Circadian control by the reduction/oxidation pathway: Catalase represses light-dependent clock gene expression in the zebrafish (original) (raw)
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Biochemical and Biophysical Research Communications, 2006
To elucidate the roles of DEC1 and DEC2, basic helix-loop-helix transcription factors, in the circadian clock of photosensitive zebrafish peripheral cells, zebrafish Dec1 and Dec2 (zDec1 and zDec2) were cloned and their functions and expression patterns were examined in BRF41, a zebrafish cell line. zDEC1 and zDEC2 have high sequence similarity to mammalian counterparts and the molecular phylogenetic analysis of the zDEC1 and zDEC2 sequences reflected the predicted pattern of species classification. zDEC1 and zDEC2 inhibited zCLOCK1:zBMAL3 mediated transcription as CRY1a. zDec1 and zDec2 mRNA showed robust circadian oscillation in BRF41 cells. However, zDec1 and zDec2 mRNA was not strongly induced by exposure to light. These results indicate that zDec1 and zDec2 are involved in the circadian clock mechanism in photosensitive zebrafish peripheral cells by suppressing CLOCK/BMAL-induced gene expression and that the feedback loops of zDEC1 and zDEC2 may be interlocked with the PER/CRY core circadian feedback loops.
Proceedings of the National Academy of Sciences, 2005
The cell cycle and the circadian clock are endogenous pacemakers, which coexist in most eukaryotic cells and share a number of conceptual features. In the zebrafish, light directly regulates the timing of both clocks, although the signaling and transcriptional pathways that convey photic information to essential nuclear regulators have yet to be deciphered. We have previously established the Z3 cell line, which recapitulates the features of zebrafish circadian clock and represents an ideal system to study lightdependent signaling and gene regulation. We conducted a search for light-responsive transcription factors and found that AP-1 DNA binding is highly induced. Light induces the expression of zWee1, a cell cycle gene essential for G2͞M transition, and zCry1a, a clock gene of the feedback regulatory loop. We have found consensus AP-1 sites in the regulatory regions of both zWee1 and zCry1a genes, and we show that light inducibility of both genes is abrogated by inhibition of AP-1 function. Light also elicits chromatin remodeling by stimulating hyperacetylation at Lys-14 of histone H3 at both zWee1 and zCry1a promoters, as assessed by chromatin immunoprecipitation assays by using anti-Fos antibody. These findings provide strong evidence that circadian and cell cycle clocks share unique light-responsive pathways in zebrafish.
From Blue Light to Clock Genes in Zebrafish ZEM-2S Cells
Melanopsin has been implicated in the mammalian photoentrainment by blue light. This photopigment, which maximally absorbs light at wavelengths between 470 and 480 nm depending on the species, is found in the retina of all classes of vertebrates so far studied. In mammals, melanopsin activation triggers a signaling pathway which resets the circadian clock in the suprachiasmatic nucleus (SCN). Unlike mammals, Drosophila melanogaster and Danio rerio do not rely only on their eyes to perceive light, in fact their whole body may be capable of detecting light and entraining their circadian clock. Melanopsin, teleost multiple tissue (tmt) opsin and others such as neuropsin and va-opsin, are found in the peripheral tissues of Danio rerio, however, there are limited data concerning the photopigment/s or the signaling pathway/s directly involved in light detection. Here, we demonstrate that melanopsin is a strong candidate to mediate synchronization of zebrafish cells. The deduced amino acid sequence of melanopsin, although being a vertebrate opsin, is more similar to invertebrate than vertebrate photopigments, and melanopsin photostimulation triggers the phosphoinositide pathway through activation of a G q/11 -type G protein. We stimulated cultured ZEM-2S cells with blue light at wavelengths consistent with melanopsin maximal absorption, and evaluated the time course expression of per1b, cry1b, per2 and cry1a. Using quantitative PCR, we showed that blue light is capable of slightly modulating per1b and cry1b genes, and drastically increasing per2 and cry1a expression. Pharmacological assays indicated that per2 and cry1a responses to blue light are evoked through the activation of the phosphoinositide pathway, which crosstalks with nitric oxide (NO) and mitogen activated protein MAP kinase (MAPK) to activate the clock genes. Our results suggest that melanopsin may be important in mediating the photoresponse in Danio rerio ZEM-2S cells, and provide new insights about the modulation of clock genes in peripheral clocks.
