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PubMed2002

Interactivating feedback loops within the mammalian clock: BMAL1 is negatively autoregulated and upregulated by CRY1, CRY2, and PER2.

Transcriptional regulation appears to be fundamental to circadian oscillations of clock gene expression. These oscillations are believed to control output rhythms. The transcriptional feedback loop and a model of interlocked loops have been proposed as the basis for these oscillations. We characterized the genomic structure of the mouse Bmal1 gene (mBmal1) and defined the mBmal1 promoter region. Transcription of mBmal1 was activated by CRY1, CRY2, and PER2, and was repressed by BMAL1-CLOCK dimers. Therefore, CRY, PER2, and BMAL1-CLOCK play bidirectional roles in transcription when they are at high levels by late day and midnight, respectively. This underlies the opposite phase of BMAL1 compared to CRY and PER. We propose that a BMAL1 negative feedback loop interlocks with the CRY and PER2 negative feedback loop by inter-activation, forming a third positive forward loop. This transcriptional model suggests a molecular basis for the maintenance of stability, persistence, and period of circadian rhythms. The transcriptional potency of CRY is predominant within the mammalian clock, suggesting a clearance mechanism for CRY in period maintenance. (c)2002 Elsevier Science (USA).

3T3 Cells↗
PubMed2005

Molecular evolution and population genetics of circadian clock genes.

This article discusses a number of common methodologies used in the field of population genetics and evolution and reviews their application within circadian rhythm research. We examine the basic principles behind phylogenetic analysis and how these can be used to illuminate clock gene evolution. We then discuss genetic variation between and within species and show how neutrality tests can reveal the signatures of selection or drift on clock genes. These tests are particularly important for moving beyond "just so" stories when discussing the evolution of clock phenotypes, and we provide relevant circadian examples. We also focus on methods that can be used to study genetic variation, such as quantitative trait loci analysis. We discuss the various bootstrapping or resampling techniques that can be applied to generate confidence intervals in the various methodologies and then examine the use of interspecific transformation studies, which can, and have, provide some useful insights, not only into clock gene evolution in particular, but "behavioral" gene evolution in general. Finally, we assess gene/protein alignments and protein structure predictions and their implicit evolutionary bases.

Animals↗
PubMed2003

vrille, Pdp1, and dClock form a second feedback loop in the Drosophila circadian clock.

The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, dCLOCK/CYCLE activates period expression, and PERIOD protein then inhibits dCLOCK/CYCLE activity. dClock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. We show here that VRI and PDP1 proteins feed back and directly regulate dClock expression. Repression of dClock by VRI is separated from activation by PDP1 since VRI levels peak 3-6 hours before PDP1. Rhythmic vri transcription is required for molecular rhythms, and here we show that the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, VRI and PDP1, together with dClock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of dClock, and probably of other genes, to generate accurate circadian rhythms.

Animals↗
PubMed2003

Comparative analysis of avian BMAL1 and CLOCK protein sequences: a search for features associated with owl nocturnal behaviour.

Animals differ widely in the phasing of their daily rhythms with respect to daily environmental rhythms. While birds are predominantly day-active, nocturnal activity is a characteristic feature of the order Strigiformes (owls). To study the evolution of owl night-activity cDNA sequences encoding the circadian core oscillator (CCO) proteins BMAL1 and CLOCK were obtained from barn owl (Tyto alba). The predicted proteins showed high sequence identity with their Galliform homologues (BMAL1: 99%; CLOCK: 95.6%). A computer-predicted chicken BMAL1 casein kinase-1 phosphorylation site is absent from T. alba BMAL1, but also absent from homologues of other six bird species (5 orders) (night-active (n=2), day-active (n=4)) indicating no evolutionary association with night activity. Sequence differences between T. alba and Galliform CLOCK frequently involved serine and threonine residues suggesting potential differences in their phosphorylation. The length of a poly-glutamine string in the CLOCK C-terminus varied between and within 25 species (6 orders) examined, however, no discernible feature distinguishing day and night active species was found. No differences were found between day (n=5) and night (n=7)-active species (12 species, 6 orders) in a region of the PER2 protein implicated in altered rhythm phasing in humans. In conclusion the avian CCO components examined showed strong evolutionary conservation. Molecular evolution associated with owl night-activity may have involved alterations in the CCO relationship with 'output' genes rather than in the molecular structure of the CCO itself.

