Tsuyoshi Hirota

Resetting Mechanism of Central and Peripheral Circadian Clocks in Mammals

Almost all organisms on earth exhibit diurnal rhythms in physiology and behavior under the control of autonomous time-measuring system called circadian clock. The circadian clock is generally reset by environmental time cues, such as light, in order to synchronize with the external 24-h cycles. In mammals, the core oscillator of the circadian clock is composed of transcription/translation-based negative feedback loops regulating the cyclic expression of a limited number of clock genes (such as Per, Cry, Bmal1, etc.) and hundreds of output genes in a well-concerted manner. The central clock controlling the behavioral rhythm is localized in the hypothalamic suprachiasmatic nucleus (SCN), and peripheral clocks are present in other various tissues. The phase of the central clock is amenable to ambient light signal captured by the visual rod-cone photoreceptors and non-visual melanopsin in the retina. These light signals are transmitted to the SCN through the retinohypothalamic tract, and transduced therein by mitogen-activated protein kinase and other signaling molecules to induce Per gene expression, which eventually elicits phase-dependent phase shifts of the clock. The central clock controls peripheral clocks directly and indirectly by virtue of neural, humoral, and other signals in a coordinated manner. The change in feeding time resets the peripheral clocks in a SCN-independent manner, possibly by food metabolites and body temperature rhythms. In this article, we will provide an overview of recent molecular and genetic studies on the resetting mechanism of the central and peripheral circadian clocks in mammals.

Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts.

The circadian clock is reset by external time cues for synchronization to environmental changes. In mammals, the light-input signalling pathway mediated by Per gene induction has been extensively studied. On the other hand, little is known about resetting mechanisms that are independent of Per induction. Here we show that activation of activin receptor-like kinase (ALK), triggered by TGF-β, activin or alkali signals, evoked resetting of the cellular clock independently of Per induction. The resetting was mediated by an immediate-early induction of Dec1, a gene whose physiological role in the function of the circadian clock has been unclear. Acute Dec1 induction was a prerequisite for ALK-mediated resetting and upregulation was dependent on SMAD3, which was phosphorylated for activation in response to the resetting stimuli. Intraperitoneal injection of TGF-β into wild-type or Dec1-deficient mice demonstrated that Dec1 has an essential role in phase-shift of clock gene expression in the kidney and adrenal gland. These results indicate that ALK–SMAD3–Dec1 signalling provides an input pathway in the mammalian molecular clock.

Chicken pineal clock genes: implication of BMAL2 as a bidirectional regulator in circadian clock oscillation.

Background In a transcription/translation‐based autoregulatory feedback loop of vertebrate circadian clock systems, a BMAL1‐CLOCK heterodimer is a positive regulator for the transcription of the negative element gene Per. The chicken pineal gland represents a photosensitive clock tissue, but the pineal clock genes constituting the oscillator loop have been less well characterized.

Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day–dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-day–dependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.

Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat‐1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1‐mediated repression of Bmal1 promoter was additive to RORE‐dependent repression by REV‐ERBα, a well‐known repressor of Bmal1 gene. In cell‐based real‐time assay, siRNA‐mediated knock‐down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1‐luciferase and Per2‐luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box‐mediated regulation of Bmal1 transcription.

Phosphorylation of mCRY2 at Ser557 in the Hypothalamic Suprachiasmatic Nucleus of the Mouse

Cryptochrome1 and 2 play a critical role in the molecular oscillations of the circadian clocks of central and peripheral tissues in mammals. Mouse Cryptochrome2 (mCRY2) is phosphorylated at Ser557 in the liver, in which the Ser557‐phosphorylated form accumulates during the night in parallel with mCRY2 protein. Phosphorylation of mCRY2 at Ser557 allows subsequent phosphorylation at Ser553 by glycogen synthase kinase‐3β (GSK‐3β), resulting in efficient degradation of mCRY2 by a proteasome pathway. In the present study, we found that mCRY2 is phosphorylated at Ser557 also in the region of the mouse brain containing the suprachiasmatic nucleus (SCN), the central circadian clock tissue. Daily fluctuation of the Ser557‐phosphorylation level in the SCN region suggests an important role of sequential phosphorylation of Ser557 and Ser553 in the rhythmic degradation of mCRY2 in both central and peripheral clocks of mice.

