Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Lint-O cooperates with L(3)mbt in target gene suppression to maintain homeostasis in fly ovary and brain.

Loss-of-function mutations in Drosophila lethal(3)malignant brain tumor [l(3)mbt] cause ectopic expression of germline genes and brain tumors. Loss of L(3)mbt function in ovarian somatic cells (OSCs) aberrantly activates germ-specific piRNA amplification and leads to infertility. However, the underlying mechanism remains unclear. Here, ChIP-seq for L(3)mbt in cultured OSCs and RNA-seq before and after L(3)mbt depletion shows that L(3)mbt genomic binding is not necessarily linked to gene regulation and that L(3)mbt controls piRNA pathway genes in multiple ways. Lack of known L(3)mbt co-repressors, such as Lint-1, has little effect on the levels of piRNA amplifiers. Identification of L(3)mbt interactors in OSCs and subsequent analysis reveals CG2662 as a novel co-regulator of L(3)mbt, termed "L(3)mbt interactor in OSCs" (Lint-O). Most of the L(3)mbt-bound piRNA amplifier genes are also bound by Lint-O in a similar fashion. Loss of Lint-O impacts the levels of piRNA amplifiers, similar to the lack of L(3)mbt. The lint-O-deficient flies exhibit female sterility and tumorous brains. Thus, L(3)mbt and its novel co-suppressor Lint-O cooperate in suppressing target genes to maintain homeostasis in the ovary and brain.

Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch.

Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase ß-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 messenger RNA (mRNA) levels were minimally affected. Nuclear PER1, CRY1, and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.

CaMKII is essential for the cellular clock and coupling between morning and evening behavioral rhythms.

Daily behavioral rhythms in mammals are governed by the central circadian clock, located in the suprachiasmatic nucleus (SCN). The behavioral rhythms persist even in constant darkness, with a stable activity time due to coupling between two oscillators that determine the morning and evening activities. Accumulating evidence supports a prerequisite role for Ca(2+) in the robust oscillation of the SCN, yet the underlying molecular mechanism remains elusive. Here, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activity is essential for not only the cellular oscillation but also synchronization among oscillators in the SCN. A kinase-dead mutation in mouse CaMKIIα weakened the behavioral rhythmicity and elicited decoupling between the morning and evening activity rhythms, sometimes causing arrhythmicity. In the mutant SCN, the right and left nuclei showed uncoupled oscillations. Cellular and biochemical analyses revealed that Ca(2+)-calmodulin-CaMKII signaling contributes to activation of E-box-dependent gene expression through promoting dimerization of circadian locomotor output cycles kaput (CLOCK) and brain and muscle Arnt-like protein 1 (BMAL1). These results demonstrate a dual role of CaMKII as a component of cell-autonomous clockwork and as a synchronizer integrating circadian behavioral activities.

The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis.

The disruption of the NRF2 (nuclear factor erythroid-derived 2-like 2)/glutathione-mediated antioxidant defense pathway is a critical step in the pathogenesis of several chronic pulmonary diseases and cancer. While the mechanism of NRF2 activation upon oxidative stress has been widely investigated, little is known about the endogenous signals that regulate the NRF2 pathway in lung physiology and pathology. Here we show that an E-box-mediated circadian rhythm of NRF2 protein is essential in regulating the rhythmic expression of antioxidant genes involved in glutathione redox homeostasis in the mouse lung. Using an in vivo bleomycin-induced lung fibrosis model, we reveal a clock "gated" pulmonary response to oxidative injury, with a more severe fibrotic effect when bleomycin was applied at a circadian nadir in NRF2 levels. Timed administration of sulforaphane, an NRF2 activator, significantly blocked this phenotype. Moreover, in the lungs of the arrhythmic Clock(Δ19) mice, the levels of NRF2 and the reduced glutathione are constitutively low, associated with increased protein oxidative damage and a spontaneous fibrotic-like pulmonary phenotype. Our findings reveal a pivotal role for the circadian control of the NRF2/glutathione pathway in combating oxidative/fibrotic lung damage, which might prompt new chronotherapeutic strategies for the treatment of human lung diseases, including idiopathic pulmonary fibrosis.

ASK family kinases mediate cellular stress and redox signaling to circadian clock.

