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

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

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

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