Clockwork Orange is a transcriptional repressor and a new Drosophila circadian pacemaker component

Abstract

Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.

Figure 1.

Identification of direct CLK targets in Drosophila S2 cells and fly heads. (A) Diagram illustrating the approach for the identification of direct CLK targets from Drosophila S2 cells and fly heads. (Dex) Dexamethasone; (GR) ligand-binding domain of the glucocorticoid receptor; (CHX) cycloheximide. Head or cells were treated for 6 h with CHX + vehicle or CHX + Dex. Heads or cells were harvested, and RNA was extracted and hybridized to oligonucleotide microarrays. Both the new Affymetrix Drosophila 2.0 Chips and the first generation of chips for Drosophila (Affymetrix Drosophila Genome) were used. (B) Top 28 direct CLK targets identified by the approach described in A. (TGT) Targetness. This index was obtained by averaging the relative stimulation by dexamethasone from S2 cells and fly heads. For example, for tim, TGT = Average{(36.75/38.3), (5.47/7.65)} = 0.83, where 36.75 is the stimulation ratio obtained for the tim probe, 38.3 is the ratio for the most stimulated probe in fly heads, and 5.47 and 7.65 are the equivalent values from S2 cells. (C) CLK protein expression activates most of the direct CLK-GR targets. Transient transfections were performed with varying amounts of pAc-Clk plasmid in S2 cells (0, 10, 30, and 100 ng). After 48 h, cells were harvested and total RNA was isolated. Microarray analysis was performed as described in Materials and Methods using the Drosophila 2.0 genomic Affymetrix chips. A heat map was generated using a normalized value as indicated in Materials and Methods. (D) Effect of CLK and PER expression on vri-Luc (Blau and Young 1999), picot-Luc, CG15095-Luc, and CG17100-Luc reporters on S2 cells. pAc-Clk and pAc-per refers to CLK- and PER-expressing plasmids, respectively (Nawathean and Rosbash 2004). In all cases, cotransfection with pCopia-Renilla Luciferase was performed to normalize for cell number, transfection efficiency, and general effects on transcription. For each condition, a normalized firefly/Renilla Luciferase value was obtained by setting the ratio obtained with the addition of pActin-Clk to 100%. The graph shows the results of a representative experiment (in which duplicates for each condition were performed). Error bars indicate the standard error of the mean (SEM). The experiment was performed three times with similar results.

Figure 2.

cwo gene expression is regulated in a circadian manner. (A) Expression profile for cwo and tim across six time points in wild-type (yw) flies. Data were obtained by Q-PCR. Expression values for each transcript and time point were generated by dividing the cwo or tim mRNA signal by the expression value for a control noncircadian mRNA (ribosomal protein 49, RP49). A value of 1 was assigned to the maximum expression across the six time points; the values in the other time points were calculated as a fraction of this maximum value. The data are the average of the normalized cwo or tim expression values for three independent RNA samples. The error bars indicate the SEM. (B) cwo mRNA levels in control (yw), Clk^Jrk, and per⁰¹ fly strains. Flies were entrained for 4 d in LD conditions and then transferred to DD. After 2 d in DD conditions, flies were harvested at CT3 and CT15. RNA from both time points for each strain was combined, and Q-PCR was performed using primers for cwo and RP49. Expression values are reported as a ratio of cwo/RP49 expression. We assigned a value of 1 to the ratio obtained for control flies and proceed as in A. The experiment was performed twice and the results were averaged. The error bars indicate the SEM. (C) cwo localization in the fly brain examined with GFP. p{cwo-gal4 (NP7442-gal4)}; UAS-GFP flies were stained with PDF antiserum. Strong overlap between the PDF staining and GFP expression can be observed in the region corresponding to the position of the pacemaker neurons (sLNv). An amplified picture of the overlap in the sLNv region is shown in the bottom right panels.

