Clockwork orange encodes a transcriptional repressor important for circadian-clock amplitude in Drosophila

Abstract

Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.

Figure 1. CG17100/clockwork orange Transcript Rhythms in Drosophila Heads Using Real-Time PCR

Quantitative real-time RT-PCR (A, B) experiments performed in light-dark (LD; A) and dark-dark conditions (DD; B). X-axis indicates either Zeitgeber Time (ZT; A), where ZT0 is lights-on, or circadian time (CT; B). For real-time experiments (A, B), relative transcript levels have been normalized with the peak level (ZT or CT13) set to 100. (C) Quantitative real-time RT-PCR analysis of cwo transcript levels in wild-type (+/+) and Clk^Jrk (Clk[Jrk]) mutants at ZT1 and ZT13. Relative transcript levels have been normalized with the WT peak (ZT13) set to 100. (D) Full-length transcript profile and domain organization of wild-type cwo. White and gray boxes indicate untranslated and protein coding regions, respectively. Positions of the various cwo transposon insertions used are shown as black triangles above the diagram. The genomic regions used for the UAS-CWO^RNAi construct is shown above the transcript profile (labelled as RNAi). Arrows over the diagram denote the location of the primer sets used in real-time PCR experiments. Asterisks indicate the physical location of canonical E-box elements (CACGTG) within the promoter and first intron, as identified by Fly Enhancer (genomeenhancer.org/fly). The figure has been drawn to scale and all units are provided in kilobases. (E,F) cwo transcript levels in wild-type (+/+; =100) and homozygous cwo mutants cwo^e (e/e) and cwo^f (f/f). Primer sets spanning either exons 1 and 2 (E) or exon 3 (F), as shown in (D), were used to measure relative transcript levels. N experiments ≥ 3. Error bars indicate SEM.

Figure 2. Circadian and Diurnal Behavior of cwo Mutants Under Constant Darkness and Light-Dark Conditions

(A-C) Behavior under constant darkness conditions of cwo^e/+ (A) (n=80), Df(3R)5495/+ (B) (n=89), and cwo^e/Df(3R)5495 (C) (n=43). White and black boxes indicate light and dark periods. Gray boxes indicates subjective day in constant darkness. (D-F) Normalized activity profiles during diurnal conditions of cwo^e/+ (D) (n=77), Df(3R)5495/+ (E) (n=79), and cwo^e/Df(3R)5495 (F) (n=35). Light and dark bars indicate activity during the light and dark phase, respectively. N experiments= 2-4. Numerical values indicate measures of morning anticipation. Error bars indicate SEM.

Figure 3. Altered Rhythmic Expression of vri, pdp1ε, and per in cwo Mutants

Quantitative real-time RT-PCR analysis of vri (A), pdp1ε (B), and per (C) expression during the first day of DD. X-axis indicates circadian time. Wild-type (+/+) levels at CT 13 (peak) are set to 100 and indicated as a closed line. cwo mutants cwo^e/cwo^f (e/f), cwo^f/Df(3R)ED5495 (f/Df), and cwo^f/cwo^f (f/f) are indicated as dashed lines. Data for f/f not shown for vri but is similar to e/f. Statistical significance (p<.05) is indicated with a “1” for comparing +/+ with e/f, “2” for comparing +/+ with f/Df, and “3” for comparing +/+ with f/f. N experiments ≥ 3 except f/Df CT5,9,17,21, where N=2. Error bars indicate SEM.

Figure 4. CWO Specifically Represses and Binds Clock Gene Promoters

(A, B) S2 cells were cotransfected with reporter plasmids (1 μg except 0.1 μg for vri-mluc) and the increasing amounts (0.25 μg and 1 μg) of expression vector for FLAG-tagged CWO (AcF/CWO) in the presence (A) or absence (B) of expression vector for V5-tagged CLK (AcV5/CLK; 10 ng for per-luc, 0.25 ng for tim-luc and pdp1-mluc, 1 ng for vri-mluc). (C) S2 cells were cotransfected with reporter plasmids (1 μg) and the expression vector for FLAG-tagged CWO or VP16 activation domain-fused CWO (1 μg). Activation fold was calculated by normalizing values to luciferase activity in the presence of reporter plasmid, which was set to 1, while repression fold by inversely normalizing them. For (A-C), N experiments = 3, and standard deviations are depicted by error bars. (D) Electrophoretic mobility shift assay for CWO binding to E boxes (CACGTG). Increasing amount (10- and 50- fold molar excess) of unlabeled competitors containing wild-type (E-box) or mutant E-box (mE-box) or 2 μg of anti-GST or anti-FLAG was preincubated with GST-fusion proteins prior to the addition of labeled probe. C1, shift by GST-CWO bHLH:DNA complex; C2, supershift by GST-bHLH:DNA:anti-GST antibody complex.