CLOCK:BMAL-Independent Circadian Oscillation of Zebrafish Cryptochrome1a Gene
Biological & Pharmaceutical Bulletin, 2009
Organisms ranging from bacteria to humans have daily rhythms driven by endogenous oscillators called circadian clocks, which regulate various biochemical, physiological, and behavioral processes. 1) Under natural conditions, circadian rhythms are entrained to a 24-h cycle by environmental time cues, of which light is the most important. Over the past few years, the molecular mechanisms responsible for these oscillations have been thoroughly investigated and specific "clock genes" that control this rhythm have been identified. The core of the clock mechanism in Drosophila, Neurospora, and mammals is commonly represented by a transcription/ translation-based negative-feedback loop that relies on positive and negative oscillator elements. 2,3) Although the organization of the negative feedback loop in Drosophila, Neurospora, and mammals is conceptually similar, its components differ among species. 3) In mammals, two basic helix-loop-helix PAS (PER-ARNT-SIM) domain-containing transcription factors, CLOCK and BMAL, constitute the positive elements. 4,5) Upon heterodimerization, the CLOCK: BMAL complex drives the transcription of the negative components of the clock machinery, two Cryptochrome genes (Cry1 and Cry2). CRYs negatively regulate their own expression, therefore setting up the rhythmic oscillations of gene expression that drive the circadian clock. 6) Zebrafish possess an intrinsic autonomous oscillator that consists of components similar to those of mammals. 7) zCLOCK and zBMAL act as positive elements and zCRYs act as negative regulators. As the result of whole-genome duplication during the evolution of the teleost lineage, the circadian oscillator of zebrafish contains duplications for most of the clock genes. Interestingly, zebrafish have four repressor types of CRYs (zCRY1a, zCRY1b, zCRY2a, and zCRY2b). Despite the structural and functional similarities seen in vitro, their expression profiles are quite different. 7) Expression of zCry1b, zCry2a and zCry2b are under the control of CLOCK:BMAL heterodimer, showing a clear circadian oscillation both in light-dark (LD) and constant dark (DD) conditions. 8) In contrast, although zCry1a exhibits a circadian oscillation in cultured cells exposed to a LD cycle, this oscillation dampens quickly after the transfer of the cells to a DD condition. 9-11) Thus, transcriptional regulation of zCry1a is believed to be CLOCK:BMAL-independent. Zebrafish oscillators in peripheral tissues and cell lines derived from zebrafish tissues display direct-light responsiveness. 12) In fact, zebrafish cultured cells constitute an attractive alternative to the mammalian system to study the complexity of the circadian clock machinery and the influence that light has on it. In zebrafish cells, light directly activates the expression of zCry1a. 10,11) Light-induced zCRY1a in turn inhibits CLOCK:BMAL-dependent transcription, thereby participating in the light entrainment of the circadian clock. 10,11) Moreover, a critical role for extracellular signalregulated kinase (ERK) signaling pathway in the circadian transcriptional regulation has been established in a variety of species. 9,13) Indeed, we have previously reported that lightinduced zCry1a expression is achieved through activation of the ERK signaling cascade, 11) showing the critical role of ERK pathway in transcriptional regulation of zCry1a gene. Here we report that the oscillation of zCry1a gene expression does not depend on CLOCK:BMAL transcriptional activation. Indeed, the abolishment of CLOCK:BMAL-transactivation capacity through the expression of a dominant negative form of zCLOCK3 (zCLOCK3-DeltaC) lacking its transactivation domain does not show any impact on the cir
Light-Dependent Regulation of Circadian Clocks in Vertebrates
Chronobiology [Working Title]
Circadian clocks are intrinsic time-tracking systems that endow organisms with a survival advantage. The core of the circadian clock mechanism is a cellautonomous and self-sustained oscillator called a cellular clock, which operates via a transcription-/translation-based negative feedback loop. Under natural conditions, circadian clocks are entrained to a 24-hour day by environmental time cues, most commonly light. In mammals, circadian clocks are regulated by cellular clocks located in the central nervous system, such as the suprachiasmatic nucleus (SCN), and in other peripheral tissues. Importantly, mammals have no photoreceptors in the peripheral tissues; therefore the effect of light on peripheral clocks is indirect. By striking contrast, zebrafish peripheral cellular clocks are directly light responsive. This characteristic of the zebrafish cellular clock has contributed to the identification of molecules and signaling pathways that are involved in the lightdependent regulation of the cellular clock. Here, selected light-dependent regulatory mechanisms of circadian clocks in mammals and zebrafish are described.