Daily rhythms of behaviors and physiologies are generated by the circadian clock, which is composed of clock genes and the encoded proteins forming transcriptional/translational feedback loops (TTFLs). The circadian clock is a self-sustained oscillator and flexibly responds to various time cues to synchronize with environmental 24-h cycles. However, the key molecule that transmits cellular stress to the circadian clockwork is unknown. Here we identified apoptosis signal-regulating kinase (ASK), a member of the MAPKKK family, as an essential mediator determining the circadian period and phase of cultured cells in response to osmotic changes of the medium. The physiological impact of ASK signaling was demonstrated by a response of the clock to changes in intracellular redox states. Intriguingly, the TTFLs drive rhythmic expression of Ask genes, indicating ASK-mediated association of the TTFLs with intracellular redox. In behavioral analysis, Ask1, Ask2, and Ask3 triple-KO mice exhibited compromised light responses of the circadian period and phase in their activity rhythms. LC-MS/MS-based proteomic analysis identified a series of ASK-dependent and osmotic stress-responsive phosphorylations of proteins, among which CLOCK, a key component of the molecular clockwork, was phosphorylated at Thr843 or Ser845 in the carboxyl-terminal region. These findings reveal the ASK-dependent stress response as an underlying mechanism of circadian clock flexibility.

The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.

Post-translational modifications (such as ubiquitination) of clock proteins are critical in maintaining the precision and robustness of the evolutionarily conserved circadian clock. Ubiquitination of the core clock transcription factor BMAL1 (brain and muscle Arnt-like 1) has recently been reported. However, it remains unknown whether BMAL1 ubiquitination affects circadian pacemaking and what ubiquitin ligase(s) is involved. Here, we show that activating UBE3A (by expressing viral oncogenes E6/E7) disrupts circadian oscillations in mouse embryonic fibroblasts, measured using PER2::Luc dynamics, and rhythms in endogenous messenger ribonucleic acid and protein levels of BMAL1. Over-expression of E6/E7 reduced the level of BMAL1, increasing its ubiquitination and proteasomal degradation. UBE3A could bind to and degrade BMAL1 in a ubiquitin ligase-dependent manner. This occurred both in the presence and absence of E6/E7. We provide in vitro (knockdown/over-expression in mammalian cells) and in vivo (genetic manipulation in Drosophila) evidence for an endogenous role of UBE3A in regulating circadian dynamics and rhythmic locomotor behaviour. Together, our data reveal an essential and conserved role of UBE3A in the regulation of the circadian system in mammals and flies and identify a novel mechanistic link between oncogene E6/E7-mediated cell transformation and circadian (BMAL1) disruption.

JNK regulates the photic response of the mammalian circadian clock.

The posttranslational regulation of mammalian clock proteins has been assigned a time-keeping function, but seems to have more essential roles. Here we show that c-Jun N-terminal kinase (JNK), identified by inhibitor screening of BMAL1 phosphorylation at Ser 520/Thr 527/Ser 592, confers dynamic regulation on the clock. Knockdown of JNK1 and JNK2 abrogates BMAL1 phosphorylation and lengthens circadian period in fibroblasts. Mice deficient for neuron-specific isoform JNK3 have altered behavioural rhythms, with longer free-running period and compromised phase shifts to light. The locomotor rhythms are insensitive to intensity variance of constant light, deviating from Aschoff's rule. Thus, JNK regulates a core characteristic of the circadian clock by controlling the oscillation speed and the phase in response to light.

Ror homolog nhr-23 is essential for both developmental clock and circadian clock in C. elegans.

Animals have internal clocks that generate biological rhythms. In mammals, clock genes such as Period form the circadian clock to generate approximately 24-h biological rhythms. In C. elegans, the clock gene homologs constitute the "developmental clock", which has an 8-h period during larval development to determine the timing of molting. Thus, the ancestral circadian clock has been believed to evolve into the oscillator with a shorter period in C. elegans. However, circadian rhythms have also been observed in adult C. elegans, albeit relatively weak. This prompts the question: if the clock gene homologs drive the developmental rhythm with 8-h period, which genes generate the circadian rhythms in C. elegans? In this study, we discovered that nhr-23, a homolog of the mammalian circadian clock gene Ror, is essential for circadian transcriptional rhythms in adult C. elegans. Interestingly, nhr-23 was also known to be essential for the molting clock. The bilaterian ancestral circadian clock genes might have evolved to function over multiple periods depending on developmental contexts rather than a single 8-h period in C. elegans.

