Circadian oscillators of Drosophila and mammals.
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Biomedical subjects
Paul E Hardin
Publications and source records attributed to Paul E Hardin.
Essential and expendable features of the circadian timekeeping mechanism.
Circadian clocks control behavioral, physiological and metabolic rhythms via one or more transcriptional feedback loops. In animals, two conserved feedback loops are thought to keep circadian time by mediating rhythmic transcription in opposite phases of the circadian cycle. Recent work in cyanobacteria nevertheless demonstrates that rhythmic transcription is dispensable for circadian timekeeping, raising the possibility that some features of the transcriptional feedback loops in animals are also expendable. Indeed, one of the two feedback loops is not necessary for circadian timekeeping in animals, but rhythmic transcription and post-translational modifications are both essential for keeping circadian time. These results not only confirm additional requirements within the animal circadian timekeeping mechanism, but also raise important questions about the function of conserved, yet expendable, features of the circadian timekeeping mechanism in animals.
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The circadian clock within the cardiomyocyte is essential for responsiveness of the heart to fatty acids.
Cells/organs must respond both rapidly and appropriately to increased fatty acid availability; failure to do so is associated with the development of skeletal muscle and hepatic insulin resistance, pancreatic beta-cell dysfunction, and myocardial contractile dysfunction. Here we tested the hypothesis that the intrinsic circadian clock within the cardiomyocytes of the heart allows rapid and appropriate adaptation of this organ to fatty acids by investigating the following: 1) whether circadian rhythms in fatty acid responsiveness persist in isolated adult rat cardiomyocytes, and 2) whether manipulation of the circadian clock within the heart, either through light/dark (L/D) cycle or genetic disruptions, impairs responsiveness of the heart to fasting in vivo. We report that both the intramyocellular circadian clock and diurnal variations in fatty acid responsiveness observed in the intact rat heart in vivo persist in adult rat cardiomyocytes. Reversal of the 12-h/12-h L/D cycle was associated with a re-entrainment of the circadian clock within the rat heart, which required 5-8 days for completion. Fasting rats resulted in the induction of fatty acid-responsive genes, an effect that was dramatically attenuated 2 days after L/D cycle reversal. Similarly, a targeted disruption of the circadian clock within the heart, through overexpression of a dominant negative CLOCK mutant, severely attenuated induction of myocardial fatty acid-responsive genes during fasting. These studies expose a causal relationship between the circadian clock within the cardiomyocyte with responsiveness of the heart to fatty acids and myocardial triglyceride metabolism.
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Circadian transcription: passing the HAT to CLOCK.
In animals, the circadian timekeeping mechanism relies on the coordinated activities of activators and repressors to control rhythmic transcription. In this issue of Cell, Doi et al. (2006) reveal that rhythms in histone acetylation are necessary for rhythmic transcription and that the histone acetyl transferase responsible is CLOCK, a key transcription factor that is essential for circadian oscillator function.
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PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription.
Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS (PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.
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Drosophila CLOCK is constitutively expressed in circadian oscillator and non-oscillator cells.
CLOCK (CLK) is a core component of the transcriptional feedback loops that comprise the circadian timekeeping mechanism in Drosophila. As a heterodimer with CYCLE (CYC), CLK binds E-boxes to activate the transcription of rhythmically expressed genes within and downstream of the circadian clock, but this activation unexpectedly occurs at times when CLK is at its lowest levels on Western blots. Recent studies demonstrate that CLK also regulates nonrhythmic gene expression and behaviors. Despite the critical roles CLK plays within and outside the circadian clock, its spatial expression pattern has not been characterized. Using a newly developed CLK antibody, the authors show that CLK is coexpressed with PERIOD (PER) in canonical oscillator cells throughout the head and body. In contrast to PER, however, the levels of CLK immunoreactivity do not cycle in intensity, CLK is detected primarily in the nucleus throughout the circadian cycle, and CLK is expressed in non-oscillator cells within the lateral and dorsal brain, including Kenyon cells, which mediate various forms of learning and memory. These results indicate that constitutive levels of nuclear CLK regulate rhythmic transcription in circadian oscillator cells and suggest that CLK contributes to other behavioral processes by regulating gene expression in non-oscillator cells.
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A Drosophila nonvisual arrestin is required for the maintenance of olfactory sensitivity.
