromatin mark facilitates the recruitment of the repressive PER complex, establishing a feedback mechanism that links the positive and negative limbs of the clock (Tamayo et al., 2015). Loss of DDB1-CUL4 or disruption of H2B monoubiquitination impairs this recruitment and weakens transcriptional feedback, underscoring the dual role of this E3 ligase complex in regulating clock protein stability and feedback timing (Tamayo et al., 2015; Tong et al., 2017; Tong et al., 2015).

Fbxl4 is a Clock-regulated E3 ubiquitin ligase that links molecular circadian oscillators to neuronal excitability and sleep timing in Drosophila (Li et al., 2017). Fbxl4 transcript levels cycle rhythmically in the large ventral lateral neurons (lLNvs) and are driven directly by Clock-cycle binding to E-box elements within the fbxl4 promoter (Li et al., 2017). Fbxl4 promotes the rhythmic ubiquitination and degradation of GABA_A receptors in lLNvs, thereby reducing GABA sensitivity and enhancing neuronal excitability during the wake phase (Li et al., 2017). Loss of fbxl4 disrupts circadian control of sleep by enhancing GABAergic inhibition in arousal-promoting lLNvs. Fbxl4 mutant flies exhibit increased daytime and nighttime sleep and shortened sleep onset latency, similar to flies overexpressing the GABA receptor RDL (Parisky et al., 2008; Agosto et al., 2008). These findings indicate that Fbxl4 promotes wakefulness by rhythmically degrading RDL in a clock dependent manner, linking the circadian clock to sleep timing.

FBXL3 and FBXL21 are closely related F-box proteins that regulate circadian rhythms by modulating the stability and degradation of CRY1 and CRY2 proteins in mammals (Abdalla et al., 2022; Busino et al., 2007). Forward genetic screens in mice identified two mutations in FBXL3–C358S (Afterhours, Afh) and I364T (Overtime, Ovtm)–that significantly lengthen circadian period (Siepka et al., 2007). These mutations impair FBXL3's interaction with CRY proteins and reduce its catalytic efficiency, leading to CRY1/2 stabilization and strong suppression of E-box-driven transcription of PER genes (Godinho et al., 2007; Siepka et al., 2007). Interestingly, the long-period phenotype in FBXL3 mutants is rescued by REV-ERBα deletion, indicating that FBXL3 also promotes circadian period determination and clock robustness by influencing REV-ERBα stability and transcriptional activity (Shi et al., 2013).

Further genetic studies revealed the Past-time (Psttm) mutation in FBXL21 shortens circadian period and counteracts the long-period phenotype of FBXL3 (Ovtm), illustrating their functional antagonism (Yoo et al., 2013). It was shown in mice that FBXL21 exhibits strong, SCN-restricted expression with pronounced diurnal and circadian rhythmicity, rising rapidly at the start of the day and declining at night–a pattern reminiscent of other CLOCK:BMAL1-driven genes such as PER1 and DBP (D-site binding protein) (Dardente et al., 2008). Although FBXL21 also binds to CRY proteins and promotes their ubiquitination, FBXL21 serves a dual role: in the nucleus, it antagonizes FBXL3 by binding and stabilizing CRY1/2, protecting them from degradation; in the cytoplasm, where FBXL3 is largely absent, FBXL21 promotes slow degradation of CRY proteins (Yoo et al., 2013). Collectively, these findings establish FBXL21 as a rhythmically expressed, clock-controlled gene that modulates circadian period of the clock by regulating CRY protein stability in opposition to FBXL3, with distinct roles in nuclear and cytoplasmic compartments (Yoo et al., 2013; Dardente et al., 2008; Hirano et al., 2013).

FBXW7 is an E3 ubiquitin ligase that plays a role in regulating circadian rhythms by targeting core clock proteins and modulators for degradation. The expression of FBXW7 itself is circadian and regulated by the transcription factor DBP, which rhythmically activates FBXW7 transcription in renal tumor models (Okazaki et al., 2014). FBXW7 contributes to circadian rhythmicity by targeting mTOR, a key regulator of circadian translation, for ubiquitin-mediated degradation, resulting in opposing oscillations between FBXW7 and mTOR protein levels and enabling rhythmic control of translational output (Okazaki et al., 2014; Cao, 2018). FBXW7 also directly modulates clock repressors CRY2 and REV-ERBα for degradation via a CDK1-dependent phosphorylation site (Thr275), relieving repression of the positive arm of the clock and enhancing circadian amplitude (Zhao et al., 2016). Hepatic deletion of FBXW7 disrupts rhythmic expression of core clock genes and alters systemic metabolic outputs, including whole-body lipid and glucose homeostasis (Zhao et al., 2016). Together, these findings establish FBXW7's roles as a post-translational regulator of the circadian clock, modulating negative feedback repressors and linking metabolic signals to molecular oscillators.