Neocortical neuronal production and maturation defects in the TcMAC21 mouse model of Down syndrome.

Down syndrome (DS) results from trisomy of human chromosome 21 (HSA21), and DS research has been conducted by the use of mouse models. We previously generated a humanized mouse model of DS, TcMAC21, which carries the long arm of HSA21. These mice exhibit learning and memory deficits, and may reproduce neurodevelopmental alterations observed in humans with DS. Here, we performed histologic studies of the TcMAC21 forebrain from embryonic to adult stages. The TcMAC21 neocortex showed reduced proliferation of neural progenitors and delayed neurogenesis. These abnormalities were associated with a smaller number of projection neurons and interneurons. Further, (phospho-)proteomic analysis of adult TcMAC21 cortex revealed alterations in the phosphorylation levels of a series of synaptic proteins. The TcMAC21 mouse model shows similar brain development abnormalities as DS, and will be a valuable model to investigate prenatal and postnatal causes of intellectual disability in humans with DS.

Diurnal shift of mouse activity by the deficiency of an ageing-related gene Lmna.

Nuclear lamina is a fundamental structure of the cell nucleus and regulates a wide range of molecular pathways. Defects of components of the nuclear lamina cause ageing-like physiological disorders, called laminopathy. Generally, ageing and diseases are often associated with perturbation of various time-of-day-dependent regulations, but it remains elusive whether laminopathy induces any changes of the circadian clock and physiological rhythms. Here, we demonstrated that deficiency of Lmna gene in mice caused an obvious shift of locomotor activities to the daytime. The abnormal activity profile was accompanied by a remarkable change in phase angle between the central clock in the suprachiasmatic nucleus (SCN) and the lung peripheral clocks, leaving the phase of the SCN clock unaffected by the mutation. These observations suggest that Lmna deficiency causes a change of the habitat from nocturnal to diurnal behaviours. On the other hand, molecular oscillation and its phase resetting mechanism were intact in both the Lmna-deficient cells and progeria-mimicking cells. Intriguingly, high-fat diet feeding extended the short lifespan and ameliorated the abnormalities of the behaviours and the phase of the peripheral clock in the Lmna-deficient mice. The present study supports the important contribution of the energy conditions to a shift between the diurnal and nocturnal activities.

Molecular encoding and synaptic decoding of context during salt chemotaxis in C. elegans.

Animals navigate toward favorable locations using various environmental cues. However, the mechanism of how the goal information is encoded and decoded to generate migration toward the appropriate direction has not been clarified. Here, we describe the mechanism of migration towards a learned concentration of NaCl in Caenorhabditis elegans. In the salt-sensing neuron ASER, the difference between the experienced and currently perceived NaCl concentration is encoded as phosphorylation at Ser65 of UNC-64/Syntaxin 1 A through the protein kinase C(PKC-1) signaling pathway. The phosphorylation affects basal glutamate transmission from ASER, inducing the reversal of the postsynaptic response of reorientation-initiating neurons (i.e., from inhibitory to excitatory), guiding the animals toward the experienced concentration. This process, the decoding of the context, is achieved through the differential sensitivity of postsynaptic excitatory and inhibitory receptors. Our results reveal the mechanism of migration based on the synaptic plasticity that conceptually differs from the classical ones.

Adiponectin regulates the circadian rhythm of glucose and lipid metabolism.

Adiponectin is a cytokine secreted from adipocytes and regulates metabolism. Although serum adiponectin levels show diurnal variations, it is not clear if the effects of adiponectin are time-dependent. Therefore, this study conducted locomotor activity analyses and various metabolic studies using the adiponectin knockout (APN (-/-)) and the APN (+/+) mice to understand whether adiponectin regulates the circadian rhythm of glucose and lipid metabolism. We observed that the adiponectin gene deficiency does not affect the rhythmicity of core circadian clock genes expression in several peripheral tissues. In contrast, the adiponectin gene deficiency alters the circadian rhythms of liver and serum lipid levels and results in the loss of the time dependency of very-low-density lipoprotein-triglyceride secretion from the liver. In addition, the whole-body glucose tolerance of the APN (-/-) mice was normal at CT10 but reduced at CT22, compared to the APN (+/+) mice. The decreased glucose tolerance at CT22 was associated with insulin hyposecretion in vivo. In contrast, the gluconeogenesis activity was higher in the APN (-/-) mice than in the APN (+/+) mice throughout the day. These results indicate that adiponectin regulates part of the circadian rhythm of metabolism in the liver.

mTOR-AKT Signaling in Cellular Clock Resetting Triggered by Osmotic Stress.