Nonvisual arrestins are a family of multifunctional adaptor molecules that regulate the activities of diverse families of receptors including G protein-coupled receptors, frizzled, and transforming growth factor-beta receptors. These activities indicate broad roles in both physiology and development for nonvisual arrestins. Drosophila melanogaster has a single nonvisual arrestin, kurtz, which is found at high levels within the adult olfactory receptor neurons (ORNs), suggesting a role for this gene in modulating olfactory sensitivity. Using heat-induced expression of a krz cDNA through development, we rescued krz(1) lethality. The resulting adults lacked detectable levels of krz in the olfactory system. The rescued krz(1) homozygotes have an incompletely penetrant antennal structural defect that was completely rescued by the neural expression of a krz cDNA. The krz(1) loss-of-function adults without visible antennal defects displayed diminished behavioral responsiveness to both aversive and attractive odors and also demonstrated reduced olfactory receptor potentials. Both the behavioral and electrophysiological phenotypes were rescued by the targeted expression of the krz cDNA within postdevelopmental ORNs. Thus, krz is required within the nervous system for antennal development and is required later in the ORNs for the maintenance of olfactory sensitivity in Drosophila. The reduced receptor potentials in krz(1) antenna indicate that nonvisual arrestins are required for the early odor-induced signaling events within the ORNs.
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The circadian timekeeping system of Drosophila.
Daily rhythms in behavior, physiology and metabolism are controlled by endogenous circadian clocks. At the heart of these clocks is a circadian oscillator that keeps circadian time, is entrained by environmental cues such as light and activates rhythmic outputs at the appropriate time of day. Genetic and molecular analyses in Drosophila have revealed important insights into the molecules and mechanisms underlying circadian oscillator function in all organisms. In this review I will describe the intracellular feedback loops that form the core of the Drosophila circadian oscillator and consider how they are entrained by environmental light cycles, where they operate within the fly and how they are thought to control overt rhythms in physiology and behavior. I will also discuss where work remains to be done to give a comprehensive picture of the circadian clock in Drosophila and likely many other organisms.
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The intrinsic circadian clock within the cardiomyocyte.
Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.
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Measuring circadian rhythms in olfaction using electroantennograms.
Circadian clocks control daily rhythms in many behavioral, physiological, and metabolic processes. Despite remarkable advances in our understanding of the circadian timekeeping mechanism and how it responds to environmental cycles, relatively little is known about how the timekeeping mechanism regulates behavior, physiology, and metabolism. One of the most extensively characterized timekeeping mechanisms is that of Drosophila melanogaster. In this species, autonomous circadian clocks are found in many neuronal and nonneuronal tissues, including essentially all sensory structures. We have shown that sensory neurons in the antenna mediate a robust rhythm in electrophysiological responses to the food odorant ethyl acetate. This article describes how rhythms in olfactory responses are measured and provides a perspective on the generality of these rhythms and their regulation by the clock.
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Circadian rhythms from multiple oscillators: lessons from diverse organisms.
The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.
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Circadian clocks in antennal neurons are necessary and sufficient for olfaction rhythms in Drosophila.
BACKGROUND: The Drosophila circadian clock is controlled by interlocked transcriptional feedback loops that operate in many neuronal and nonneuronal tissues. These clocks are roughly divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and are thought to control other rhythmic outputs. We previously showed that peripheral oscillators are required to mediate rhythmic olfactory responses in the antenna, but the identity and relative autonomy of these peripheral oscillators has not been defined. RESULTS: Targeted ablation of lateral neurons by using apoptosis-promoting factors and targeted clock disruption in antennal neurons with newly developed dominant-negative versions of CLOCK and CYCLE show that antennal neurons, but not central clock cells, are necessary for olfactory rhythms. Targeted rescue of antennal neuron oscillators in cyc(01) flies through wild-type CYCLE shows that these neurons are also sufficient for olfaction rhythms. CONCLUSIONS: Antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila. These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation.
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Simulation of Drosophila circadian oscillations, mutations, and light responses by a model with VRI, PDP-1, and CLK.
