A recessive mutant of Drosophila Clock reveals a role in circadian rhythm amplitude

Abstract

The transcription factor Clock (Clk) plays a critical role in animal circadian rhythms. Genetic studies de®ning its function have relied on two dominant negative alleles, one in Drosophila and one in mice. Here we describe a novel recessive allele of Drosophila Clock, Clk ar . Homozygous Clk ar¯i es are viable and behaviorally arrhythmic. The Clk ar phenotype is caused by a splice site mutation that severely disrupts splicing and reduces Clk activity. Despite the behavioral arrhythmicity, molecular oscillations are still detectable in Clk ar¯i es. Transcription analysis indicates potent effects of Clk ar on levels and amplitude of transcriptional oscillations. Taken together with other data, we propose that Clk makes a major contribution to the strength and amplitude of circadian rhythms. Keywords: amplitude/circadian rhythms/Clock/mutant allele/transcription IntroductionThe persistence of daily rhythmic behaviors without external temporal cues is a manifestation of circadian clocks. Although only approximating 24 h periodicity under constant conditions, these clocks achieve precise 24 h cycles by synchronizing to solar light cycles. Circadian pacemakers facilitate anticipation of these and other environmental events, and rhythmicity present throughout the animal and plant kingdoms punctuates its value to species ®tness.The investigation of fruit¯y (Drosophila melanogaster) clocks has revealed many evolutionarily conserved pacemaker components (Allada et al., 2001). Studies on the biochemistry of the timekeeping mechanism have focused on transcriptional regulation. It is believed that the basic helix±loop±helix (bHLH) transcription factors CLOCK (CLK) and CYCLE (CYC) directly bind to upstream E boxes (CACGTG) and activate transcription of the period (per) and timeless (tim) genes (Hao et al., 1997;Allada et al., 1998;Darlington et al., 1998;Rutila et al., 1998;Wang et al., 2001). This view is based on strong biochemical evidence and on the phenotype of one semidominant allele of Clk. PER and TIM proteins subsequently feed back and inhibit transcriptional activation by CLOCK and CYCLE (Darlington et al., 1998;Lee et al., 1998Lee et al., , 1999. A similar feedback loop exists in mammals, including humans. Of note, circadian transcription studies in vivo have relied heavily on two dominant negative (antimorphic) alleles of Clock, one in Drosophila (Clk Jrk ) and one in mouse (King et al., 1997a,b;Allada et al., 1998).Further studies have implicated a second feedback loop in circadian timing. Like per and tim, Clk and cry RNA levels also oscillate with respect to time of day Darlington et al., 1998;Emery et al., 1998). However, these oscillations are antiphase to those of per and tim, suggesting that they are indirect targets of the Clk±cyc system. This is consistent with the levels of the Clk and cry RNAs in Clk Jrk and cyc 0 mutants; they are high, whereas the levels of per and tim RNAs are low (Emery et al., 1998;Glossop et al., 1999). It has been proposed that these genes, per and tim on the one hand and Clk on ...

Clk post-transcriptional control denoises circadian transcription both temporally and spatially

“…To minimize the effects of an endogenous WT Clk gene, we performed the experiment in the background of the Clk hypomorphic mutant Clk AR (ref. 29). In these transgenic lines, the ClkWT transgene rescued the arrhytmicity of the Clk AR mutation much more effectively (475%) than the ClkSV40 transgene (o40%).…”

“…This is based on the fact that the circadian behaviour of clk AR mutants can be rescued using the cry-GAL4 driver and a UAS-Clk transgene 29 and that there are negligible effects on expression of CLK under the per or tim promoters (ARK flies 21 ). However, the same UAS-Clk transgene expressed under control of other gal4 drivers like tim-gal4 leads to developmental lethality 29 and the behavioural deficits of Clk AR mutants are not rescued by Clk ARK transgenes (our own unpublished observation). Moreover, addition of extra Clk gene copies leads to a copy-numberdependent shortening of the period 28 , demonstrating that CLK levels cannot be regulated solely at the post-translational level.…”

“…The CantonS (CS) strain used as wild type is CSIso3H (Bloomington Stock Centre, Indiana). Clk AR mutants were described 29 . ClkSV40 flies were constructed by injecting ClkSV40 transgene into yw embryos (service provided by Best Gene).…”

“…In addition, it is well established that the levels or activity of CLK markedly alter the circadian period and amplitude [27][28][29] . Moreover, uncontrolled expression of CLK leads to death.…”

“…Indeed, CLK expression in non-circadian cells is enough to generate ectopic circadian clocks in the fly brain 30 . In addition, CLK-driven transcription may have a role in the development of the circadian system, as Clk and cyc fly mutants display abnormalities in the morphology of circadian neurons 29,31 . These data demonstrate that post-translational control alone may not be enough to buffer big changes in CLK levels.…”