C-Terminal Binding Protein (CtBP) Activates the Expression of E-Box Clock Genes with CLOCK/CYCLE in <i>Drosophila</i>

<div><p>In <i>Drosophila</i>, CLOCK/CYCLE heterodimer (CLK/CYC) is the primary activator of circadian clock genes that contain the E-box sequence in their promoter regions (hereafter referred to as “E-box clock genes”). Although extensive studies have investigated the feedback regulation of clock genes, little is known regarding other factors acting with CLK/CYC. Here we show that Drosophila C-terminal binding protein (dCtBP), a transcriptional co-factor, is involved in the regulation of the E-box clock genes. <i>In vivo</i> overexpression of dCtBP in clock cells lengthened or abolished circadian locomotor rhythm with up-regulation of a subset of the E-box clock genes, <i>period</i> (<i>per</i>), <i>vrille</i> (<i>vri</i>), and <i>PAR domain protein 1ε</i> (<i>Pdp1ε</i>). Co-expression of dCtBP with CLK <i>in vitro</i> also increased the promoter activity of <i>per</i>, <i>vri</i>, <i>Pdp1ε</i> and <i>cwo</i> depending on the amount of dCtBP expression, whereas no effect was observed without CLK. The activation of these clock genes <i>in vitro</i> was not observed when we used mutated dCtBP which carries amino acid substitutions in NAD⁺ domain. These results suggest that dCtBP generally acts as a putative co-activator of CLK/CYC through the E-box sequence.</p></div

Free-running periods of <i>dCtBP</i>-overexpressing and knockdown flies.

<p>N_R: Number of rhythmic flies recorded.</p><p>N_A: Number of arrhythmic flies recorded.</p>a<p>significantly different from the period of the flies carrying the <i>tim(UAS)-Gal4</i> as a control (t test, <i>P</i><0.05).</p>b<p>significantly different from the period of the flies carrying the <i>UAS</i> sequence as a control (t test, <i>P</i><0.05).</p

Responses to odors in taste neurons expressing <i>Ors</i> under different Gal4 drivers.

<p>a. Taste neurons expressing <i>Or22a</i>+<i>Or83b</i> in sugar-sensing neurons (driven by <i>Gr5a</i>-Gal4) respond to sugar and to butyl acetate (first two traces: smaller amplitude spikes); taste neurons expressing <i>Or22a</i>+<i>Or83b</i> in bitter-sensing neurons (driven by <i>Gr66a</i>-Gal4) respond to caffeine and to butyl acetate (lower two traces: larger amplitude spikes). Gray bar = 2 s stimulus. Horizontal black bar: 1 s; vertical bar: 0.2 mV. b. Comparison of the responses of altered GRNs to butyl acetate depending on the driver, <i>Gr5a</i>-Gal4 (“sugar cells”) or <i>Gr66a</i>-Gal4 (“bitter cells”) (mean±S.E.M.; n = 12, 17 trials). There is a small difference in response intensity to butyl acetate in <i>Gr5a</i>- and <i>Gr66a</i>-GRNs (*: P = 0.039, Student's t-test).</p

Behavioral choices expressed by flies between a control agar disk and disk treated with butyl acetate.

<p>Ordinates: ratio of the density of presence of flies on odorant-treated or non-treated food (mean±S.E.M.; number of trails for each is noted in the figure). Abscissa: Wild-type (<i>w</i>¹¹¹⁸) and flies expressing <i>Or22a</i>, <i>Or83b</i> or <i>Or22a</i>+<i>Or83b</i> under the control of <i>Gr5a</i>- or <i>Gr66a</i>-Gal4. Flies expressing both <i>Or22a</i> and <i>Or83b</i> showed an altered preference to butyl acetate as compared to the wild-type (<i>w</i>¹¹¹⁸). Depending on the GRNs that express the <i>Ors</i>, flies exhibited the opposite preferences to the same odorant. BA = Butyl acetate. * P<0.05, ** P<0.01 with the Tukey-Kramer multiple comparisons test.</p

Expression level of an output gene, <i>takeout</i>, in <i>dCtBP</i>-overexpressing flies.

<p>Relative mRNA levels of <i>takeout</i> were measured at ZT1 and ZT13 using a quantitative PCR assay (Q-PCR). The blue, red and green bars represent the <i>tim(UAS)-Gal4</i>, <i>UAS-dCtBP-2</i> and <i>dCtBP</i> overexpression flies, respectively. The expression level in <i>dCtBP</i> overexpression flies was significantly different from that in <i>tim(UAS)-Gal4</i> (a: t test, <i>P<</i>0.05) and that of <i>UAS-dCtBP-2</i> (b: t test, <i>P<</i>0.05) at both phases. RNAs were sampled three times at each point and error bars represent S.E.M.</p

Electrophysiological responses of <i>Drosophila</i> i-type taste sensilla to sugar and bitter substances.