Amino Acid Sequence↗
PubMed2002

Dec1 and Dec2 are regulators of the mammalian molecular clock.

The circadian rhythms in mammals are regulated by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. Four clock-gene families have been found to be involved in a transcription-translation feedback loop that generates the circadian rhythm at the intracellular level. The proteins Clock and Bmal1 form a heterodimer which activates the transcription of the Per gene from the E-box elements in its promoter region. Protein products of Per act together with Cry proteins to inhibit Per transcription, thus closing the autoregulatory feedback loop. We found that Dec1 and Dec2, basic helix-loop-helix transcription factors, repressed Clock/Bmal1-induced transactivation of the mouse Per1 promoter through direct protein-protein interactions with Bmal1 and/or competition for E-box elements. Dec1 and Dec2 are expressed in the suprachiasmic nucleus in a circadian fashion, with a peak in the subjective day. A brief light pulse induced Dec1 but not Dec2 expression in the suprachiasmic nucleus in a phase-dependent manner. Dec1 and Dec2 are regulators of the mammalian molecular clock, and form a fifth clock-gene family.

ARNTL Transcription Factors↗
PubMed2001

Circadian activity rhythm in methamphetamine-treated Clock mutant mice.

It is well established that the Clock gene is essential for expressing circadian activity rhythms in mammals under constant darkness. The Clock gene product is a positive component of a molecular feedback loop which is assumed to generate the circadian rhythm. On the other hand, chronic treatment of methamphetamine (MAP) induces locomotor activity rhythm in a circadian domain, which is independent of the suprachiasmatic nucleus (SCN) and is driven by a pacemaker outside the SCN. However, it is not known whether the pacemaker outside the SCN possesses a similar molecular mechanism to that in the SCN. Here we show that MAP restores locomotor activity rhythm in arrhythmic homozygous Clock mutant (Clock/Clock) mice under constant darkness. This result indicates that the Clock mutation does not affect the MAP-induced locomotor rhythm.

Animals↗
PubMed2001

Biological clock in total darkness: the Clock/MOP3 circadian system of the blind subterranean mole rat.

Blind subterranean mole rats retain a degenerated, subcutaneous, visually blind but functionally circadian eye involved in photoperiodic perception. Here we describe the cloning, sequence, and expression of the circadian Clock and MOP3 cDNAs of the Spalax ehrenbergi superspecies in Israel. Both genes are relatively conserved, although characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of humans and mice, and is different in amino acid composition from that of rats. We also show that MOP3 is a bona fide partner of Spalax Clock and that the Spalax Clock/MOP3 dimer is less potent than its human counterpart in driving transcription. We suggest that this reduction in transcriptional activity may be attributed to the Spalax Clock glutamine-rich domain, which is unique in its amino acid composition compared with other studied mammalian species. Understanding Clock/MOP3 function could highlight circadian mechanisms in blind mammals and their unique pattern as a result of adapting to life underground.

ARNTL Transcription Factors↗
PubMed2003

Molecular mechanism of mammalian circadian clock.

Circadian rhythms in behaviors and physiological phenomena of plants and animals have long been well known, but the frameworks of the molecular mechanism of circadian clocks have become clearer only within the last decade. A transcription-translation feedback loop has been shown to be an essential component of the clock, and this mechanism seems to be conserved over a wide range of species. The transcriptional activation by a Clock:Bmal1 heterodimer and the inhibition by Cryptochrome and Period are believed to provide the framework of the feedback loop in mammals. Posttranslational modifications such as phosphorylation, nuclear entry and degradation have also been demonstrated to be necessary for the oscillation. Complex auxiliary loops have also been found, and these are thought to contribute to the stabilization of the feedback loop. The molecular mechanisms by which the circadian clock is adjusted to external conditions such as daily light-dark cycles, and by which the oscillation of the feedback loop is transferred to the peripheral organs are also discussed.

Animals↗
PubMed2004

Clock gene mRNA and protein rhythms in the pineal gland of mice.