Figure 3.

cwo is a new clock component. (A, top) Schematic of e4027 and f5073 insertions in the first intron of cwo. Arrows indicate the position of primer pairs for Q-PCR. (Bottom) Q-PCR from control (5073/+), 5073/5073, and 4027/4027 fly heads at ZT3 and ZT15 using the primer pair shown in the top part. (B) Behavioral analysis of fly strains deficient for cwo and control flies. “D” refers to a deletion that includes the cwo locus (see text). (C) Comparison of circadian locomotor behavior of control (5073/+; top panels) and cwo-deficient flies (5073/5073; bottom panels). In each case, the behavior is shown in average actograms (left) and autocorrelation analysis (right) (Levine et al. 2002). (DD1–DD4) The first 4 d in DD; (after DD4) the locomotor activity after 4 d in DD. For each interval of time (DD1–DD4 and DD5–DD8), 5073/5073 flies can be divided in two groups according to their behavioral rhythms (top for rhythmic flies and bottom for arrhythmic flies). The results illustrate a model experiment (n = 28 for control flies and n = 24 for 5073 flies).

Figure 4.

cwo is responsible for the slow clock in LD and DD conditions. (A) Expression of cwo in the circadian neurons can rescue the long rhythms present in cwo-deficient flies. (B) Comparison of the circadian locomotor behavior of control (5073/+; top panels) and cwo-deficient flies (5073/5073; bottom panels) in LD conditions. Two standard days are shown, with timing indicated by alternating white and gray background areas, with white representing the illuminated interval of LD (ZT0–ZT12), and gray representing the dark period (ZT12–ZT24). The arrows indicate the phase of morning or evening anticipation for each fly strain. (Control) 5073/+ flies. (C) PRC for control and 5073 flies. The time onset of the photic stimuli was plotted on the X-axis (ZT, in hours). The phase response was plotted on the Y-axis as the difference (in hours) from the phase of untreated flies. The experiment was repeated twice, and the error bars represent the SEM.

Figure 5.

mRNA oscillation of the direct CLK targets is selectively affected in cwo-deficient flies. (A) Amplitude of oscillation for the core clock genes in 5073 flies. The data were obtained by Affymetrix oligonucleotide microarray. The maximum and minimum values were selected, and the amplitude of oscillation was defined as the ratio of these two values (N = 3 for ZT3 and N = 2 for ZT15). The amplitude is expressed as the percentage of control (yw). Error bars indicate the SEM. (B) Profile for tim, Pdp1, vri, and per mRNAs. Results were obtained by microarrays. (C) Normalized profile for vri and tim mRNA. Results were obtained by microarray, and the value for each time point was normalized to the maximum value across the six time points. (D) Comparison of trough values for tim, vri, and per for control and 5073 flies as measured by oligonucleotide microarray (N = 3). Error bars indicate the SEM. (E) Average mRNA levels for clock-relevant mRNAs. Data were obtained by oligonucleotide microarrays. The six time-point values were averaged for control and cwo-deficient flies and expressed as a percentage of control expression.

Figure 6.

CWO binds to E-boxes and functionally cooperates with PER in inhibiting CLK–CYC-mediated transcription. (A) Effect of cwo expression (150 ng of cwo-expressing plasmid, pAc-cwo) on the transcription of pActin-Luc, vri-Luc, tim-Luc, CG15095-Luc, and cwo-Luc. Expression was measured and normalized as indicated for Figure 1D. A representative experiment is shown. Two experiments with duplicates for each condition were performed with similar results. Error bars represent the SEM. (B) Comparison of CYC and CWO amino acid composition in the bHLH domain. (Red) Zones with identical amino acid composition; (green) areas with similar charge amino acids. (C) Effect of cwoVP16 overexpression (0, 25, or 100 ng of pAc-cwoVP16) on the transcription driven by tim-Luc and timΔTer1ΔTer2m-Luc (McDonald et al. 2001). A representative experiment is shown. Two experiments with duplicates for each condition were performed with similar results. Error bars represent the SEM. (D) Effects of PER and CWO expression on CLK-mediated expression. “Clk, No Per” indicates that 10 ng of pAc-Clk was cotransfected with 0, 100, or 200 ng of pAc-cwo and the correspondent reporters. “Clk + Per” indicates that the transfection was performed as before, except 50 ng of pAc-per were also cotransfected. In both cases, the expression was obtained after normalization to the control Copia-Renilla Luciferase value. A value of 1 was assigned to the maximum value in this condition. A representative experiment is shown. Three experiments with duplicates for each condition were performed with similar results. Error bars represent the SEM. (E) PER repression of CLK-mediated transcription in the presence (100 ng of pAc-cwo) or absence of CWO. Expression was calculated and expressed as in Figure 1D. A representative experiment is shown. Two experiments with duplicates for each condition were performed with similar results.

Figure 7.

Proposed role of CWO in the circadian molecular circuit.