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.

Singularity response reveals entrainment properties in mammalian circadian clock.

Entrainment is characterized by phase response curves (PRCs), which provide a summary of responses to perturbations at each circadian phase. The synchronization of mammalian circadian clocks is accomplished through the receipt of a variety of inputs from both internal and external time cues. A comprehensive comparison of PRCs for various stimuli in each tissue is required. Herein, we demonstrate that PRCs in mammalian cells can be characterized using a recently developed estimation method based on singularity response (SR), which represents the response of desynchronized cellular clocks. We confirmed that PRCs can be reconstructed using single SR measurements and quantified response properties for various stimuli in several cell lines. SR analysis reveals that the phase and amplitude after resetting are distinguishable among stimuli. SRs in tissue slice cultures reveal tissue-specific entrainment properties. These results demonstrate that SRs can be employed to unveil entrainment mechanisms with diverse stimuli in multiscale mammalian clocks.

The circadian clock in the piriform cortex intrinsically tunes daily changes of odor-evoked neural activity.

The daily activity in the brain is typically fine-tuned by the circadian clock in the local neurons as well as by the master circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. In the olfactory response, odor-evoked activity in the piriform cortex (PC) and olfactory behavior retain circadian rhythmicity in the absence of the SCN, yet how the circadian rhythm in the PC is achieved independently of the SCN remains elusive. Here, to define neurons regulating the circadian rhythm of the odor-evoked activity in the PC, we knocked out the clock gene Bmal1 in a host of specific neurons along the olfactory circuit. We discovered that Bmal1 knockout in the PC largely abolishes the circadian rhythm of the odor-evoked activity. We further showed that isolated PC exhibits sustained circadian rhythms of the clock gene Per2 expression. Quantitative PCR analysis revealed that expression patterns of multiple genes involved in neural activity and synaptic transmission exhibit circadian rhythm in the PC in a BMAL1-dependent manner. Our findings indicate that BMAL1 acts intrinsically in the PC to control the circadian rhythm of the odor-evoked activity in the PC, possibly through regulating expression patterns of multiple genes involved in neural activity and transmission.

Diurnal variation in declarative memory and the involvement of SCOP in cognitive functions in nonhuman primates.

Cognitive functions depend on the time of day in various organisms. Previously, we found that 24-h recognition memory performance of nocturnal mice changes diurnally through SCOP protein-dependent regulation. It remains unknown whether diurnal change and SCOP-dependent regulation of memory performance are conserved across species with diurnal/nocturnal habits. We tested whether the memory performance of diurnal Japanese macaques depends on the time of day. The memory association between bitter taste of drinking water and the nozzle color of the water bottle was established during the light period of the day to evaluate of memory performance for macaques. Here we found diurnal variation of declarative memory in Japanese macaques. The middle of the daytime is the most effective time for memory performance during the light period. To assess whether SCOP is involved in declarative memory performance, we interfered with SCOP expression by using lentiviral vector expressing shRNA against Scop in the hippocampus of Japanese macaques. Scop knockdown in the hippocampus abrogated the memory performance in the middle of the daytime. Our results implicate that SCOP in the hippocampus is necessary for the diurnal rhythm of the memory system and that the SCOP-dependent memory regulation system may be conserved in mammals.

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.

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.

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.

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.

Molecular connections between circadian clock and health/ageing.

For decades, considerable efforts have been expended for solving the molecular mechanisms of disease progression. An important clue to tackle this question is the circadian clock. Recent findings have uncovered previously unknown molecular connections between circadian clock and disease incidence, consequently causing the ageing process. Furthermore, 'chronotherapy' is emerging as a new concept of optimizing the time of the day for drug administration according to target gene expressions in order to maximize therapeutic efficacy and minimize the side effects. This concept will help cure patients and prevent them from suffering evitable pain and side effects. This JB special issue 'Molecular connections between circadian clock and health/aging' discusses how the circadian clocks link to health and ageing from molecular to organismal levels.

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.

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.

[Photoreceptors for Entrainment of Circadian Rhythms].

In mammals, the intrinsic circadian clock regulates various physiological rhythms, including sleep-wake cycles. These circadian rhythms can be entrained to daily 24-hour light-dark cycles by virtue of photoreception within the retina. The photo-entrainment of circadian rhythms is predominantly mediated by intrinsically photosensitive retinal ganglion cells (ipRGCs). In this article, we review the mechanisms of action of ipRGC photoreception, the retinal circuit involving ipRGCs, and the heterogeneity of ipRGCs in mice.

Foxq2 determines blue cone identity in zebrafish.

Most vertebrate lineages retain a tetrachromatic visual system, which is supported by a functional combination of spectrally distinct multiple cone photoreceptors, ultraviolet (UV), blue, green, and red cones. The blue cone identity is ensured by selective expression of blue (sws2) opsin, and the mechanism is poorly understood because sws2 gene has been lost in mammalian species such as mouse, whose visual system has been extensively studied. Here, we pursued loss-of-function studies on transcription factors expressed predominantly in zebrafish cone photoreceptors and identified Foxq2 as a blue cone­specific factor driving sws2 gene expression. Foxq2 has dual functions acting as an activator of sws2 transcription and as a suppressor of UV (sws1) opsin transcription in blue cones. A wide range of vertebrate species retain both foxq2 and sws2 genes. We propose that Foxq2-dependent sws2 expression is a prevalent regulatory mechanism that was acquired at the early stage of vertebrate evolution.

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.

Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms.

Circadian rhythms are based on biochemical oscillations generated by clock genes/proteins, which independently evolved in animals, fungi, plants, and cyanobacteria. Temperature compensation of the oscillation speed is a common feature of the circadian clocks, but the evolutionary-conserved mechanism has been unclear. Here, we show that Na+/Ca2+ exchanger (NCX) mediates cold-responsive Ca2+ signaling important for the temperature-compensated oscillation in mammalian cells. In response to temperature decrease, NCX elevates intracellular Ca2+, which activates Ca2+/calmodulin-dependent protein kinase II and accelerates transcriptional oscillations of clock genes. The cold-responsive Ca2+ signaling is conserved among mice, Drosophila, and Arabidopsis The mammalian cellular rhythms and Drosophila behavioral rhythms were severely attenuated by NCX inhibition, indicating essential roles of NCX in both temperature compensation and autonomous oscillation. NCX also contributes to the temperature-compensated transcriptional rhythms in cyanobacterial clock. Our results suggest that NCX-mediated Ca2+ signaling is a common mechanism underlying temperature-compensated circadian rhythms both in eukaryotes and prokaryotes.

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.

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).