The E3 ubiquitin ligase HRD1, encoded by the Synoviolin 1 gene, has been identified as a post-translational regulator of the core circadian transcription factor BMAL1 in mammals. Co-immunoprecipitation and immunofluorescence assays demonstrated that HRD1 physically interacts with BMAL1 and enhances its K48-linked polyubiquitination, leading to proteasome-mediated degradation without altering BMAL1 mRNA levels (Guo et al., 2020). BMAL1 forms heterodimers with CLOCK to drive the transcription of core clock-controlled genes such as PER'S, CRY'S and DBP (Brown, 2016; Mendoza-Viveros et al., 2017), and consistently, HRD1 overexpression significantly reduces PER1 and DBP mRNA levels, suggesting downstream transcriptional suppression via BMAL1 destabilization (Guo et al., 2020). Further supporting this, luciferase assays revealed that while HRD1 does not affect BMAL1 promoter activity, it significantly represses PER1 promoter activity in the presence of BMAL1 and CLOCK, suggesting that HRD1 regulates the expression of PER1 and DBP indirectly by reducing BMAL1 protein levels (Guo et al., 2020). A role for the Drosophila ortholog of HDR1, called septin interacting protein 3 (sip3), in circadian regulation has not been identified.

jetlag, mentioned previously, is an F-box protein with leucine-rich repeats that functions as a substrate adaptor within SCF-type E3 ubiquitin ligase complexes and plays a critical role in light-dependent resetting of the Drosophila circadian clock (Koh et al., 2006; Peschel et al., 2006). Initially identified through mutant screens for flies exhibiting persistent rhythmic behavior under constant light, jet was found to mediate the photic degradation of the core clock protein timeless, thereby facilitating circadian entrainment (Koh et al., 2006; Peschel et al., 2009). Loss-of-function jet mutants show impaired phase shifts in response to light pulses, slower re-entrainment to shifted light-dark cycles, and stabilized tim protein levels under light conditions (Koh et al., 2006). These phenotypes were rescued by reintroducing wild-type jet in clock neurons. In cultured cells, co-expression of jet and cry is sufficient to recapitulate light-induced degradation of tim, suggesting that jet transduces cry-dependent light input to tim (Koh et al., 2006).

Beyond tim, jet has also been shown to mediate light-induced degradation of cry itself, indicating a broader role in light-responsive circadian protein turnover (Peschel et al., 2009). Yeast two-hybrid and in vivo studies demonstrate that jet binds to cry in a light-induced manner, and that jet deficiency leads to cry accumulation in fly heads and cultured cells (Peschel et al., 2009). The relative abundance of tim influences cry stability, as tim competitively inhibits cry degradation, indicating that jet targets these proteins sequentially depending on their relative affinities (Peschel et al., 2009). Structural studies have further revealed that light exposure induces a conformational change in cry that enhances its interaction with jet, enabling ubiquitin-mediated degradation (Peschel et al., 2009; Ozturk et al., 2011). Collectively, these findings establish jet as a central component of the UPS in circadian phototransduction, regulating the timely degradation of both tim and cry to ensure precise clock resetting in response to environmental light cues.

The E3 ligase MDM2 ubiquitinates PER2 at conserved lysine residues in mammalian cells, promoting its proteasomal degradation and regulating circadian period length (Liu et al., 2018). MDM2 is a well-known oncogene, frequently overexpressed in cancers where it promotes degradation of the tumor suppressor p53 (Lee and Gu, 2010; Cheok et al., 2011). PER2 forms complexes with both MDM2 and p53 but can also bind MDM2 independently (Liu et al., 2018). Importantly, the circadian interplay between PER2 and p53 forms a regulatory feedback loop in which p53 suppresses PER2 expression, while PER2 stabilizes p53 by inhibiting MDM2-dependent ubiquitination, linking circadian rhythm to cellular stress responses and tumor suppression (Gotoh et al., 2015; Fagiani et al., 2022).

