Dimerization and nuclear entry of mPER proteins in mammalian cells

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner

Methylation deficiency disrupts biological rhythms from bacteria to humans

メチル化と体内時計が生命誕生以来の密な関係にあることを発見 --生命の起源に学ぶヒト障害の新治療法--. 京都大学プレスリリース. 2020-05-27.The methyl cycle is a universal metabolic pathway providing methyl groups for the methylation of nuclei acids and proteins, regulating all aspects of cellular physiology. We have previously shown that methyl cycle inhibition in mammals strongly affects circadian rhythms. Since the methyl cycle and circadian clocks have evolved early during evolution and operate in organisms across the tree of life, we sought to determine whether the link between the two is also conserved. Here, we show that methyl cycle inhibition affects biological rhythms in species ranging from unicellular algae to humans, separated by more than 1 billion years of evolution. In contrast, the cyanobacterial clock is resistant to methyl cycle inhibition, although we demonstrate that methylations themselves regulate circadian rhythms in this organism. Mammalian cells with a rewired bacteria-like methyl cycle are protected, like cyanobacteria, from methyl cycle inhibition, providing interesting new possibilities for the treatment of methylation deficiencies

Activation of AMPA Receptors in the Suprachiasmatic Nucleus Phase-Shifts the Mouse Circadian Clock In Vivo and In Vitro

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN

Quantification of Circadian Rhythms in Single Cells

Bioluminescence techniques allow accurate monitoring of the circadian clock in single cells. We have analyzed bioluminescence data of Per gene expression in mouse SCN neurons and fibroblasts. From these data, we extracted parameters such as damping rate and noise intensity using two simple mathematical models, one describing a damped oscillator driven by noise, and one describing a self-sustained noisy oscillator. Both models describe the data well and enabled us to quantitatively characterize both wild-type cells and several mutants. It has been suggested that the circadian clock is self-sustained at the single cell level, but we conclude that present data are not sufficient to determine whether the circadian clock of single SCN neurons and fibroblasts is a damped or a self-sustained oscillator. We show how to settle this question, however, by testing the models' predictions of different phases and amplitudes in response to a periodic entrainment signal (zeitgeber)

Cigarette smoking, genetic polymorphisms and colorectal cancer risk: the Fukuoka Colorectal Cancer Study

Background: It is uncertain whether smoking is related to colorectal cancer risk. Cytochrome P-450 CYP1A1, glutathione-S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are important enzymes in the metabolism of tobacco carcinogens, and functional genetic polymorphisms are known for these enzymes. We investigated the relation of cigarette smoking and related genetic polymorphisms to colorectal cancer risk, with special reference to the interaction between smoking and genetic polymorphism. Methods: We used data from the Fukuoka Colorectal Cancer Study, a population-based case-control study, including 685 cases and 778 controls who gave informed consent to genetic analysis. Interview was conducted to assess lifestyle factors, and DNA was extracted from buffy coat. Results: In comparison with lifelong nonsmokers, the odds ratios (OR) of colorectal cancer for <400, 400-799 and ≥800 cigarette-years were 0.65 (95 % confidence interval [CI], 0.45-0.89), 1.16 (0.83-1.62) and 1.14 (0.73-1.77), respectively. A decreased risk associated with light smoking was observed only for colon cancer, and rectal cancer showed an increased risk among those with ≥400 cigarette-years (OR 1.60, 95 % CI 1.04-2.45). None of the polymorphisms under study was singly associated with colorectal cancer risk. Of the gene-gene interactions studied, the composite genotype of CYP1A1*2A or CYP1A1*2C and GSTT1 polymorphisms was associated with a decreased risk of colorecta

Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals

マウスとハエに共通にみられる体温の日内リズムを制御する仕組み. 京都大学プレスリリース. 2018-02-14.Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila. DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms

Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour.

体内時計を調節するオーファンGPCRの同定 －生体リズム調整薬の開発に期待－. 京都大学プレスリリース. 2016-02-18.G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz