Temperature Regulates Transcription in the Zebrafish Circadian Clock

It has been well-documented that temperature influences key aspects of the circadian clock. Temperature cycles entrain the clock, while the period length of the circadian cycle is adjusted so that it remains relatively constant over a wide range of temperatures (temperature compensation). In vertebrates, the molecular basis of these properties is poorly understood. Here, using the zebrafish as an ectothermic model, we demonstrate first that in the absence of light, exposure of embryos and primary cell lines to temperature cycles entrains circadian rhythms of clock gene expression. Temperature steps drive changes in the basal expression of certain clock genes in a gene-specific manner, a mechanism potentially contributing to entrainment. In the case of the per4 gene, while E-box promoter elements mediate circadian clock regulation, they do not direct the temperature-driven changes in transcription. Second, by studying E-box-regulated transcription as a reporter of the core clock mechanism, we reveal that the zebrafish clock is temperature-compensated. In addition, temperature strongly influences the amplitude of circadian transcriptional rhythms during and following entrainment by light–dark cycles, a property that could confer temperature compensation. Finally, we show temperature-dependent changes in the expression levels, phosphorylation, and function of the clock protein, CLK. This suggests a mechanism that could account for changes in the amplitude of the E-box-directed rhythm. Together, our results imply that several key transcriptional regulatory elements at the core of the zebrafish clock respond to temperature

Zirkadianen Expression und Promotor Analyse des Zebrafisch Period-4 gens

In most organisms, light plays a key role in the synchronization of the circadian timing system with the environmental day–night cycle. Light pulses that phase-shift the circadian clock also induce the expression of period genes in vertebrates. Here, we report the cloning of a zebrafish period gene, zfperiod4, which is repressed by light. High amplitude rhythms of zfperiod4 expression are detected under light dark (LD) cycles in zebrafish larvae as well as in the zebrafish cell line PAC-2 that contains a directly light-entrainable clock. The expression of zfperiod4 is detected during the first day of development and we show that the presence of a LD cycle is essential to subsequently establish a robust circadian rhythm in gene expression. We have developed a transient and stable transfection protocol for PAC-2 cells. In this way we have established an in vivo luciferase reporter assay for zfperiod4 expression in this cell line. High-definition bioluminescence traces have enabled us to accurately measure phase-shifting of the clock by light. We have also exploited this model to study how four E-box elements in the zfperiod4 promoter regulate expression. Mutagenesis reveals that the integrity of these four E-boxes is crucial for maintaining low basal expression together with robust rhythmicity and repression by light. In the context of a minimal heterologous promoter, the E-box elements also direct a robust circadian rhythm of expression that is significantly phase-advanced compared with the original zfperiod4 promoter and lacks the light-repression property. These results reveal flexibility in the phase and light responsiveness of E-box-directed rhythmic expression, depending on the promoter context. Finally, a preliminary pharmacological analysis implicates the involvement of the MAP Kinase, cAMP, and PKC signaling pathways in the maintainance of the amplitude as well as entrainment of circadian clock rhythms.In den meisten Organismen spielt Licht eine Schlüsselrolle bei der Synchronisierung der zirkadianen Zeitgebung mit dem Tag-Nacht Zyklus der Umwelt. Lichtpulse, die eine Phasenverschiebung der Uhr verursachen, induzieren ebenfalls die Expression von period-Genen in Vertebraten. In dieser Arbeit beschreiben wir die Klonierung eines period-Genes des Zebrafisches, zfperiod4, das durch Licht reprimiert wird. Unter einem Licht-Dunkel Zyklus oszilliert die Expression von zfperiod4 mit grosser Amplitude, sowohl in Zebrafischlarven als auch in der Zebrafishzellinie PAC-2, deren Uhr direkt durch Licht synchronisiert werden kann. Die Expression von zfperiod4 beginnt während des ersten Entwicklungstages, und wir zeigen, dass Licht-Dunkel-Zyklen notwendig sind, um einen robust oszillierenden Expressionsrhythmus in den folgenden Tagen zu etablieren. Wir haben ein Protokoll für transiente und stabile Transfektionen der PAC-2-Zellinie entwickelt, was die Herstellung eines in vivo-Luciferase-Reportergensystems für die Expression von zfperiod4 ermöglichte. Die präzisen Biolumineszenzdaten dieses Systems ermöglichten es uns, die Phasenverschiebung der zirkadianen Uhr durch Licht genau zu bestimmen. Wir haben dieses Modell weiterhin benutzt, um zu untersuchen, wie vier “E-box”-Elemente im zfperiod4-Promoter die Expression regulieren. Mutagenese-Experimente haben gezeigt, dass die Vollständigkeit dieser “E-box”-Elemente entscheidend für den Erhalt eines niedrigen basalen Expressionsniveaus sowie für eine robuste Oszillation und die Repression durch Licht sind. Im Kontext eines minimalen heterologen Promotors können die “E-box”-Elemente eine robuste zirkadiane Oszillation steuern, die aber verglichen mit dem ursprünglichen zfperiod4 Promotor deutlich phasenverschoben ist und nicht mehr durch Licht reprimiert werden kann. Diese Ergebnisse zeigen eine Flexibilität in der Phasen- und Lichtempfindlichkeit von “E-Box”-gesteuerter rhythmischer Expression in Abhängigkeit vom Promotorkontext. Schliesslich impliziert eine vorläufige pharmakologische Studie die Signaltransduktionswege der MAP-Kinase, von cAMP und der PKC in der Erhaltung der Amplitude sowie der Synchronisierung der Rhythmen der zirkadianen Uhr

A stochastic oscillator model simulates the entrainment of vertebrate cellular clocks by light

The circadian clock is a cellular mechanism that synchronizes various biological processes with respect to the time of the day. While much progress has been made characterizing the molecular mechanisms underlying this clock, it is less clear how external light cues influence the dynamics of the core clock mechanism and thereby entrain it with the light–dark cycle. Zebrafish-derived cell cultures possess clocks that are directly light-entrainable, thus providing an attractive laboratory model for circadian entrainment. Here, we have developed a stochastic oscillator model of the zebrafish circadian clock, which accounts for the core clock negative feedback loop, light input, and the proliferation of single-cell oscillator noise into population-level luminescence recordings. The model accurately predicts the entrainment dynamics observed in bioluminescent clock reporter assays upon exposure to a wide range of lighting conditions. Furthermore, we have applied the model to obtain refitted parameter sets for cell cultures exposed to a variety of pharmacological treatments and predict changes in single-cell oscillator parameters. Our work paves the way for model-based, large-scale screens for genetic or pharmacologically-induced modifications to the entrainment of circadian clock function

Rhythmic Clock Gene Expression under LD and Temperature Cycles

<div><p>Graphical summary of RPA assays are described:</p> <p>(A) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d either in a light (12 h) or dark (12 h) cycle at a constant temperature (25.3 °C).</p> <p>(B) <i>Per4</i> (solid line) and <i>cry3</i> mRNA expression (dashed line) in zebrafish larvae raised for 6 d in DD, under a temperature cycle of 4 °C (23.5 °C/11 h, 27.5 °C/11 h, plus 1 h for each heating and cooling phase). RNA samples were harvested during the seventh day (ZT0 is defined as the beginning of the heating and light periods).</p> <p>(C and D) Equivalent analysis of <i>clock1</i> (solid line) and <i>cry2a</i> (dashed line) expression in (C) LD, and (D) temperature cycle larvae.</p> <p>(E) <i>Per2</i> expression was assayed in LD (dashed line) or temperature cycle (ΔT) larvae (solid line). By linear regression analysis, the slope of the ΔT trace has no significant deviation from zero (R² = 0.033 and <i>p</i> = 0.66, F-test). The LD cycle curve fits to a 6th-order polynomial regression model (R² = 0.96 and Runs test for deviation from model <i>p</i> = 0.99).</p> <p>In each case, zeitgeber time is plotted on the <i>x</i>-axis while the relative expression levels (percentage) are plotted on the <i>y</i>-axis. <i>β-actin</i> levels were used to standardize the results. The highest band intensity in each experiment was arbitrarily defined as 100%, and then all other values were expressed as a percentage of this value. All experiments were performed in triplicate, and error bars denote the standard deviation.</p></div

Model for Temperature Regulation of the <i>per4</i> Promoter

<div><p>(A) Temperature steps entrain the phase of the clock by driving expression levels of <i>per4</i> and other clock genes via a hypothetical enhancer element X. Temperature decreases result in expression increases, and vice versa. Although E-boxes ultimately mediate regulation of the <i>per4</i> promoter by the entrained clock, they do not participate in the temperature-driven response.</p> <p>(B) Temperature influences the amplitude of rhythmic <i>per4</i> expression that has been entrained by LD cycles in two ways: (1) by determining the amplitude of E-box-directed rhythmic expression, via changes in CLK protein levels, phosphorylation, and E-box binding, and (2) by driving expression changes through element X (see panel A). The promoter integrates these two regulatory mechanisms. The temperature-dependent amplitude of E-box-directed rhythmic expression would be predicted to involve the core feedback loops of the clock itself and, according to mathematical models, might thereby underlie temperature compensation.</p></div