morgue is a unique ubiquitin pathway protein in Drosophila notable for its dual-domain structure containing an F-box and a non-catalytic E2-conjugase domain (Hays et al., 2002; Wing et al., 2002). It physically associates with SkpA, a core component of SCF-type E3 ligases, and binds K48-linked polyubiquitin chains, implicating it in proteasomal degradation (Zhou et al., 2013). In the circadian clock, morgue has emerged as a regulator of light input and behavioral rhythmicity under constant light. Overexpression of morgue in the tim-expressing neurons, but not in Pdf-expressing neurons, confers resistance to constant light-induced arrhythmicity and sustains robust per oscillations, specifically in DN1 neurons (Murad et al., 2007). This suggests that morgue supports rhythmicity in a neuron-specific manner, independently of the canonical LNv pacemakers. Moreover, behavioral phase shifts to light are blunted in morgue-overexpressing flies, reminiscent of cry hypomorphs, pointing to a possible inhibitory role for morgue in the cry signaling pathway (Murad et al., 2007; Busza et al., 2004).

SIAH2 (Seven in Absentia 2) is a RING-type E3 ubiquitin ligase that promotes the ubiquitination and proteasomal degradation of REV-ERBα and REB-ERBβ, two core repressors in the circadian clock (DeBruyne et al., 2015). In cultured cells, SIAH2 loss stabilizes REV-ERBs, disrupting rhythmicity, altering downstream gene expression, and lengthening the circadian period, highlighting the role of SIAH2 in clock timing via UPS-mediated turnover (DeBruyne et al., 2015). In vivo, SIAH2 deletion modestly affects expression of other clock genes such as BMAL1 and PER2 but does not significantly alter REV-ERBα protein rhythms, likely due to compensatory E3 ligases (Mekbib et al., 2022). Interestingly, SIAH2 deficiency in female mice leads to a phase-advanced circadian liver transcriptome and altered lipid rhythms, revealing an intriguing sex-specific role for UPS regulation in circadian rhythms, whose cause remains unknown (Mekbib et al., 2022).

The Drosophila Cullin-RING E3 ligase component slmb, encoded by slmb, and its mammalian orthologs β-TrCP1 and β-TrCP2 (F-box proteins within the SCF complex) are critical regulators of circadian timing through their control of per and tim protein stability (Grima et al., 2002; Shirogane et al., 2005). In flies, slmb targets phosphorylated per for ubiquitin-mediated degradation, a process initiated by dbt-dependent phosphorylation at Ser47 (Chiu et al., 2008; Grima et al., 2002). Slmb mutants fail to anticipate light-to-dark transitions under light/dark (LD) conditions and display completely arrhythmic behavior under constant darkness (DD), indicating disrupted circadian control (Grima et al., 2002). Remarkably, rhythmicity was rescued by restoring slmb in Pdf-positive neurons, highlighting its essential role in the clock network (Grima et al., 2002). Interestingly, slmb expression is not rhythmic, indicating how non-rhythmic E3 ligases can still exert strong influence over rhythmic biological processes. For instance, although slmb levels remain constant, its activity becomes critical at specific phases of the cycle such as late night when degradation of per and tim relieves transcriptional repression and resets the clock (Grima et al., 2002; Ko et al., 2002). Slmb's constant presence may allow it to act flexibly at multiple points in the cycle, illustrating how stable components of the UPS can contribute to temporal regulation in a circadian context.

In mammals, β-TrCP1 and β-TrCP2 similarly recognize, and target phosphorylated PER proteins, particularly following casein kinase 1 (CK1) phosphorylation, for degradation (Eide et al., 2005). Both β-TrCP1 and β-TrCP2 bind PER1/2, and expression of a mutant β-TrCP lacking the F-box domain prevents PER2 degradation (Eide et al., 2005; Shirogane et al., 2005). Disruption of β-TrCP–PER interactions in cells lengthens period or dampens rhythms, while inducible knockout of β-TrCP2 in mice leads to disrupted behavioral rhythms and variable period lengths under DD (Reischl et al., 2007; D'Alessandro et al., 2017). β-TrCP1/2 also regulates the degradation of other circadian proteins such as DEC1, further emphasizing their broad role in maintaining circadian period and amplitude (Kim et al., 2014).

