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

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

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

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

<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

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

Mated <i>Drosophila melanogaster</i> females consume more amino acids during the dark phase

<div><p>To maintain homeostasis, animals must ingest appropriate quantities, determined by their internal nutritional state, of suitable nutrients. In the fruit fly <i>Drosophila melanogaster</i>, an amino acid deficit induces a specific appetite for amino acids and thus results in their increased consumption. Although multiple processes of physiology, metabolism, and behavior are under circadian control in many organisms, it is unclear whether the circadian clock also modulates such motivated behavior driven by an internal need. Differences in levels of amino acid consumption by flies between the light and dark phases of the day:night cycle were examined using a capillary feeder assay following amino acid deprivation. Female flies exhibited increased consumption of amino acids during the dark phase compared with the light phase. Investigation of mutants lacking a functional <i>period</i> gene (<i>per</i>⁰), a well-characterized clock gene in <i>Drosophila</i>, found no difference between the light and dark phases in amino acid consumption by <i>per</i>⁰ flies. Furthermore, increased consumption of amino acids during the dark phase was observed in mated but not in virgin females, which strongly suggested that mating is involved in the rhythmic modulation of amino acid intake. Egg production, which is induced by mating, did not affect the rhythmic change in amino acid consumption, although egg-laying behavior showed a <i>per</i>⁰-dependent change in rhythm. Elevated consumption of amino acids during the dark phase was partly induced by the action of a seminal protein, sex peptide (SP), on the sex peptide receptor (SPR) in females. Moreover, we showed that the increased consumption of amino acids during the dark phase is induced in mated females independently of their internal level of amino acids. These results suggest that a post-mating SP/SPR signal elevates amino acid consumption during the dark phase <i>via</i> the circadian clock.</p></div

A post-mating signal elevates amino acid consumption during the dark phase.

<p>(A) The experimental scheme for the CAFE assays. Each L phase is shown by a white box and each D phase by a gray box. (B and C) Amino acid consumption during L (orange bars) and D (blue bars) phases was quantified using no-choice CAFE assays with the following strains: virgin CS females and CS females mated with CS or <i>SP</i>⁰/Δ¹³⁰ males (B; n = 3 or 4 trials); virgin <i>Df(1)Exel6234</i> (shown as Δ<i>SPR</i>) females and <i>Df(1)Exel6234</i> females mated with CS males (B; n = 4 trials); and virgin <i>ovo</i>^<i>D1</i>/CS females and <i>ovo</i>^<i>D1</i>/CS females mated with CS males (C; n = 4 trials). Intake per single fly is shown. Error bars indicate SEM. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for comparisons between L and D phases for each type of female in (B) and (C) using the Student’s <i>t</i>-test. <i>p</i> > 0.05 for all comparisons during L phase among females in (B) using one-way ANOVA. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for all comparisons during D phase among females in (B) using one-way ANOVA followed by <i>post hoc</i> Bonferroni/Dunn test. *<i>p</i> < 0.05 and **<i>p</i> < 0.01 for comparisons between <i>ovo</i>^<i>D1</i>/CS virgin and mated females in (C) using the Student’s <i>t</i>-test.</p

Mated females increase amino acid consumption during the dark phase without amino acid deprivation.

<p>(A) The experimental scheme for the CAFE assays. Flies were tested without deprivation of amino acids. Each L phase is shown by a white box and each D phase by a gray box. (B) Amino acid consumption during L (orange bars) and D (blue bars) phases was quantified using two-choice CAFE assays between 50 mM glucose and 50 mM glucose containing 1/5 amino acid mixture in virgin and mated CS females (n = 6 trials). Intake of 50 mM glucose alone was subtracted from intake of 50 mM glucose containing 1/5 amino acid mixture, and the value was subsequently divided by the number of flies in each vial. The mean value is shown as “estimated amino acid intake”. Error bars indicate SEM. *<i>p</i> < 0.05 for comparisons between L and D phases and between virgin and mated females using the Student’s <i>t</i>-test.</p