Global rise of potential health hazards caused by blue light-induced circadian disruption in modern aging societies

Mammals receive light information through the eyes, which perform two major functions: image forming vision to see objects and non-image forming adaptation of physiology and behavior to light. Cone and rod photoreceptors form images and send the information via retinal ganglion cells to the brain for image reconstruction. In contrast, nonimage-forming photoresponses vary widely from adjustment of pupil diameter to adaptation of the circadian clock. nonimage-forming responses are mediated by retinal ganglion cells expressing the photopigment melanopsin. Melanopsin-expressing cells constitute 1–2% of retinal ganglion cells in the adult mammalian retina, are intrinsically photosensitive, and integrate photic information from rods and cones to control nonimage-forming adaptation. Action spectra of ipRGCs and of melanopsin photopigment peak around 480 nm blue light. Understanding melanopsin function lets us recognize considerable physiological effects of blue light, which is increasingly important in our modern society that uses light-emitting diode. Misalignment of circadian rhythmicity is observed in numerous conditions, including aging, and is thought to be involved in the development of age-related disorders, such as depression, diabetes, hypertension, obesity, and cancer. The appropriate regulation of circadian rhythmicity by proper lighting is therefore essential. This perspective introduces the potential risks of excessive blue light for human health through circadian rhythm disruption and sleep deprivation. Knowing the positive and negative aspects, this study claims the importance of being exposed to light at optimal times and intensities during the day, based on the concept of the circadian clock, ultimately to improve quality of life to have a healthy and longer life.

In Vivo Role of Phosphorylation of Cryptochrome 2 in the Mouse Circadian Clock

ABSTRACT The circadian clock is finely regulated by posttranslational modifications of clock components. Mouse CRY2, a critical player in the mammalian clock, is phosphorylated at Ser557 for proteasome-mediated degradation, but its in vivo role in circadian organization was not revealed. Here, we generated CRY2(S557A) mutant mice, in which Ser557 phosphorylation is specifically abolished. The mutation lengthened free-running periods of the behavioral rhythms and PER2::LUC bioluminescence rhythms of cultured liver. In livers from mutant mice, the nuclear CRY2 level was elevated, with enhanced PER2 nuclear occupancy and suppression of E-box-regulated genes. Thus, Ser557 phosphorylation-dependent regulation of CRY2 is essential for proper clock oscillation in vivo.

Heat Shock Factors Modulate Circadian Rhythms

Temperature changes have a variety of effects on physiological processes including the circadian clock that generates diurnal rhythms of sleep/wake behavior, hormone release, metabolism, and so on. Even in homeothermic mammals, body temperature fluctuates in a circadian manner that transmits the time information to peripheral tissues and cells. The body temperature rhythms cause cyclic activation of the transcription factor HSF1 and its targets such as HSP genes and a clock gene Per2, resulting in adjustment of the circadian oscillation in peripheral cells for synchronization. Loss of function of HSF1 therefore leads to reduced synchronization of the clock against temperature changes. HSF1 inhibition also slows down the speed of the clock oscillation and impairs the mechanism that maintains the oscillation speed constant under varying temperature. In the chick pineal gland, a photosensitive clock tissue, HSF and HSP genes are activated by light pulse at a specific time of the day, suggesting a role of the HSF pathway in light-dependent synchronization of the circadian clock. Together, HSF has substantial roles in modulating the circadian clock function in response to environmental changes.

Nuclear receptor HNF4A trans-represses CLOCK:BMAL1 and acts as a core component of tissue-specific circadian networks

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly trans-represses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed co-occupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose and amino acid homeostasis. Taken together, we establish that HNF4A defines a novel feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways. Significance Interlocked feedback loops promote robustness and stability in a system and are a feature of circadian clocks in both animal and plants. The mammalian circadian clock is known to consist of two transcriptional feedback loops, relying on the transcriptional activity of the master complex CLOCK:BMAL1 and the feedback regulation by its target genes. Our research extends this knowledge by establishing a novel feedback loop in peripheral circadian oscillators and highlights the underlying mechanisms mediated by the unappreciated CLOCK:BMAL1 trans-repression activity of the circadian nuclear receptor HNF4A.