Probing the relative importance of molecular oscillations in the circadian clock

Abstract

Circadian ( approximately 24 hr) rhythms of behavior and physiology are driven by molecular clocks that are endogenous to most organisms. The mechanisms underlying these clocks are remarkably conserved across evolution and typically consist of auto-regulatory loops in which specific proteins (clock proteins) rhythmically repress expression of their own genes. Such regulation maintains 24-hr cycles of RNA and protein expression. Despite the conservation of these mechanisms, however, questions are now being raised about the relevance of different molecular oscillations. Indeed, several studies have demonstrated that oscillations of some critical clock genes can be eliminated without loss of basic clock function. Here, we describe the multiple levels at which clock gene/protein expression and function can be rhythmically regulated-transcription, protein expression, post-translational modification, and localization-and speculate as to which aspect of this regulation is most critical. While the review is focused on Drosophila, we include some discussion of mammalian clocks to indicate the extent to which the questions concerning clock mechanisms are similar, regardless of the organism under study.

Figure 1.—

Model of the Drosophila circadian clock based on interlocking transcriptional feedback loops. CLK and CYC form a heterodimer and bind to E-box elements of the circadian clock genes per and tim and activate their transcription during the day and early evening; as per and tim mRNAs peak, PER and TIM proteins accumulate, form a PER–TIM complex, and translocate into the nucleus to repress their own transcription during the late night. During the day, PER and TIM are degraded by light-dependent and independent pathways, thus allowing a new cycle of transcription to start. In another transcription-based loop, CLK–CYC activate transcription of vri and Pdp1ɛ; as VRI and PDP1ɛ proteins accumulate, they translocate into the nucleus to inhibit and activate Clk transcription, respectively. Both VRI and PDP1ɛ bind to E4BP4 sites in the Clk promoter. PDP1ɛ accumulation lags behind that of VRI, resulting in rhythmic Clk transcription.

Figure 2.—

Model of the Drosophila circadian clock depicting the importance of post-translational modifications. Clock genes such as per and tim and other clock-controlled genes (CCGs) are activated by CLK–CYC during the day, and their transcription peaks in the early night. The PER–TIM complex forms during the second half of the night and translocates into the nucleus to repress CLK–CYC activity. A balance of kinase and phosphatase activity regulates the stability of PER, TIM, and CLK and most likely the nuclear entry of PER and TIM. Casein kinases DBT and CKII phosphorylate PER and the glycogen synthesis kinase SGG phosphorylates TIM. PP2A and PP1 dephosphorylate both PER and TIM. For the sake of simplicity, each is shown here acting only on the primary target (PP2A on PER and PP1 on TIM). According to this model, critical steps of the timekeeping process are controlled by post-translational modifications of key clock proteins. Note that nuclear expression of SGG and PP1 has not been experimentally determined.