Zebrafish circadian clocks: cells that see light
In the classical view of circadian clock organization, the daily rhythms of most organisms were thought to be regulated by a central, 'master' pacemaker, usually located within neural structures of the animal. However, with the results of experiments performed in zebrafish, mammalian cell lines and, more recently, mammalian tissues, this view has changed to one where clock organization is now seen as being highly decentralized. It is clear that clocks exist in the peripheral tissues of animals as diverse as Drosophila, zebrafish and mammals. In the case of Drosophila and zebrafish, these tissues are also directly lightresponsive. This light sensitivity and direct clock entrainability is also true for zebrafish cell lines and earlystage embryos. Using luminescent reporter cell lines containing clock gene promoters driving the expression of luciferase and single-cell imaging techniques, we have been able to show how each cell responds rapidly to a single light pulse by being shifted to a common phase, equivalent to the early day. This direct light sensitivity might be related to the requirement for light in these cells to activate the transcription of genes involved in DNA repair. It is also clear that the circadian clock in zebrafish regulates the timing of the cell cycle, demonstrating the wide impact that this light sensitivity and daily rhythmicity has on the biology of zebrafish.
Journal of Neuroendocrinology, 2005
In zebrafish, the pineal gland is a photoreceptive organ that contains an intrinsic circadian oscillator and exhibits rhythmic arylalkylamine-N-acetyltransferase (zfaanat2) mRNA expression. In the present study, we investigated the role of light and of a clock gene, zperiod2 (zper2), in the development of this rhythm. Analysis of zfaanat2 mRNA expression in the pineal gland of 3-day-old zebrafish embryos after exposure to different photoperiodic regimes indicated that light is required for proper development of the circadian clock-controlled rhythmic expression of zfaanat2, and that a 1-h light pulse is sufficient to initiate this rhythm. Analysis of zper2 mRNA expression in zebrafish embryos exposed to different photoperiodic regimes indicated that zper2 expression is transiently up-regulated by light but is not regulated by the circadian oscillator. To establish the association between light-induced zper2 expression and light-induced clock-controlled zfaanat2 rhythm, zPer2 knock-down experiments were performed. The zfaanat2 mRNA rhythm, induced by a 1-h light pulse, was abolished in zPer2 knock-down embryos. These experiments indicated that light-induced zper2 expression is crucial for establishment of the clock-controlled zfaanat2 rhythm in the zebrafish pineal gland.
Current Biology, 2002
The signaling pathways that couple light photoreception to entrainment of the circadian clock have yet to be deciphered. Two prominent groups of candidates for the circadian photoreceptors are opsins (e.g., melanopsin) and blue-light photoreceptors (e.g., cryptochromes). We have previously showed that the zebrafish is an ideal model organism in which to study circadian regulation and light response in peripheral tissues. Here, we used the light-responsive zebrafish cell line Z3 to dissect the response of the clock gene zPer2 to light. We show that the MAPK (mitogen-activated protein kinase) pathway is essential for this response, although other signaling pathways may also play a role. Moreover, action spectrum analyses of zPer2 transcriptional response to monochromatic light demonstrate the involvement of a blue-light photoreceptor. The Cry1b and Cry3 cryptochromes constitute attractive candidates as photoreceptors in this setting. Our results establish a link between blue-light photoreceptors, probably cryptochromes, and the MAPK pathway to elicit light-induced transcriptional activation of clock genes.
Light Directs Zebrafish period2 Expression via Conserved D and E Boxes
PLoS Biology, 2009
For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly lightsensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer lightregulated transcription in zebrafish cells. The LRM contains E-and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E-and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light and how the regulation of clock gene expression by light has evolved in vertebrates.
Analysis of a Gene Regulatory Cascade Mediating Circadian Rhythm in Zebrafish
PLoS Computational Biology, 2013
In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow lightinduced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms.