The inner workings of an ancient biological clock.

Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth's rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.

TIMELESS promotes reprogramming of glucose metabolism in oral squamous cell carcinoma.

BACKGROUND: Oral squamous cell carcinoma (OSCC), the predominant malignancy of the oral cavity, is characterized by high incidence and low survival rates. Emerging evidence suggests a link between circadian rhythm disruptions and cancer development. The circadian gene TIMELESS, known for its specific expression in various tumors, has not been extensively studied in the context of OSCC. This study aims to explore the influence of TIMELESS on OSCC, focusing on cell growth and metabolic alterations. METHODS: We analyzed TIMELESS expression in OSCC using western blot, immunohistochemistry, qRT-PCR, and data from The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia (CCLE). The role of TIMELESS in OSCC was examined through clone formation, MTS, cell cycle, and EdU assays, alongside subcutaneous tumor growth experiments in nude mice. We also assessed the metabolic impact of TIMELESS by measuring glucose uptake, lactate production, oxygen consumption, and medium pH, and investigated its effect on key metabolic proteins including silent information regulator 1 (SIRT1), hexokinase 2 (HK2), pyruvate kinase isozyme type M2 (PKM2), recombinant lactate dehydrogenase A (LDHA) and glucose transporter-1 (GLUT1). RESULTS: Elevated TIMELESS expression in OSCC tissues and cell lines was observed, correlating with reduced patient survival. TIMELESS overexpression enhanced OSCC cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). Conversely, TIMELESS knockdown inhibited these cellular and metabolic processes, an effect mirrored by manipulating SIRT1 levels. Additionally, SIRT1 was positively associated with TIMELESS expression. The expression of SIRT1, HK2, PKM2, LDHA and GLUT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS. CONCLUSION: TIMELESS exacerbates OSCC progression by modulating cellular proliferation and metabolic pathways, specifically by enhancing glycolysis and reducing oxidative phosphorylation, largely mediated through the SIRT1 pathway. This highlights TIMELESS as a potential target for OSCC therapeutic strategies.

The cross-talk between leptin and circadian rhythm signaling proteins in physiological processes: a systematic review.

BACKGROUND: Today, modern lifestyles and disrupted sleep patterns cause circadian clock rhythm impairments that are associated with altered leptin levels, which subsequently affect a wide range of physiological processes and have significant health burdens on societies. Nevertheless, there has been no systematic review of circadian clock genes and proteins, leptin, and related signaling pathways. METHODS: Accordingly, we systematically reviewed circadian clock proteins, leptin, and molecular mechanisms between them by searching Pubmed, Scopus, ProQuest, Web of Sciences, and Google Scholar until September 2022. After considering the inclusion and exclusion criteria, 20 animal studies were selected. The risk of bias was assessed in each study. RESULTS: The results clarified the reciprocal interconnected relationship between circadian clock genes and leptin. Circadian clock genes regulate leptin expression and signaling via different mechanisms, such as CLOCK-BMAL1 heterodimers, which increase the expression of PPARs. PPARs induce the expression of C/EBPα, a key factor in upregulating leptin expression. CLOCK-BMAL1 also induces the expression of Per1 and Rev-erb genes. PER1 activates mTORC1 and mTORC1 enhances the expression of C/EBPα. In addition, REV-ERBs activate the leptin signaling pathway. Also, leptin controls the expression of circadian clock genes by triggering the AMPK and ERK/MAPK signaling pathways, which regulate the activity of PPARs. Moreover, the roles of these molecular mechanisms are elucidated in different physiological processes and organs. CONCLUSIONS: Crosstalk between circadian clock genes and leptin and their affecting elements should be considered in the selection of new therapeutic targets for related disorders, especially obesity and metabolic impairments.

Roles for the Synechococcus elongatus RNA-Binding Protein Rbp2 in Regulating the Circadian Clock.

The cyanobacterial circadian oscillator, consisting of KaiA, KaiB, and KaiC proteins, drives global rhythms of gene expression and compaction of the chromosome and regulates the timing of cell division and natural transformation. While the KaiABC posttranslational oscillator can be reconstituted in vitro, the Kai-based oscillator is subject to several layers of regulation in vivo. Specifically, the oscillator proteins undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localized as a focus at or near the pole of the cell at night. Here, we report that the CI domain of KaiC, when in a hexameric state, is sufficient to target KaiC to the pole. Moreover, increased ATPase activity of KaiC correlates with enhanced polar localization. We identified proteins associated with KaiC in either a localized or diffuse state. We found that loss of Rbp2, found to be associated with localized KaiC, results in decreased incidence of KaiC localization and long-period circadian phenotypes. Rbp2 is an RNA-binding protein, and it appears that RNA-binding activity of Rbp2 is required to execute clock functions. These findings uncover previously unrecognized roles for Rbp2 in regulating the circadian clock and suggest that the proper localization of KaiC is required for a fully functional clock in vivo.