Aims: The circadian clock oscillates in a cell-autonomous manner with a period of ∼24 h, and the phase is regulated by various time cues such as light and temperature through multiple clock input pathways. We previously found that osmotic and oxidative stress strongly affected the circadian period and phase of cellular rhythms, and triple knockout of apoptosis signal-regulating kinase (ASK) family members, Ask1, Ask2, and Ask3, abolished the phase shift (clock resetting) induced by hyperosmotic pulse treatment. We aimed at exploring a key molecule(s) and signaling events in the clock input pathway dependent on ASK kinases. Results: The phase shift of the cellular clock induced by the hyperosmotic pulse treatment was significantly reduced by combined deficiencies of the clock(-related) genes, Dec1, Dec2, and E4 promoter-binding protein 4 (also known as Nfil3) (E4bp4). In addition, liquid chromatography mass/mass spectrometry (LC-MS/MS)-based proteomic analysis identified hyperosmotic pulse-induced phosphorylation of circadian locomotor output cycles caput (CLOCK) Ser845 in an AKT-dependent manner. We found that AKT kinase was phosphorylated at Ser473 (i.e., activated) in response to the hyperosmotic pulse experiments. Inhibition of mechanistic target of rapamycin (mTOR) kinase by Torin 1 treatment completely abolished the AKT activation, suppressed the phosphorylation of CLOCK Ser845, and blocked the clock resetting induced by the hyperosmotic pulse treatment. Innovation and Conclusions: We conclude that mTOR-AKT signaling is indispensable for the CLOCK Ser845 phosphorylation, which correlates with the clock resetting induced by the hyperosmotic pulse treatment. Immediate early induction of the clock(-related) genes and CLOCK carboxyl-terminal (C-terminal) region containing Ser845 also play important roles in the clock input pathway through redox-sensitive ASK kinases. Antioxid. Redox Signal. 37, 631-646.

Rhythmic transcription of Bmal1 stabilizes the circadian timekeeping system in mammals.

In mammals, the circadian clock consists of transcriptional and translational feedback loops through DNA cis-elements such as E-box and RRE. The E-box-mediated core feedback loop is interlocked with the RRE-mediated feedback loop, but biological significance of the RRE-mediated loop has been elusive. In this study, we established mutant cells and mice deficient for rhythmic transcription of Bmal1 gene by deleting its upstream RRE elements and hence disrupted the RRE-mediated feedback loop. We observed apparently normal circadian rhythms in the mutant cells and mice, but a combination of mathematical modeling and experiments revealed that the circadian period and amplitude of the mutants were more susceptible to disturbance of CRY1 protein rhythm. Our findings demonstrate that the RRE-mediated feedback regulation of Bmal1 underpins the E-box-mediated rhythm in cooperation with CRY1-dependent posttranslational regulation of BMAL1 protein, thereby conferring the perturbation-resistant oscillation and chronologically-organized output of the circadian clock.

Circadian Phosphorylation of CLOCK and BMAL1.

Daily rhythms of behaviors and physiologies are driven by transcriptional-translational negative feedback loops of clock genes and encoded clock proteins (Bass and Takahashi Science 330:1349-1354, 2010; Brown et al. Dev Cell 22:477-487, 2012). Posttranslational modifications of clock proteins, including protein phosphorylation, play an essential role for normal oscillation of the circadian clock through regulation of their activities, stabilities, interactions, and intracellular localization (Gallego and Virshup Nat Rev Mol Cell Biol 8:139-148, 2007; Hirano et al. Nat Struct Mol Biol 23:1053-1060, 2016). In this chapter, we describe detailed methods for quantitative analysis of phosphorylation levels of clock proteins, particularly focusing on circadian phosphorylation of CLOCK, BMAL1, and their complex (Yoshitane et al. Mol Cell Biol 29:3675-3686, 2009).