Temperature Steps Regulate Clock Gene Expression Levels

<div><p>(A) Larvae were raised in DD at 21 °C for 7 d and then shifted to 29 °C and harvested at the indicated times relative to the temperature shift (h). Controls remained at 21 °C and were harvested in parallel with the temperature shift larvae. RPA analysis of the indicated genes was then performed. “t” represents a tRNA control sample.</p> <p>(B) As in (A), except that 5-d-old larvae were shifted from 29 °C to 21 °C, and controls remained at 29 °C.</p> <p>All data are representative of at least three independent experiments.</p></div

Temperature Influences CLK Protein Expression and Function

<div><p>(A) In vitro luciferase assays of transiently transfected PAC2 cells. The combinations of CLK (Clk) and BMAL (Bml) expression vectors cotransfected with the 4x Ebox (−7) reporter plasmid are indicated for each assay result. Control cells were transfected with the reporter plasmid or with the pGL3 Control plasmid alone. Values represent the mean fold difference between luciferase activities measured in 30 °C and 20 °C, 60 h after transfection. All assays were standardized for transfection efficiency using a β-galactosidase assay. The results are based on four independent experiments, and error bars indicate the standard deviation.</p> <p>(B) Electrophoretic mobility shift assay of nuclear extracts from PAC-2 cells cultured at 20 °C or 30 °C on a LD cycle, and harvested at ZT3, 9, 15, and 21 (lanes 1 to 8). Three specific complexes are indicated by A, B, and an asterisk. Supershift assays of a ZT15, 30 °C extract (+Ab), used either a dopamine transporter antibody (Control) or a mouse clk antibody (Clock) (lanes 9 and 10). Complexes indicated by A, B, and an asterisk are all efficiently competed by a 25-, 50-, and 100-fold excess of cold E-box probe (lanes 12, 13, and 14, respectively, and compare with lane 11), but not with a 100-fold excess of a CRE probe (compare lane 15 with lane 11).</p> <p>(C) Western blotting assay using the anti-mouse CLK antibody of the same nuclear extracts tested in the electrophoretic mobility shift assay analysis of panel B. The migration of a 100-kDa marker band is shown. Below are shown western blotting results for the same extracts using an anti-mouse CREB antibody as a loading control.</p> <p>(D) Western blot assay of CLK protein in 30 °C extracts prepared at ZT9 or ZT21 (time points representing the trough and peak, respectively, of the CLK protein rhythm). Samples were prepared with (+) or without (−) treatment with alkaline phosphatase prior to electrophoresis and transfer. In panels B, C, and D, data are representative of at least three independent experiments.</p></div

Temperature Compensation and the Amplitude of E-box-Directed Rhythmic Expression

<div><p>(A) Bioluminescence profile of 4xE-box (−7) reporter cells held at 20 °C under a LD cycle and then transferred to DD conditions. Plates were counted once per hour and maintained in robotic stacking units between assays, where they were illuminated.</p> <p>(B) Equivalent experiment to panel A, with cells maintained at 30 °C.</p> <p>(C) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 20 °C under LD cycle and DD conditions.</p> <p>(D) Bioluminescence traces from 1.7-kb WT <i>per4</i> reporter cells maintained at 30 °C under LD cycle and DD conditions.</p> <p>(E) RPA analysis of <i>per4</i> expression in WT PAC-2 cells held at 20 °C and 30 °C under an LD cycle for 3 d. RNA extracts were prepared on the fourth day at 3-h intervals during one 24-h cycle. Time 0 represents ZT 0: the onset of the light period. A white and black bar above the autoradiograph indicates the duration of the light and dark periods. RPA results with a β-actin loading control are also shown. “t” represents a tRNA control sample.</p> <p>(F) A bar graph shows quantification of the peak (ZT3) and trough (ZT15) <i>per4</i> expression values at 20 °C and 30 °C plotted as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030351#pbio-0030351-g001" target="_blank">Figure 1</a>, with error bars representing the standard deviation of three independent experiments.</p> <p>All bioluminescence traces represent the mean values of 16 independent wells. Each panel is representative of at least three independent experiments.</p></div