Synchronization of the circadian clock to the environment tracked in real time.

The circadian system of the cyanobacterium Synechococcus elongatus PCC 7942 relies on a three-protein nanomachine (KaiA, KaiB, and KaiC) that undergoes an oscillatory phosphorylation cycle with a period of ~24 h. This core oscillator can be reconstituted in vitro and is used to study the molecular mechanisms of circadian timekeeping and entrainment. Previous studies showed that two key metabolic changes that occur in cells during the transition into darkness, changes in the ATP/ADP ratio and redox status of the quinone pool, are cues that entrain the circadian clock. By changing the ATP/ADP ratio or adding oxidized quinone, one can shift the phase of the phosphorylation cycle of the core oscillator in vitro. However, the in vitro oscillator cannot explain gene expression patterns because the simple mixture lacks the output components that connect the clock to genes. Recently, a high-throughput in vitro system termed the in vitro clock (IVC) that contains both the core oscillator and the output components was developed. Here, we used IVC reactions and performed massively parallel experiments to study entrainment, the synchronization of the clock with the environment, in the presence of output components. Our results indicate that the IVC better explains the in vivo clock-resetting phenotypes of wild-type and mutant strains and that the output components are deeply engaged with the core oscillator, affecting the way input signals entrain the core pacemaker. These findings blur the line between input and output pathways and support our previous demonstration that key output components are fundamental parts of the clock.

A multi-organoid platform identifies CIART as a key factor for SARS-CoV-2 infection.

COVID-19 is a systemic disease involving multiple organs. We previously established a platform to derive organoids and cells from human pluripotent stem cells to model SARS-CoV-2 infection and perform drug screens1,2. This provided insight into cellular tropism and the host response, yet the molecular mechanisms regulating SARS-CoV-2 infection remain poorly defined. Here we systematically examined changes in transcript profiles caused by SARS-CoV-2 infection at different multiplicities of infection for lung airway organoids, lung alveolar organoids and cardiomyocytes, and identified several genes that are generally implicated in controlling SARS-CoV-2 infection, including CIART, the circadian-associated repressor of transcription. Lung airway organoids, lung alveolar organoids and cardiomyocytes derived from isogenic CIART-/- human pluripotent stem cells were significantly resistant to SARS-CoV-2 infection, independently of viral entry. Single-cell RNA-sequencing analysis further validated the decreased levels of SARS-CoV-2 infection in ciliated-like cells of lung airway organoids. CUT&RUN, ATAC-seq and RNA-sequencing analyses showed that CIART controls SARS-CoV-2 infection at least in part through the regulation of NR4A1, a gene also identified from the multi-organoid analysis. Finally, transcriptional profiling and pharmacological inhibition led to the discovery that the Retinoid X Receptor pathway regulates SARS-CoV-2 infection downstream of CIART and NR4A1. The multi-organoid platform identified the role of circadian-clock regulation in SARS-CoV-2 infection, which provides potential therapeutic targets for protection against COVID-19 across organ systems.

KaiC-like proteins contribute to stress resistance and biofilm formation in environmental Pseudomonas species.

KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologues of this protein are widespread among nonphotosynthetic bacteria, but the function, interaction network, and mechanism of action of these proteins are still largely unknown. Here, we focus on KaiC homologues found in environmental Pseudomonas species. Using bioinformatics, we describe the distribution of this protein family in the genus and reveal a conserved interaction network comprising a histidine kinase and response regulator. We characterize experimentally the only KaiC homologue present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both species. KaiC homologues are found in different phosphorylation states and physically interact with a cognate histidine kinase and response regulator. In contrast with cyanobacterial counterparts, the expression and phosphorylation of KaiC homologues do not correlate with light variations under 12:12 light: dark cycles in either Pseudomonas species, and KaiC itself is not required to support a light-driven behaviour in P. putida. Overall, this suggests that KaiC homologues in Pseudomonas species are involved in environmental stress resistance but not in responses to diurnal rhythms.

Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm.

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. Elucidating this temperature compensation is essential for understanding the KaiABC circadian clock, but its mechanism has remained a mystery. We analyzed the KaiABC temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model explained the experimentally observed responses of the oscillation phase to the temperature shift or the ADP-concentration change and suggested that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

KidA, a multi-PAS domain protein, tunes the period of the cyanobacterial circadian oscillator.