In vertebrates, the rhythmic transcription of clock genes, regulated by their own gene products, provides the basis for self-sustaining circadian clockworks. These endogenous clocks are lost in most mammalian tissues, but not in the central pacemaker of the hypothalamic suprachiasmatic nucleus (SCN). An interesting model system to understand this phylogenetic shift in function of clock gene products is the rodent pineal gland, as its intrinsic clockwork was replaced during evolution by an input-dependent oscillator. By means of immunohistochemistry, immunoblotting and real time PCR, we investigated the day/night expression profiles of all major clock genes and their products in the pineal gland of one melatonin-proficient and one melatonin-deficient mouse strain. All clockwork elements known to be indispensable for a sustained rhythm generation in the SCN were also found in the pineal organ of both mouse strains. Only mPer1 mRNA and PER1 protein accumulation coincides with timecourses of many other pineal genes and their products, which are cyclicAMP inducible. Here, presented data together with the known mechanisms for regulation of the mPer1 gene in the rodent pineal gland forward the idea that in this tissue PER1 may have a trigger function for initiating the cycles of the clockwork's transcriptional/translational feedback loops.

Animals↗
PubMed2000

Neuroanatomy of cells expressing clock genes in Drosophila: transgenic manipulation of the period and timeless genes to mark the perikarya of circadian pacemaker neurons and their projections.

Subsets of brain neurons expressing the clock genes period (per) and timeless (tim) are involved in the generation of circadian behavioral rhythms. However, current knowledge of projection patterns of these neurons is limited to those immunoreactive to an antibody against a crustacean neuropeptide. The GAL4-expression system was utilized to visualize neuronal processes from all per and tim-expressing neurons in the central nervous system. Each of two types of GAL4-driver fusion genes, per-gal4 or tim-gal4, was combined in transgenic flies with marker genes-lacZ, and sequences encoding green fluorescent protein or TAU protein-under the control of the GAL4-responsive element UAS. This allowed visualization of the cytoplasm of GAL4-expressing cells. Thus, neurites of clock neurons in the adult brain as well as those of larvae and pupae were revealed. Among the anatomical patterns revealed by per-gal4- or tim-gal4-driven marker expression were a previously unknown, dorsally located neuronal cluster, along with the projections of these cells and of other dorsal neurons characterized in earlier studies only by the location of their perikarya. The similarity of projections from PER- or TIM-containing neurons during development to those in the adult implies that these features of mature clock neurons are established by the larval stages. Neurons that have never been identified as PER- or TIM-immunoreactive were also visualized in this assay system, indicating promoter activity of the clock genes in these cells and suggesting that their products cannot accumulate to detectable levels in certain neurons.

Animals↗
PubMed2006

Dact1 presomitic mesoderm expression oscillates in phase with Axin2 in the somitogenesis clock of mice.

During segmentation (somitogenesis) in vertebrate embryos, somites form in a rostral-to-caudal sequence according to a species-specific rhythm called the somitogenesis clock. The expression of genes participating in somitogenesis oscillates in the presomitic mesoderm (PSM) in time with this clock. We previously reported that the Dact1 gene (aka Dpr1/Frd1/ThyEx3), which encodes a Dishevelled-binding intracellular regulator of Wnt signaling, is prominently expressed in the PSM as well as in a caudal-rostral gradient across the somites of mouse embryos. This observation led us to examine whether Dact1 expression oscillates in the PSM. We have found that Dact1 PSM expression does indeed oscillate in time with the somitogenesis clock. Consistent with its known signaling functions and with the "clock and wavefront" model of signal regulation during somitogenesis, the oscillation of Dact1 occurs in phase with the Wnt signaling component Axin2, and out of phase with the Notch signaling component Lfng.

Animals↗
PubMed2007

Start the clock! Circadian rhythms and development.

The contribution of timing cues from the environment to the coordination of early developmental processes is poorly understood. The day-night cycle represents one of the most important, regular environmental changes that animals are exposed to. A key adaptation that allows animals to anticipate daily environmental changes is the circadian clock. In this review, we aim to address when a light-regulated circadian clock first emerges during development and what its functions are at this early stage. In particular, do circadian clocks regulate early developmental processes? We will focus on results obtained with Drosophila and vertebrates, where both circadian clock and developmental control mechanisms have been intensively studied.