STIP1 homology U-box-containing protein 1 STUB1, also known as C terminus of HSP70-interacting protein (CHIP), is a multifunctional protein with both chaperone activity and U-box-dependent E3 ubiquitin ligase activity (Ballinger et al., 1999; Rosser et al., 2007). STUB1 plays a central role in protein quality control by linking molecular chaperones to the UPS, playing a critical role in proteostasis (VanPelt and Page, 2017). It targets a range of substrates, including misfolded or oxidized proteins, tau, polyglutamine-expanded proteins, and key signaling factors (Lee et al., 2013; Jana et al., 2005; Seo et al., 2016; Xin et al., 2005; Hatakeyama et al., 2004; Murata et al., 2001). In circadian regulation, STUB1 was identified as a selective BMAL1-binding protein in a mass spectrometry screening, with no observed interaction with CLOCK, indicating a specific role in targeting BMAL1 (Ullah et al., 2020). Overexpression of wild-type STUB1, but not of a catalytically inactive mutant, leads to reduced BMAL1 protein levels, confirming its role in regulating BMAL1 stability through K48-linked polyubiquitination and proteasomal degradation (Ullah et al., 2020). Under oxidative stress, STUB1 translocates to the nucleus where it enhances BMAL1 degradation and modulates circadian-linked cellular senescence (Ullah et al., 2020). These findings underscore STUB1 as a post-translational regulator of the clock via linking the UPS to circadian timing and stress adaptation.

Tumor Necrosis Factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ubiquitin ligase, has recently been identified as a key regulator of circadian timing through its modulation of DBP, a clock-controlled transcription factor that drives rhythmic gene expression via D-box elements (Masuda et al., 2024). DBP exhibits strong circadian oscillation at the protein level, and this rhythmicity is shown to be regulated post-translationally through K48-linked polyubiquitination by TRAF7, in cooperation with E2 enzymes UBE2G1 and UBE2T. In cells, Masuda et al. (2024) revealed overexpression of TRAF7 promotes DBP degradation, while TRAF7 knockdown up-regulated DBP and disrupts its time-of-day–dependent oscillation. TRAF7 also shortens the circadian period, underscoring its broader role in shaping clock dynamics (Masuda et al., 2024). These findings position TRAF7 as an important link between the UPS and circadian regulation through its targeted destabilization of DBP.

In addition to TRAF7, the E3 ligase TRAF2 has also been implicated in clock protein regulation. Initially identified as a CRY1-interacting protein in a high-throughput yeast two-hybrid screen (Rual et al., 2005), TRAF2 was later shown to bind directly to BMAL1, reducing its abundance without affecting CRY1 levels (Chen et al., 2018a). This interaction is mediated though TRAF2's zinc finger domain rather than the canonical TRAF domain, and deletion of its RING domain stabilized BMAL1, confirming the importance of its ubiquitin ligase activity (Chen et al., 2018a). TRAF2 overexpression enhances BMAL1 ubiquitination and proteasomal degradation, thereby attenuating E-box-mediated transcription and dampening PER1 oscillations (Chen et al., 2018a). Together, TRAF7 and TRAF2 highlight how multiple TRAF family E3 ligases influence circadian output by targeting distinct transcriptional regulators.

UBE3A, an E3 ubiquitin ligase previously implicated in Angelman syndrome and HPV-related tumorigenesis, has recently emerged as a conserved regulator of the circadian clock through its post-translational control of BMAL1 stability (Gossan et al., 2014; Mabb et al., 2011). Experimental activation of UBE3A–achieved via expression of the HPV E6/E7 oncogenes–in murine fibroblasts significantly disrupted circadian oscillations, reduced BMAL1 protein levels, and increased BMAL1 ubiquitination (Gossan et al., 2014). These effects were dependent on UBE3A's ligase activity and were observed both with and without oncogene expression, indicating an endogenous role for UBE3A in clock regulation.

In Drosophila, both overexpression and knockdown of Ube3a in central clock neurons resulted in pronounced alterations in circadian locomotor rhythms, indicating that tight regulation of Ube3a levels is essential for proper pacemaker function across species (Gossan et al., 2014; Wu et al., 2008). Furthermore, flies harboring a global null mutation of Ube3a exhibited significant circadian deficits, supporting the notion that Ube3a is an integral component of the Drosophila circadian clock (Wu et al., 2008). These findings, together with the widespread expression of UBE3A in mammalian tissues including the SCN, suggest a conserved role for UBE3A in circadian timekeeping across species (Tian et al., 2011; Wu et al., 2008; Gossan et al., 2014).