The cyanobacterial clock presents a unique opportunity to understand the biochemical basis of circadian rhythms. The core oscillator, composed of the KaiA, KaiB, and KaiC proteins, has been extensively studied, but a complete picture of its connection to the physiology of the cell is lacking. To identify previously unknown components of the clock, we used KaiB locked in its active fold as bait in an immunoprecipitation/mass spectrometry approach. We found that the most abundant interactor, other than KaiC, was a putative diguanylate cyclase protein predicted to contain multiple Per-Arnt-Sim (PAS) domains, which we propose to name KidA. Here we show that KidA directly binds to the fold-switched active form of KaiB through its N-terminal PAS domains. We found that KidA shortens the period of the circadian clock both in vivo and in vitro and alters the ability of the clock to entrain to light-dark cycles. The dose-dependent effect of KidA on the clock period could be quantitatively recapitulated by a mathematical model in which KidA stabilizes the fold-switched form of KaiB, favoring rebinding to KaiC. Put together, our results show that the period and amplitude of the clock can be modulated by regulating the access of KaiB to the fold-switched form.

Coupling of distant ATPase domains in the circadian clock protein KaiC.

The AAA+ family member KaiC is the central pacemaker for circadian rhythms in the cyanobacterium Synechococcus elongatus. Composed of two hexameric rings of adenosine triphosphatase (ATPase) domains with tightly coupled activities, KaiC undergoes a cycle of autophosphorylation and autodephosphorylation on its C-terminal (CII) domain that restricts binding of clock proteins on its N-terminal (CI) domain to the evening. Here, we use cryogenic-electron microscopy to investigate how daytime and nighttime states of CII regulate KaiB binding on CI. We find that the CII hexamer is destabilized during the day but takes on a rigidified C2-symmetric state at night, concomitant with ring-ring compression. Residues at the CI-CII interface are required for phospho-dependent KaiB association, coupling ATPase activity on CI to cooperative KaiB recruitment. Together, these studies clarify a key step in the regulation of cyanobacterial circadian rhythms by KaiC phosphorylation.

Highly sensitive tryptophan fluorescence probe for detecting rhythmic conformational changes of KaiC in the cyanobacterial circadian clock system.

KaiC, a core protein of the cyanobacterial circadian clock, consists of an N-terminal CI domain and a C-terminal CII domain, and assembles into a double-ring hexamer upon binding with ATP. KaiC rhythmically phosphorylates and dephosphorylates its own two adjacent residues Ser431 and Thr432 at the CII domain with a period of ∼24 h through assembly and disassembly with the other clock proteins, KaiA and/or KaiB. In this study, to understand how KaiC alters its conformation as the source of circadian rhythm, we investigated structural changes of an inner-radius side of the CII ring using time-resolved Trp fluorescence spectroscopy. A KaiC mutant harboring a Trp fluorescence probe at a position of 419 exhibited a robust circadian rhythm with little temperature sensitivity in the presence of KaiA and KaiB. Our fluorescence observations show a remarkable environmental change at the inner-radius side of the CII ring during circadian oscillation. Crystallographic analysis revealed that a side chain of Trp at the position of 419 was oriented toward a region undergoing a helix-coil transition, which is considered to be a key event to allosterically regulate the CI ring that plays a crucial role in determining the cycle period. The present study provides a dynamical insight into how KaiC generates circadian oscillation.

RadA, a Key Gene of the Circadian Rhythm of Escherichia coli.

Circadian rhythms are present in almost all living organisms, and their activity relies on molecular clocks. In prokaryotes, a functional molecular clock has been defined only in cyanobacteria. Here, we investigated the presence of circadian rhythms in non-cyanobacterial prokaryotes. The bioinformatic approach was used to identify a homologue of KaiC (circadian gene in cyanobacteria) in Escherichia coli. Then, strains of E. coli (wild type and mutants) were grown on blood agar, and sampling was made every 3 h for 24 h at constant conditions. Gene expression was determined by qRT-PCR, and the rhythmicity was analyzed using the Cosinor model. We identified RadA as a KaiC homologue in E. coli. Expression of radA showed a circadian rhythm persisting at least 3 days, with a peak in the morning. The circadian expression of other E. coli genes was also observed. Gene circadian oscillations were lost in radA mutants of E. coli. This study provides evidence of molecular clock gene expression in E. coli with a circadian rhythm. Such a finding paves the way for new perspectives in antibacterial treatment.

Regulation mechanisms of the dual ATPase in KaiC.