Animals↗
PubMed2003

Development of PDF-immunoreactive cells, possible clock neurons, in the housefly Musca domestica.

Even though the housefly Musca domestica shows clear circadian rhythms in its behavioural and physiological processes, a circadian pacemaker system controlling these rhythms has not yet been described morphologically in this species. In M. domestica, neurons immunoreactive to pigment-dispersing factor (PDF), a neurotransmitter/neuromodulator of circadian information arising from a circadian clock and transmitted to target cells, are similar in their number and distribution to the PDF neurons of Drosophila melanogaster. In D. melanogaster these neurons co-localize PER protein and have been identified as clock neurons in that species. Here we report PDF-immunoreactive cells in the housefly's brain during postembryonic development in the larval and pupal stages, as well as in the adult fly soon after eclosion. In the housefly's brain, there are three groups of PDF-immunoreactive neurons: two groups with small (sPDFMe) and large (lPDFMe) cell bodies in the proximal medulla of the optic lobe; and one group in the dorsal protocerebrum (PDFD). Three out of four sPDFMe can be detected during the first hour of larval development, but the fourth sPDFMe is observed in the larva only from 48 hours after hatching, along with five lPDFMe neurons, seen first as two subgroups, and three out of four PDFD neurons. During postembryonic development these neurons show changes in their structure and immunoreactivity. New PDF neurons are observed during pupal development but these neurons mostly do not survive into adulthood. In the adult fly's brain, the PDF neurons have also been examined in double-labelled preparations made with a second antibody directed against the product of one of several clock genes: period (per), timeless (tim), or cryptochrome (cry). Among them, only immunoreactivity to CRY-like protein has been detected in the brain of M. domestica and has shown a daily rhythm in its concentration, as examined immunocytochemically. CRY was co-localized with PDF in the sPDFMe of the housefly's brain fixed during the day. The possibility that the sPDFMe neurons are the housefly's clock neurons is discussed.

Animals↗
PubMed2001

Photoreception and circadian clock system of the chicken pineal gland.

Chicken pinealocytes contain three major components of the circadian clock system: 1) a self-sustained oscillator, 2) a photic-input pathway to the oscillator, and 3) an overt output represented by the rhythmic production of melatonin. Even under cultured conditions of isolated pineal gland or dissociated pinealocytes, the input-oscillator-output functions are well maintained. Because of these experimental advantages, chicken pineal gland has been one of the best models for the study of the circadian clock system. Since the finding of a pineal-specific photoreceptive molecule, pinopsin, we have characterized the endogenous phototransduction pathway in the pinealocytes. On the other hand, despite the long history of chick pineal research, the molecular mechanism underlying the pineal clock oscillation has been largely unknown. Our recent characterization of the chick pineal clock genes strongly suggests that they constitute a transcription/translation-based autoregulatory feedback loop, which is very similar to that generating circadian rhythmicity in mammalian SCN.

Animals↗
PubMed2001

A model for a network of phosphorylation-dephosphorylation cycles displaying the dynamics of dominoes and clocks.

We consider a model for a network of phosphorylation-dephosphorylation cycles coupled through forward and backward regulatory interactions, such that a protein phosphorylated in a given cycle activates the phosphorylation of a protein by a kinase in the next cycle as well as the dephosphorylation of a protein by a phosphatase in a preceding cycle. The network is cyclically organized in such a way that the protein phosphorylated in the last cycle activates the kinase in the first cycle. We study the dynamics of the network in the presence of both forward and backward coupling, in conditions where a threshold exists in each cycle in the amount of protein phosphorylated as a function of the ratio of kinase to phosphatase maximum rates. We show that this system can display sustained (limit-cycle) oscillations in which each cycle in the pathway is successively turned on and off, in a sequence resembling the fall of a series of dominoes. The model thus provides an example of a biochemical system displaying the dynamics of dominoes and clocks (Murray & Kirschner, 1989). It also shows that a continuum of clock waveforms exists of which the fall of dominoes represents a limit. When the cycles in the network are linked through only forward (positive) coupling, bistability is observed, while in the presence of only backward (negative) coupling, the system can display multistability or oscillations, depending on the number of cycles in the network. Inhibition or activation of any kinase or phosphatase in the network immediately stops the oscillations by bringing the system into a stable steady state; oscillations resume when the initial value of the kinase or phosphatase rate is restored. The progression of the system on the limit cycle can thus be temporarily halted as long as an inhibitor is present, much as when a domino is held in place. These results suggest that the eukaryotic cell cycle, governed by a network of phosphorylation-dephosphorylation reactions in which the negative control of cyclin-dependent kinases plays a prominent role, behaves as a limit-cycle oscillator impeded in the presence of inhibitors. We contrast the case where the sequence of domino-like transitions constitutes the clock with the case where the sequence of transitions is passively coupled to a biochemical oscillator operating as an independent clock.