UBE2O, a unique ubiquitin-conjugating enzyme with hybrid E2/E3 ligase activity (Ullah et al., 2019), has emerged as a regulator of the circadian clock. In HEK293T cells, UBE2O overexpression leads to a dose-dependent reduction of endogenous BMAL1, while its knockdown increases BMAL1 protein levels (Chen et al., 2018b). UBE2O physically associates with BMAL1 in mouse Neuro2a cells and whole brain tissue, but does not associate with CLOCK, demonstrating its specificity for BMAL1 (Chen et al., 2018b). Functionally, UBE2Ooverexpression attenuates BMAL1-driven transcription and dampens downstream circadian outputs, while its silencing increases PER2 rhythm amplitude, confirming its role in maintaining clock dynamics (Chen et al., 2018b). Beyond circadian regulation, UBE2O dysfunction is implicated in diseases such as cancer, diabetes, and obesity, linking its disruption to broader physiological consequences (Maffeo and Cilloni, 2024).

Deubiquitinases (DUBs) are enzymes that remove ubiquitin moieties from target proteins, reversing the action of E3 ubiquitin ligases and thereby shaping ubiquitin signaling with high accuracy (Srikanta and Cermakian, 2021; Stojkovic et al., 2014). In circadian biology, DUBs serve as key modulators of clock protein stability, ensuring the robustness and proper timing of transcriptional-translational feedback loops that drive 24-h rhythms (Harris-Gauthier et al., 2022). Recent work has highlighted their emerging importance in both central and peripheral clocks, where they fine-tune the amplitude, period, and phase of circadian gene expression (Harris-Gauthier et al., 2022; Table 2). We summarize the current status of knowledge in the field regarding DUB involvement in circadian regulation in the next few sections, with DUBs listed alphabetically by gene name.

MAGEL2 plays an important role in modulating clock protein ubiquitination by acting in concrete with both E3 ligases and deubiquitinases. Among its known binding partners are E3 ligases TRIM27 and RNF41, and the deubiquitinases USP7 and USP8, which it recruits to regulate the ubiquitination and stability of diverse targets (Tacer and Potts, 2017; Hao et al., 2013; Wijesuriya et al., 2017). MAGEL2 was shown to modulate the stability and activity of key circadian regulators including CLOCK, BMAL1, and PER2 (Devos et al., 2011). In addition, MAGEL2 modulates the ubiquitination and stability of CRY1 and CRY2 through interactions with USP7 and FBXL proteins (Hirano et al., 2016; Ansar et al., 2019). MAGEL2, in cooperation with E3 ligases and deubiquitinases, fine-tunes CRY1 protein levels through a ubiquitin-mediated degradation pathway, contributing to the temporal control of CRY1 stability and ensuring proper progression of the negative feedback loop that sustains circadian rhythms (Carias et al., 2020).

The biological importance of MAGEL2-mediated regulation of clock proteins is underscored by the circadian phenotypes observed in MAGEL2 knock-out mice. These animals exhibit fragmented activity and mistimed daytime behavior under light/dark cycles (Kozlov et al., 2007), supporting a role for MAGEL2 in maintaining circadian output. MAGEL2 mRNA is rhythmically expressed in the SCN, particularly in vasopressin-expressing neurons that are critical for circadian outputs, and this rhythmicity persists even in constant darkness (Kozlov et al., 2007). Moreover, MAGEL2 expression is disrupted in mice carrying mutations in the core circadian regulator CLOCK (Carias et al., 2020). MAGEL2 is the only gene among those disrupted in Prader-Willi syndrome with expression and function linked to excessive daytime sleepiness and night waking, and while it is also mutated in Schaaf-Yang syndrome, circadian features in this disorder remain poorly characterized due to the small number of diagnosed individuals (Fountain et al., 2017; Schaaf et al., 2013). Together, these findings highlight MAGEL2 and its interaction with E3 ligases and deubiquitinases in circadian rhythm control.