KaiC is a dual adenosine triphosphatase (ATPase), with one active site in its N-terminal domain and another in its C-terminal domain, that drives the circadian clock system of cyanobacteria through sophisticated coordination of the two sites. To elucidate the coordination mechanism, we studied the contribution of the dual-ATPase activities in the ring-shaped KaiC hexamer and these structural bases for activation and inactivation. At the N-terminal active site, a lytic water molecule is sequestered between the N-terminal domains, and its reactivity to adenosine triphosphate (ATP) is controlled by the quaternary structure of the N-terminal ring. The C-terminal ATPase activity is regulated mostly by water-incorporating voids between the C-terminal domains, and the size of these voids is sensitive to phosphoryl modification of S431. The up-regulatory effect on the N-terminal ATPase activity inversely correlates with the affinity of KaiC for KaiB, a clock protein constitutes the circadian oscillator together with KaiC and KaiA, and the complete dissociation of KaiB from KaiC requires KaiA-assisted activation of the dual ATPase. Delicate interactions between the N-terminal and C-terminal rings make it possible for the components of the dual ATPase to work together, thereby driving the assembly and disassembly cycle of KaiA and KaiB.

Elucidation of master allostery essential for circadian clock oscillation in cyanobacteria.

Spatiotemporal allostery is the source of complex but ordered biological phenomena. To identify the structural basis for allostery that drives the cyanobacterial circadian clock, we crystallized the clock protein KaiC in four distinct states, which cover a whole cycle of phosphor-transfer events at Ser431 and Thr432. The minimal set of allosteric events required for oscillatory nature is a bidirectional coupling between the coil-to-helix transition of the Ser431-dependent phospho-switch in the C-terminal domain of KaiC and adenosine 5'-diphosphate release from its N-terminal domain during adenosine triphosphatase cycle. An engineered KaiC protein oscillator consisting of a minimal set of the identified master allosteric events exhibited a monophosphorylation cycle of Ser431 with a temperature-compensated circadian period, providing design principles for simple posttranslational biochemical circadian oscillators.

Overall structure of fully assembled cyanobacterial KaiABC circadian clock complex by an integrated experimental-computational approach.

In the cyanobacterial circadian clock system, KaiA, KaiB and KaiC periodically assemble into a large complex. Here we determined the overall structure of their fully assembled complex by integrating experimental and computational approaches. Small-angle X-ray and inverse contrast matching small-angle neutron scatterings coupled with size-exclusion chromatography provided constraints to highlight the spatial arrangements of the N-terminal domains of KaiA, which were not resolved in the previous structural analyses. Computationally built 20 million structural models of the complex were screened out utilizing the constrains and then subjected to molecular dynamics simulations to examine their stabilities. The final model suggests that, despite large fluctuation of the KaiA N-terminal domains, their preferential positionings mask the hydrophobic surface of the KaiA C-terminal domains, hindering additional KaiA-KaiC interactions. Thus, our integrative approach provides a useful tool to resolve large complex structures harboring dynamically fluctuating domains.

Multimeric structure enables the acceleration of KaiB-KaiC complex formation induced by ADP/ATP exchange inhibition.

Circadian clocks tick a rhythm with a nearly 24-hour period in a variety of organisms. In the clock proteins of cyanobacteria, KaiA, KaiB, and KaiC, known as a minimum circadian clock, the slow KaiB-KaiC complex formation is essential in determining the clock period. This complex formation, occurring when the C1 domain of KaiC hexamer binds ADP molecules produced by the ATPase activity of C1, is considered to be promoted by accumulating ADP molecules in C1 through inhibiting the ADP/ATP exchange (ADP release) rather than activating the ATP hydrolysis (ADP production). Significantly, this ADP/ATP exchange inhibition accelerates the complex formation together with its promotion, implying a potential role in the period robustness under environmental perturbations. However, the molecular mechanism of this simultaneous promotion and acceleration remains elusive because inhibition of a backward process generally slows down the whole process. In this article, to investigate the mechanism, we build several reaction models of the complex formation with the pre-binding process concerning the ATPase activity. In these models, six KaiB monomers cooperatively and rapidly bind to C1 when C1 binds ADP molecules more than a given threshold while stabilizing the binding-competent conformation of C1. Through comparison among the models proposed here, we then extract three requirements for the simultaneous promotion and acceleration: the stabilization of the binding-competent C1 by KaiB binding, slow ADP/ATP exchange in the binding-competent C1, and relatively fast ADP/ATP exchange occurring in the binding-incompetent C1 in the presence of KaiB. The last two requirements oblige KaiC to form a multimer. Moreover, as a natural consequence, the present models can also explain why the binding of KaiB to C1 reduces the ATPase activity of C1.