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation.

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

Post-transcriptional repression of circadian component CLOCK regulates cancer-stemness in murine breast cancer cells.

Disruption of the circadian clock machinery in cancer cells is implicated in tumor malignancy. Studies on cancer therapy reveal the presence of heterogeneous cells, including breast cancer stem-like cells (BCSCs), in breast tumors. BCSCs are often characterized by high aldehyde dehydrogenase (ALDH) activity, associated with the malignancy of cancers. In this study, we demonstrated the negative regulation of ALDH activity by the major circadian component CLOCK in murine breast cancer 4T1 cells. The expression of CLOCK was repressed in high-ALDH-activity 4T1, and enhancement of CLOCK expression abrogated their stemness properties, such as tumorigenicity and invasive potential. Furthermore, reduced expression of CLOCK in high-ALDH-activity 4T1 was post-transcriptionally regulated by microRNA: miR-182. Knockout of miR-182 restored the expression of CLOCK, resulted in preventing tumor growth. Our findings suggest that increased expression of CLOCK in BCSCs by targeting post-transcriptional regulation overcame stemness-related malignancy and may be a novel strategy for breast cancer treatments.

Preferential inhibition of BMAL2-CLOCK activity by PER2 reemphasizes its negative role and a positive role of BMAL2 in the circadian transcription.

In the molecular oscillatory mechanism governing circadian rhythms, positive regulators, including CLOCK and BMAL1, transactivate Per and Cry genes through E-box elements, and translated PER and CRY proteins negatively regulate their own transactivation. Like BMAL1, its paralog BMAL2 dimerizes with CLOCK to activate the E-box-dependent transcription, but the role of BMAL2 in the circadian clockwork is still elusive. Here we characterized BMAL2 function in NIH3T3 cells and found that the cellular rhythms monitored by Bmal1 promoter-driven bioluminescence signals were blunted by RNA interference-mediated suppression of Bmal2 as well as that of Bmal1. Transcription assays with a 2.1-kb mPer1 promoter revealed that CRY2 inhibited the transactivation mediated by BMAL1-CLOCK more strongly than that by BMAL2-CLOCK. In contrast, PER2 showed a stronger inhibitory effect on BMAL2-CLOCK than on BMAL1-CLOCK. The molecular link between BMAL2 and PER2 was further strengthened by the fact that PER2 exhibited a greater affinity for BMAL2 than for BMAL1 in co-immunoprecipitation experiments. These results indicate a functional partnership between BMAL2 and PER2 and reemphasize the negative role of PER2 in the circadian transcription. As a broad spectrum function, BMAL2-CLOCK activated transcription from a variety of SV40-driven reporters harboring various E/E'-box-containing sequences present in the upstream regions of clock and clock-controlled genes. Importantly, the efficiencies of BMAL2-CLOCK-mediated transactivation relative to that achieved by BMAL1-CLOCK were dependent heavily on the E-box-containing sequences, supporting distinguishable roles of the two BMALs. Collectively, it is strongly suggested that BMAL2 plays an active role in the circadian transcription.

Functional D-box sequences reset the circadian clock and drive mRNA rhythms.

The circadian clock drives gene expression rhythms, leading to daily changes in physiology and behavior. In mammals, Albumin D-site-Binding Protein (DBP) rhythmically activates transcription of various genes through a DNA cis-element, D-box. The DBP-dependent transactivation is repressed by competitive binding of E4BP4 to the D-box. Despite the elaborate regulation, physiological roles of the D-box in the circadian clockwork are still elusive. Here we identified 1490 genomic regions recognized commonly by DBP and E4BP4 in the mouse liver. We comprehensively defined functional D-box sequences using an improved bioinformatics method, MOCCS2. In RNA-Seq analysis of E4bp4-knockout and wild type liver, we showed the importance of E4BP4-mediated circadian repression in gene expression rhythms. In addition to the circadian control, we found that environmental stimuli caused acute induction of E4BP4 protein, evoking phase-dependent phase shifts of cellular circadian rhythms and resetting the clock. Collectively, D-box-mediated transcriptional regulation plays pivotal roles in input and output in the circadian clock system.