<p>a. Schematic diagram of the recording setup. Electrical signals were recorded from an electrolytically sharpened tungsten electrode inserted through the cuticle at the base of a taste sensillum. To stimulate the GRNs, the tip of the sensillum was capped during 2–5 s with a glass capillary filled with a stimulating solution. The i-type sensilla house only two GRNs, which elicit spikes of different amplitudes that were separated using custom software routines (23) as shown in 1c and 1e. The sugar-sensing GRN expresses <i>Gr5a</i> and the bitter-sensitive GRN expresses <i>Gr66a </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002610#pone.0002610-Hiroi1" target="_blank">[40]</a>. b. Sample recording with 100 mM sucrose (stimulus = 2 s horizontal grey bar). c. Sucrose elicits a response only in the cell that fires spikes of smaller amplitude (lower trace); superimposed spikes (left column) and time-series extracted (central trace and bars) after software spike separation. d. Sample recording with 1 mM caffeine. e. Caffeine elicits a response only in the cell that produces spikes of larger amplitude. Vertical bars = 0.3 mV.</p

The actograms of <i>dCtBP</i>-knockdown and -overexpressing flies.

<p>Typical locomotor activity in the control (upper left), <i>dCtBP</i>-knockdown flies (upper right), and <i>dCtBP-</i>overexpressing flies (lower panels). The number in parentheses represents the free-running period of the corresponding flies. Adult flies were entrained to a 12-h light:12-h dark cycle (LD) for 3 days, and then kept in constant darkness (DD). Horizontal bars in white and black indicate times of light and dark, respectively, in LD. Vertical bar in white: LD; vertical bar in black: DD.</p

Temporal <i>dCtBP</i> expression in control and <i>dCtBP</i>-overexpressing flies.

<p>A: Temporal expression profile of <i>dCtBP</i> (blue) and <i>Clk</i> (red) in the head of adult control flies measured by quantitative PCR assay (Q-PCR). ZT1 and ZT13 correspond to 1 h from the onset of light-on and -off conditions in LD, respectively. <i>dCtBP</i> expression reveals a circadian rhythm peaking at the end of night phase. Cross indicates significant difference with trough level of <i>Clk</i> at ZT17 (Tukey’s test, <i>P</i><0.05). Asterisks indicate a significant difference with the trough level of <i>dCtBP</i> at ZT9 (Tukey’s test, <i>P</i><0.05). RNAs were sampled three times at each point, and error bars represent S.E.M. B: The expression level of <i>dCtBP</i> at ZT1 and ZT13 in control flies (white) and <i>dCtBP-</i>overexpressing flies (black). <i>dCtBP</i> expression was higher in <i>dCtBP</i>-overexpressing flies than control flies at each phase (*: t test, <i>P<</i>0.05). RNAs were sampled three times at each point, and error bars represent S.E.M. (n  = 3).</p

Expression levels of core clock genes in <i>dCtBP</i>-overexpressing flies.

<p>Relative mRNA levels of the indicated genes at the peak and trough phases were measured using a quantitative PCR assay (Q-PCR). Expression levels of <i>per, vri</i>, and <i>Pdp1ε</i> were higher in the <i>dCtBP</i> overexpression flies (black) than in control (white) at the peak phase. <i>dCtBP</i> overexpression decreased the expression levels of <i>cwo</i> at the trough phase. Asterisks indicate a significant difference from control values (t test, <i>P<</i>0.05). RNAs were sampled three times at each point, and error bars represent S.E.M.</p

<i>dCtBP</i> regulates transcription of known clock genes with CLK/CYC.

<p>Relative luciferase activities of <i>per-luc</i>, <i>tim-luc</i>, <i>vri-luc, Pdp1-luc</i>, and <i>cwo-luc</i> in the presence of 0 (–) or 100 (+) ng <i>pAc5.1-dCtBP</i> alone, or 0 (–), 100 (+), 400 (++) ng <i>pAc5.1-dCtBP</i> (<i>dCtBP</i>), or 400 (++) ng <i>pAc5.1-dCtBP-G183A/G186A</i> (<i>dCtBP -DM</i>) in conjunction with 100 ng <i>pAct-Clk</i> are represented. The luciferase activity was normalized by the activity of <i>Renilla</i> luciferase as a control reporter, and then the activity was normalized by the activity of <i>pAct-Clk</i> alone. RLU means relative luminescence unit. <i>dCtBP</i> regulates the promoter activity of core clock genes. The difference between values without <i>Clk</i> was calculated by t test. The difference between the values with <i>Clk</i> was calculated by the Tukey’s test, and asterisks indicate significant differences between two values (*<i>P<</i>0.05 and **<i>P<0.01</i>). These experiments were performed independently three times (or four in some cases) and error bars represent S.E.M.</p