Speed control: cogs and gears that drive the circadian clock

Abstract

In most organisms, an intrinsic circadian (~24-h) timekeeping system drives rhythms of physiology and behavior. Within cells that contain a circadian clock, specific transcriptional activators and repressors reciprocally regulate each other to generate a basic molecular oscillator. A mismatch of the period generated by this oscillator with the external environment creates circadian disruption, which can have adverse effects on neural function. Although several clock genes have been extensively characterized, a fundamental question remains: how do these genes work together to generate a ~24-h period? Period-altering mutations in clock genes can affect any of multiple regulated steps in the molecular oscillator. In this review, we examine the regulatory mechanisms that contribute to setting the pace of the circadian oscillator.

Figure 1. Simplified model of a transcriptional feedback oscillator in eukaryotic cells

(a) The activator promotes transcription of the repressor gene, and accumulating repressor proteins feedback to inhibit repressor gene transcription. A series of post-transcriptional and translational modifications are imposed to delay the accumulation of repressor proteins. In this simple model, a stronger activator, earlier feedback repression, or faster dissipation of the repression results in a short period, whereas a weaker activator, delayed or prolonged repression renders a long period. Post-translational modifications regulate the stability, cellular localization and activity of the repressor protein. The Activator also promotes transcription of other modulator genes, whose products positively and negatively feedback to regulate Activator gene transcription. (b) Such feedback regulation generates oscillations of clock molecules and of transcriptional activity. Cyclic activation and repression of clock gene transcription are interlocked (dark blue dotted line) so that the strength, timing and duration of one phase affects the other. We speculate that the overall duration of these processes forms the basis of period determination.

Figure 2. A basic molecular framework for circadian timing is conserved from fungi to flies and humans

(a) In Drosophila, CLK-CYC heterodimer activates transcription of per, tim. Accumulated PER and TIM proteins heterodimerize and feedback to repress CLK-CYC activity. CLK-CYC also activates transcription of vri and Pdp1ε, whose products feedback to repress and activate Clk transcription, respectively. (b) In mammals, the CYC homolog BMAL1 heterodimerizes with Clock to activate transcription of Per, Cry, Ror and rev-erb. PER forms a repressor complex with Cryptochrome (CRY), instead of TIM. There are three PERs (PER1, PER2 and PER3), of which PER2 may be most important, and two CRYs (CRY1 and CRY2), whose functions are not fully redundant. RORs and REV-ERBs feedback to regulate transcription of Clock, Bmal1 and Cry [30, 32] (also see review in [145]). (c) In Neurospora, White collar (WC1/2) activates transcription of the repressor gene frequency (frq). Accumulated FRQ protein interacts with an RNA helicase, FRH, to repress WC1/2 activity. FRQ protein also positively regulates wc1/2 transcription (see review in [155] ). Similar negative and positive feedback regulation also exists in prokaryotes, although transcriptional feedback is not necessary to sustain the cyanobacteria circadian clock (see review in [4]).

Figure 3. Model for the core negative feedback loop of the Drosophila circadian clock

The circadian activator CLK-CYC promotes transcription of its target genes, the circadian repressors that include per, tim and cwo, during the day (1). mRNA expression of these genes peaks in the early evening, followed by accumulation of the proteins at night. DBT phosphorylates and promotes PER degradation, and this process is counterbalanced by PP2A (2). TIM mainly localizes to the cytoplasm in the early part of the night due to an active CRM/exportin mediated nuclear export mechanism (3). SGG phosphorylates and may also decrease TIM stability, while PP1 stabilizes TIM in the cytoplasm (4). Around midnight, accumulated PER and TIM proteins form heterodimers (5) and start to enter the nucleus. Nuclear translocation is facilitated by CK2 (perhaps also PP2A) and SGG phosphorylation of PER and TIM, respectively. The PER/TIM/DBT complex may dissociate briefly before nuclear translocation (6). In the late night, nuclear PER is progressively phosphorylated by DBT and reaches maximum repressor activity on the CLK-CYC heterodimer (7). CWO also binds to the E-box sequence of CLK-CYC target genes to help terminate their transcription. At daybreak, hyperphosphorylated PER is targeted by SLIMB for proteasomal degradation (8). TIM protein is degraded by light-dependent and independent mechanisms (question mark). The degradation of this PER-TIM repressor allows CLK-CYC to start a new cycle of transcription and feedback repression. Note that the only kinase for which a circadian time course of interaction with PER has been determined is DBT. Timing of clock protein association with other kinases and phosphatases is speculated (question mark) based upon cell culture and genetic assays. Neurospora and mammals have similar mechanisms (see review in [145, 156]).