A model of Drosophila circadian rhythm generation was developed to represent feedback loops based on transcriptional regulation of per, Clk (dclock), Pdp-1, and vri (vrille). The model postulates that histone acetylation kinetics make transcriptional activation a nonlinear function of [CLK]. Such a nonlinearity is essential to simulate robust circadian oscillations of transcription in our model and in previous models. Simulations suggest that two positive feedback loops involving Clk are not essential for oscillations, because oscillations of [PER] were preserved when Clk, vri, or Pdp-1 expression was fixed. However, eliminating positive feedback by fixing vri expression altered the oscillation period. Eliminating the negative feedback loop in which PER represses per expression abolished oscillations. Simulations of per or Clk null mutations, of per overexpression, and of vri, Clk, or Pdp-1 heterozygous null mutations altered model behavior in ways similar to experimental data. The model simulated a photic phase-response curve resembling experimental curves, and oscillations entrained to simulated light-dark cycles. Temperature compensation of oscillation period could be simulated if temperature elevation slowed PER nuclear entry or PER phosphorylation. The model makes experimental predictions, some of which could be tested in transgenic Drosophila.
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Transcription regulation within the circadian clock: the E-box and beyond.
The circadian oscillator is composed of transcriptional feedback loops in organisms ranging from cyanobacteria to humans. These transcriptional feedback loops are so named because transcriptional regulators accumulate to high levels and then feed back to control their own genes' transcription, thus generating a self-sustaining rhythm in gene expression. In insects and vertebrates, the genes that encode these feedback regulators are remarkably well conserved and function to control not 1 but 2 feedback loops. These feedback loops control rhythmic transcription in opposite phases of the circadian cycle, yet they are interlocked because they share a number of components. In this review, the author will compare transcriptional regulatory mechanisms within the Drosophila and mammalian feedback loops and outline remaining questions concerning transcriptional regulation within and downstream of these feedback loops.
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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.
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VRILLE feeds back to control circadian transcription of Clock in the Drosophila circadian oscillator.
The Drosophila circadian oscillator consists of interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLK and CYCLE (CYC) activate per and tim transcription at the same time as they repress Clk transcription, thus controlling the opposite cycling phases of these transcripts. CLK-CYC directly bind E box elements to activate transcription, but the mechanism of CLK-CYC-dependent repression is not known. Here we show that a CLK-CYC-activated gene, vrille (vri), encodes a repressor of Clk transcription, thereby identifying vri as a key negative component of the Clk feedback loop in Drosophila's circadian oscillator. The blue light photoreceptor encoding cryptochrome (cry) gene is also a target for VRI repression, suggesting a broader role for VRI in the rhythmic repression of output genes that cycle in phase with Clk.
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Central and peripheral circadian oscillators in Drosophila.
Drosophila circadian oscillators comprise interlocked period (per)/timeless (tim) and Clock (Clk) transcriptional/translational feedback loops. Within these feedback loops, CLOCK (CLK) and CYCLE (CYC) bind E-box elements to activate per and tim transcription, and we now show that at the same time CLK-CYC repress Clk by activating the transcriptional repressor vrille (vri), thus accounting for the opposite cycling phases of these transcripts and identifying vri as the negative component of the Clk-feedback-loop. The core oscillator mechanism is assumed to be the same for oscillators in different tissues. However, we have shown that CRYPTOCHROME (CRY) has a light-independent function in the oscillator that controls olfaction rhythms, suggesting that CRY may function within the oscillator mechanism itself as it does in mammals. These olfaction rhythms require the function of 'peripheral' oscillators which are distinct from the 'central' lateral neuron (LN) oscillators that mediate locomotor activity rhythms. Preliminary results show that antennal oscillator cells are sufficient and LNs are not necessary for olfaction rhythms, indicating that unlike the situation in mammals, the central oscillator has little impact on the olfaction rhythm oscillator under these conditions.
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Central and peripheral circadian oscillator mechanisms in flies and mammals.
Circadian oscillators are cell-autonomous time-keeping mechanisms that reside in diverse tissues in many organisms. In flies and mice, the core molecular components that sustain these oscillators are highly conserved, but the functions of some of these components appear to have diverged significantly. One possible reason for these differences is that previous comparisons have focused primarily on the central oscillator of the mouse and peripheral oscillators in flies. Recent research on mouse and Drosophila peripheral oscillators shows that the function of the core components between these organisms may be more highly conserved than was first believed, indicating the following: (1) that central and peripheral oscillators in flies do not necessarily have the same molecular mechanisms; (2) that mammalian central oscillators are regulated differently from peripheral oscillators; and (3) that different peripheral oscillators within and across species show striking similarities. The core feedback loop in peripheral oscillators might therefore be functionally well conserved, and central oscillators could be specialized versions of a basic oscillator design.
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