Animals↗
PubMed2007

Even a stopped clock tells the right time twice a day: circadian timekeeping in Drosophila.

"Even a stopped clock tells the right time twice a day, and for once I'm inclined to believe Withnail is right. We are indeed drifting into the arena of the unwell... What we need is harmony. Fresh air. Stuff like that" "Bruce Robinson (1986, ref. 1)". Although a stopped Drosophila clock probably does not tell the right time even once a day, recent findings have demonstrated that accurate circadian time-keeping is dependent on harmony between groups of clock neurons within the brain. Furthermore, when harmony between the environment and the endogenous clock is lost, as during jet lag, we definitely feel unwell. In this review, we provide an overview of the current understanding of circadian rhythms in Drosophila, focussing on recent discoveries that demonstrate how approximately 100 neurons within the Drosophila brain control the behaviour of the whole fly, and how these rhythms respond to the environment.

Adaptation, Biological↗
PubMed2001

The polyglutamine motif is highly conserved at the Clock locus in various organisms and is not polymorphic in humans.

Circadian rhythms play a central role in diverse physiological phenomena and the recent years have witnessed the identification of a number of genes responsible for the maintenance of these rhythms. One of these is the Clock gene, which was first identified in mouse and subsequently in a large number of organisms, including humans. The human Clock gene has been proposed as a possible candidate for disorders affected by alterations of circadian rhythm, including bipolar disorder and schizophrenia. This gene contains a highly conserved polyglutamine motif, that in humans is coded for by CAG repeats. In view of the involvement of CAG repeat expansion in a number of neuro-psychiatric disorders, we have sought to determine the polymorphism status of CAG repeats at the Clock locus in humans. Our analysis of 190 unrelated individuals, who included patients suffering from bipolar disorder and schizophrenia, indicated that the repeat, which consisted of 6 CAG triplets, was not polymorphic in humans. An analysis of the repeat in non-human primates and other organisms revealed that the glutamine stretch is shortest in humans and baboons, and longest in Drosophila and zebrafish. A study of various Drosophila species revealed that the repeat number is highly polymorphic, ranging from 25 to 33 pure glutamine repeats. Unlike most other microsatellites, the CAG repeat stretch at the Clock locus in humans is smaller than its homologues in non-human primates. We propose that glutamine repeat size is functionally important in this gene and thus tightly regulated. The variation in repeat number is probably deleterious to the individual, resulting in the maintenance of a short and invariable repeat structure in the human population.

Amino Acid Sequence↗
PubMed2002

Molecular machinery of the circadian clock in mammals.

The discovery of clock genes and the general principle of their oscillation has stimulated research on biological clocks and this research has had a major impact on the field of life sciences. The mammalian circadian core oscillator is thought to be composed of an autoregulatory transcription-(post)translation-based feedback loop involving a set of clock genes. The real time monitoring of clock gene oscillation at the levels of genes, cells, tissues, and systems will clarify the issue of how the time signal is born and how it is integrated into the organismic level. Investigations of circadian systems in various organisms employ multiple methods including ethology, physiology, neuroscience, molecular biology, cell biology and genetics. The circadian system has thus become a unique example in the elucidation of the general principles of how genes control cellular, systemic and behavioral functions.

Animals↗