A genome-wide CRISPR/Cas9 knockdown screen identified ubiquitin-specific protease 1 (USP1) as a novel regulator that positively modulates BMAL1 protein levels (Hu et al., 2024). Overexpression of wild-type USP1, but not its catalytically inactive mutant, led to increased BMAL1 protein abundance, whereas USP1 knockdown via shRNA or CRISPR knockout significantly reduced BMAL1 levels in U2OS cells (Hu et al., 2024). Pharmacological inhibition of USP1 similarly reduced BMAL1 protein levels both in vitro and in mouse tissues, further confirming its role in regulating BMAL1 stability (Hu et al., 2024). USP1 modulates BMAL1 at the post-transcriptional level, as its silencing did not affect BMAL1 mRNA levels. Furthermore, reduced levels or inhibition of USP1 resulted in decreased expression of several BMAL1 target genes, including CRY1, CRY2, PER1, PER2, and DBP, along with reduced protein levels of BMAL1, CRY1, and CRY2 (Hu et al., 2024).

Interestingly, USP1 displays a strong tissue-specific expression. In the mouse heart, USP1 expression is markedly higher than the other BMAL1 deubiquitinases, USP2 and USP9X, while in the hypothalamus USP9X was much higher than the others (Hu et al., 2024). This suggests that distinct DUBs may control BMAL1 stability in a tissue-specific manner, with USP1 playing a dominant role in cardiac circadian regulation.

One of the most well characterized DUBs in circadian biology is USP2. It is rhythmically expressed across multiple tissues including the suprachiasmatic nucleus (SCN), liver, and retina and its deletion disrupts clock gene expression in both central and peripheral oscillators (Harris-Gauthier et al., 2022; Scoma et al., 2011). USP2 exists in multiple isoforms, with evidence that USP2a stabilizes CRY1, while USP2b interacts with BMAL1 and PER1, modulating their localization and stability (Scoma et al., 2011; Tong et al., 2012).

Cellular overexpression of USP2 enhances PER1-mediated repression on CLOCK/BMAL1 transcriptional activity by promoting PER nuclear retention, and consistently, USP2 knockout mice exhibit increased cytoplasmic PER1 and dampened PER2 and REV-ERBα rhythmicity (Yang et al., 2014). More recently, Srikanta and Cermakian (2021), Srikanta et al. (2025) demonstrated that USP2 deletion in mice impairs synchrony among SCN neurons, dampens molecular rhythms, and alters behavioral circadian outputs, reinforcing USP2's role in maintaining coherence within the central clock network (Srikanta et al., 2025).

USP7, also known as HAUSP (Herpes virus-associated ubiquitin-specific protease), has been implicated in circadian rhythms by stabilizing CRY1 and CRY2 proteins through deubiquitination (Hirano et al., 2016; Papp et al., 2015). While reducing USP7 activity via RNA interference or pharmacological inhibition lengthened circadian period in MEFs and U2OS cells (Papp et al., 2015), other studies reported the opposite effect, with knockdown shortening and overexpression lengthening the period (Hirano et al., 2016). These discrepancies may stem from cell type differences, CRY paralog-specific roles, or redundancy with other DUBs (Maier et al., 2009; Baggs et al., 2009).

Importantly, Papp et al. (2015) demonstrated that genotoxic stress induces CRY1 phosphorylation and USP7-mediated stabilization, shifting clock phase and increasing the CRY1/CRY2 ratio, which helps coordinate circadian and DNA damage responses (Papp et al., 2015). USP7 itself is negatively regulated by MAGEL2, an E3 ligase mentioned above which is highly expressed in the SCN and associated with sleep disorders and circadian rhythm disruptions in both humans and mice (Carias et al., 2020; Kozlov et al., 2007; Lee et al., 2003), suggesting that USP7 may influence mammalian sleep–wake regulation as part of a broader transcriptional complex. Circadian roles for the fly Usp7 have not yet been defined.