Dimer dissociation is a key energetic event in the fold-switch pathway of KaiB.

Cyanobacteria possesses the simplest circadian clock, composed of three proteins that act as a phosphorylation oscillator: KaiA, KaiB, and KaiC. The timing of this oscillator is determined by the fold-switch of KaiB, a structural rearrangement of its C-terminal half that is accompanied by a change in the oligomerization state. During the day, KaiB forms a stable tetramer (gsKaiB), whereas it adopts a monomeric thioredoxin-like fold during the night (fsKaiB). Although the structures and functions of both native states are well studied, little is known about the sequence and structure determinants that control their structural interconversion. Here, we used confinement molecular dynamics (CCR-MD) and folding simulations using structure-based models to show that the dissociation of the gsKaiB dimer is a key energetic event for the fold-switch. Hydrogen-deuterium exchange mass spectrometry (HDXMS) recapitulates the local stability of protein regions reported by CCR-MD, with both approaches consistently indicating that the energy and backbone flexibility changes are solely associated with the region that fold-switches between gsKaiB and fsKaiB and that the localized regions that differentially stabilize gsKaiB also involve regions outside the dimer interface. Moreover, two mutants (R23C and R75C) previously reported to be relevant for altering the rhythmicity of the Kai clock were also studied by HDXMS. Particularly, R75C populates dimeric and monomeric states with a deuterium incorporation profile comparable to the one observed for fsKaiB, emphasizing the importance of the oligomerization state of KaiB for the fold-switch. These findings suggest that the information necessary to control the rhythmicity of the cyanobacterial biological clock is, to a great extent, encoded within the KaiB sequence.

Stx2 Induces Differential Gene Expression and Disturbs Circadian Rhythm Genes in the Proximal Tubule.

Shiga toxin-producing Escherichia coli (STEC) causes proximal tubular defects in the kidney. However, factors altered by Shiga toxin (Stx) within the proximal tubules are yet to be shown. We determined Stx receptor Gb3 in murine and human kidneys and confirmed the receptor expression in the proximal tubules. Stx2-injected mouse kidney tissues and Stx2-treated human primary renal proximal tubular epithelial cell (RPTEC) were collected and microarray analysis was performed. We compared murine kidney and RPTEC arrays and selected common 58 genes that are differentially expressed vs. control (0 h, no toxin-treated). We found that the most highly expressed gene was GDF15, which may be involved in Stx2-induced weight loss. Genes associated with previously reported Stx2 activities such as src kinase Yes phosphorylation pathway activation, unfolded protein response (UPR) and ribotoxic stress response (RSR) showed differential expressions. Moreover, circadian clock genes were differentially expressed, suggesting Stx2-induced renal circadian rhythm disturbance. Circadian rhythm-regulated proximal tubular Na+-glucose transporter SGLT1 (SLC5A1) was down-regulated, indicating proximal tubular functional deterioration, and mice developed glucosuria confirming proximal tubular dysfunction. Stx2 alters gene expression in murine and human proximal tubules through known activities and newly investigated circadian rhythm disturbance, which may result in proximal tubular dysfunctions.

Cortical diurnal rhythms remain intact with microglial depletion.

Microglia are subject to change in tandem with the endogenously generated biological oscillations known as our circadian rhythm. Studies have shown microglia harbor an intrinsic molecular clock which regulates diurnal changes in morphology and influences inflammatory responses. In the adult brain, microglia play an important role in the regulation of condensed extracellular matrix structures called perineuronal nets (PNNs), and it has been suggested that PNNs are also regulated in a circadian and diurnal manner. We sought to determine whether microglia mediate the diurnal regulation of PNNs via CSF1R inhibitor dependent microglial depletion in C57BL/6J mice, and how the absence of microglia might affect cortical diurnal gene expression rhythms. While we observe diurnal differences in microglial morphology, where microglia are most ramified at the onset of the dark phase, we do not find diurnal differences in PNN intensity. However, PNN intensity increases across many brain regions in the absence of microglia, supporting a role for microglia in the regulation of PNNs. Here, we also show that cortical diurnal gene expression rhythms are intact, with no cycling gene changes without microglia. These findings demonstrate a role for microglia in the maintenance of PNNs, but not in the maintenance of diurnal rhythms.

Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5.

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes (Bmal1, Per2, Nr1d1, and Dbp) and an autophagy-related gene (Atg5) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of Bmal1, Per2, and Dbp in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, Nr1d1 knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of Bmal1-/- mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited Atg5 transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.