Figure 4. Dual role of phosphorylation in regulating the stability and activity of circadian repressor proteins

(a) In Drosophila, newly synthesized PER is unstable, mainly due to DBT and CK2 mediated degradation [57, 68]. Progressive phosphorylation of the PER short domainby DBT creates a resistor that acts to slow down phosphorylation of PER at other sites, and hence stabilize PER. Inhibiting phosphorylation of this PER short domain by deletion or mutation facilitates phosphorylation of downstream and other sites, and hence faster PER degradation [66, 70]. Similar mechanisms may exist in mammals. (b) Human PER2 protein is phosphorylated at some unknown sites by CK1 and other kinases, which promote PER2 degradation. Phosphorylation of S662 promotes phosphorylation of neighboring sites by CK1, which generates a phosphor-cluster to inhibit phosphorylation of other sites responsible for PER2 degradation. However, current models regarding the FASPS mutation (S662G) are still unresolved [92-94]. It appears that the phosphoryation state of S662 also affects PER2 nuclear localization and Per2 transcription. (c) In Neurospora, early phosphorylation of the C-terminus of the FREQUENCY (FRQ) protein inhibits phosphorylation of the PEST-1 domain and other sites, and thus slows down FRQ degradation. Deletion, or mutations that inhibit phosphorylation of the C-terminus, result in faster degradation and a shorter circadian period [157].

Figure 5. Hypothetical model for circadian period determination in Drosophila

We propose that the speed of a circadian oscillator is regulated by multiple temporally gated events: circadian transcription of repressor genes, posttranscriptional regulation of their mRNAs, translation and cytoplasmic accumulation of repressor proteins, nuclear accumulation and retention of repressors, and clearance of repressors from the nucleus. In a 24 h cycle, the repressor protein PER remains in the nucleus for 6-8 h after daybreak (time 0). Premature clearance of PER/TIM results in an earlier de-repression of CLK-CYC and thus a shorter circadian period, while extended presence of PER/TIM proteins slows it down. Progressive degradation of hyperphosphorylated PER/TIM enables gradual increase of CLK-CYC activity during the day, hence peak accumulation of per/tim mRNA around dusk. Newly translated PER protein is presumably targeted by DBT for degradation, thus it creates a delay between per mRNA and PER protein. Around midnight, accumulated PER and TIM start to translocate into the nucleus. Earlier nuclear accumulation of PER/TIM shortens circadian period, while delayed accumulation slows down the circadian clock. Nuclear PER is progressively phosphorylated by DBT, which maximizes its repressor activity in the late night and early morning (time 24). In the morning, hyperphosphorylated PER is gradually cleared from the nucleus by proteasome mediated degradation, thus allowing a new cycle to start (time 0). Some of these events maybe interlocked, while others are independent of each other. A dynamic assembly of these processes may form the basis of circadian period determination.

Figure I

Mismatch of the endogenous 24 h circadian period with the environmental 20 h light:dark cycle results in morphological changes of medial prefrontal cortex (PFC) neurons in mice. (a) A layer III cell of prelimbic medial PFC neuron labelled with Lucifer yellow. Circadian disrupted mice showed shrunken apical dendrites in the PFC (b) but normal basal dendrites (c). Reproduced, with permission, from [158].