Recent studies have revealed a link between ubiquitin-specific protease 13 (USP13) and circadian rhythm regulation through its interaction with BMAL1. USP13 was identified as a downstream effector of TDP-43, a protein implicated in several neurodegenerative disorders including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD) (Chen and Mitchell, 2021; Josephs et al., 2014; Neumann et al., 2006). TDP-43 exhibits rhythmic expression, and its knockdown disrupts circadian behavior, alters the expression of core clock genes including BMAL1, CLOCK, CRY1, and PER2, and weakens cognition and balancing abilities in mice (Gu et al., 2025). Mechanistically, Gu et al. (2025) found TDP-43 knockdown induces aberrant splicing and downregulation of USP13, which in turn leads to increased ubiquitination and degradation of BMAL1. Increasing the amount of USP13 in HEK-293T cells increased BMAL1 protein in a dose-dependent manner, supporting USP13s role in BMAL1 stabilization via directly modulating its ubiquitination (Gu et al., 2025). Furthermore, USP13 itself is rhythmically expressed, and disruption of its expression perturbs the normal oscillation of BMAL1 protein levels (Gu et al., 2025). These findings position USP13 as a key post-translational regulator, linking deubiquitination of BMAL1 to broader rhythms in physiology and highlighting its potential as a molecular target in circadian dysfunction associated with neurodegenerative conditions.

In Drosophila, DUBs such as Usp8 and non-stop (USP22 in humans) have been shown to influence circadian rhythms through transcriptional regulation. Usp8, identified as rhythmically expressed in the Drosophila brain and more specifically clock neurons, directly deubiquitinates Clock to suppress its transcriptional activity within the Clock-cycle complex, thereby reducing expression of target genes like per and tim (McDonald and Rosbash, 2001; Kula-Eversole et al., 2010; Luo et al., 2012). Flies with Usp8 knockdown exhibited disrupted locomotor activity rhythms and displayed either arrhythmic or lengthened periods, displaying the role of Usp8 in maintaining circadian timing (Luo et al., 2012). There is currently no reported role for mammalian USP8 in circadian regulation.

USP9X has been implicated in regulation of the positive arm of the molecular clock by deubiquitinating and stabilizing BMAL1 (Zhang et al., 2018). Reducing USP9X lowered the expression of BMAL1 target genes PER2 and CRY1 in mouse neuroblastoma cells, but in U2OS cells, knockdown only slightly reduced rhythm amplitude without changing the period (Zhang et al., 2018). These differences may reflect tissue-specific interactions or compensation by other DUBs, highlighting the need for further study of USP9X and other DUBs roles in circadian regulation across tissues.

USP14 has been implicated in stabilizing PER proteins, as expression of a dominant-negative USP14 in HEK293 cells decreases levels of PER1 and PER2, and in MEFs leads to reduced PER2 half-life and a dose-dependent shortening of circadian period, indicating USP14's potential role in regulating the timing of the negative feedback loop (D'Alessandro et al., 2017; D'Alessandro et al., 2015). Interestingly, a recent study showed Usp14 down-regulation corrects sleep and circadian dysfunction of a Drosophila model of Parkinson's disease, suggesting it may modulate neurodegeneration-related disruptions in biological time (Favaro et al., 2024).

non-stop (not; also known as USP22 in humans), the deubiquitinase module of the Spt-Ada-Gcn5 acetyltransferase (SAGA) chromatin-modifying complex, modulates transcription through histone H2B deubiquitination (Mohan et al., 2014). not knockdown in clock neurons lengthened locomotor periods, reduced rhythm robustness, and decreased expression of several clock genes, including tim, per, and Clock (Mahesh et al., 2020; Bu et al., 2020). Furthermore, loss of not led to a marked increase in histone H2B ubiquitination at the tim and Pdp1ε gene loci (Bu et al., 2020). This effect was amplified by simultaneous knockdown of Nipped-A (the Drosophila ortholog of the human schizophrenia-associated gene TRAPP), the histone acetyltransferase component of the SAGA complex (Bu et al., 2020; Mahesh et al., 2020). Nipped-A knockdown similarly results in a lengthened circadian period (1–3 h across independent RNAi lines), reduced rhythm power, and decreased expression of tim and Pdp1ε, specifically through increased H2B ubiquitination at their promoter regions (Bu et al., 2020; Mahesh et al., 2020).

Genetic manipulation demonstrates that not and Nipped-A function together to regulate circadian timing. Overexpression of not can rescue the period-lengthening phenotype caused by Nipped-A deficiency, whereas double knockdown of not and Nipped-A synergistically exacerbates circadian defects (Bu et al., 2020). Although the mammalian not ortholog, USP22, has not yet been directly linked to clock regulation, the finding that H2B ubiquitination at mammalian clock gene loci was found to regulate clock gene transcription in mouse livers supports a conserved role for the SAGA DUBm in circadian rhythm control (Tamayo et al., 2015).